The Daily Mail and General Trust said Monday it expects to shed a thousand jobs in its Northcliffe Media unit, which produces more than 110 local publications.
The trust said advertising revenues at Northcliffe Media were expected to be down 37 percent in the current quarter, worse than the group's overall drop of 24 percent.
The number of job cuts planned in Northcliffe are double the level the group announced in November, and …
It's our one-man's opinion that the reported hanging of Lt. Col.William R. Higgins by mad dog Shiite Hezbollah terrorists points uphow futile has been our intelligence in Lebanon. American hostageshave been held there for years, yet we still have not been able topinpoint their location. If our intelligence had garnered suchinformation, a U.S. commando raid, such as the one Israel pulled offin seizing Sheik Abdul Karim Obeid would have been possible.
UP TO NOW, THE WHITE HOUSE explanation always has been, "We'dlike to send a rescue mission, but we don't know where the hostagesare." Why don't we know, after all these years?
AND WE MUST ASK WHY the terrorists in …
| All Times EDT | |
|---|---|
| DIVISION SERIES | |
| American | League |
| N.Y. Yankees vs. Detroit | |
| Friday, Sept. 30 |
Detroit 1, N.Y. Yankees 1
| Sunday, Oct. 2 |
|---|
Detroit 5, N.Y. Yankees 3
| Monday, Oct. 3 |
|---|
Detroit 5, N.Y. Yankees 4
| Tuesday, Oct. 4 |
|---|
N.Y. Yankees 10, Detroit 1
| Thursday, Oct. 6 |
|---|
Detroit 3, N.Y. Yankees 2, …
Greece's unemployment rate in the third quarter of 2009 jumped to a four-year high of 9.3 percent, the national statistics service said Thursday.
The July-September figure for last year was 7.2 percent. In the second quarter of 2009, unemployment …
The Patchway Workforce should remain steadfast where it rightfullybelongs. I am sure that the announcement that Concorde was not beingused to its full capacity was not altogether surprising in view ofthe present state of air travel.
In that context, too, I wasn't surprised that engine manufacturerRolls Royce was having to make adjustments to its workforce here inBristol and I would presume in its other divisions too.
The days are long since gone when manufacturers bore the brunt ofdownfalls from their own resources.
Now, it seems, if the products are not being sold or the servicesthey provide are not being made use of, there is only one solution -reduce the …
NASA's Ice, Cloud and Land Elevation Satellite (ICESat) lifted off from Vandenberg Air Force Base in California on 12 January.
"The ICESat spacecraft was right where we expected and is performing great. The whole team is thrilled to be having such a wonderful start to our mission," says Jim Watzin, the ICESat project manager at NASA's Goddard Space Flight Center in Greenbelt, Maryland.
ICESat will eventually orbit approximately 373 miles above Earth. It is the latest in a series of Earth Observing System spacecraft, following the recent launchings of the Terra and Aqua satellites.
ICESat was designed to answer questions concerning many aspects of the Earth's climate …
Daisuke Matsuzaka lasted one inning against St. Louis _ the shortest outing of his brief career _ in his return from the disabled list on Saturday. The Red Sox starter, who was placed on the DL on May 30 because of a mild rotator cuff strain, was charged with seven runs in one-plus inning.
Matsuzaka, who left his start May 27 in Seattle with the injury, gave up four runs in the first inning, allowing five hits _ including Aaron Miles' first homer of the season.
He loaded the bases in …
The best-case scenario for the Bears is that they find a team totrade down with in the top 10 of the NFL draft.
That was easy to infer from listening to coach Lovie Smith onThursday. The Bears know what they can get with the No. 4 pick, butthey apparently like the multiple opportunities they would have ifthey slide down a few spots.
There are only three guys going ahead of us," Smith said. We knowat the very worst we can stay there and get a good player that wefeel comfortable with."
Like it or not, staying at No. 4 might be what the Bears wind updoing Saturday. While the Washington Redskins are calling everyone,including the Bears, their intention appears to …
Kurabo Industries Ltd. developed a new type of yarn by combining a two-layer yarn (polyester core is covered with "Supima" long staple cotton) with muraito (a type of uneven yarn). This yarn possesses easy body moisture absorbing and quick drying properties, and is soft to the skin. …
Ford Motor Co. said Friday it is seeking a 2 billion Canadian dollars ($1.59 billion) standby credit line from the Canadian government, and General Motors Corp. and Chrysler LLC are seeking undisclosed amounts as well as the Detroit Three struggle with a worsening economy.
Ford, based in Dearborn, Michigan, said it was seeking the credit line _ "to be used only if the current economic crisis worsens" _ but isn't asking for any money up front.
Chrysler, with headquarters in Auburn Hills, Michigan, wouldn't comment on the amount it was asking for, saying only that the figure was proportional to the $34 billion that the three automakers were asking …
KANSAS CITY, Mo. (AP) — There was no apology from Baylor's Brittney Griner to Texas Tech's Jordan Barncastle at Big 12 media day. In fact, the two didn't talk or even see each other.
Barncastle was on the receiving end of the punch from Griner that went viral last March. The 6-foot-8 dunking sensation broke Barncastle's nose after the two tussled in the lane during a game in Lubbock, Texas.
Griner, who was suspended two games, issued a general apology for her actions, but she hasn't told Barncastle directly that she's sorry.
"I have not crossed paths with her," Barncastle said Wednesday. "The Baylor team went in the early session this morning. Maybe by coincidence, maybe …
The City of Los Angeles is still trying to figure out its violence problem. The mayor of Los Angeles has gone to Washington (twice) to seek more federal funds; gone to Sacramento to get the Govenator to help him terminate violence in Los Angeles. The request? More money for more cops. Every time L.A. is put to the test of how to make the city safer, it gives the same answer. More resources for suppression. The city usually gets the money. But the city doesn't get any safer. Gang membership doesn't go down. After more than twenty years of being near (or at) the top of America's Most Violent City list, you would think the city would develop another answer-a more innovative response with a greater basis for resolution. Nope. The only answer that city officials can come up with is more cops, more weaponry, more jails, more strict "lock 'em up and throw 'em away" laws-in essence, more suppression. Even after they're given another answer-one that makes more sense, one that has produced greater results in other cities and certainly could produce greater results in L.A., city officials have not been responsive to doing anything different than what they've done before.
They say the definition of insanity is "doing the same thing and expecting a different result". More than a month after the Rice Report was presented to a Los Angeles City Council Ad Hoc Committee, not a single recommendation in the report has been followed. In fact, nothing has been done, and the only response to request for help, has been suppression support. Suppression alone can't do it. There is another answer. The question is, why won't city leaders pursue other answers beyond'what we know is not working? Solutions to youth violence don't have to be an "either/or" proposition. It now appears evident that the city just wants to treat it like one. Los Angeles will never pass the violence prevention test for as long as it only has one answer, suppression, to a problem that requires a broader, more compre-hensive approach. So, what gives?
There are really no more excuses for Los Angeles' lack of responsiveness to violence. Arty. Connie Rice did what you asked her to do-study the problem, bring back some possible solutions. Well, she more than studied the problem. She deciphered the city's organizational dysfunction. One that spends $82 million dolors to address 700 gangs and 40,000 gangs members with 61 interventionists. The city budgets a total of $958 million between the city council, the mayor, LAUSD and county government spread over some 78 departments and programs whose org chart looks like a maze of madness instead of a municipal approach to violence prevention. Most of the money never escapes the bureaucracy. As a result, Los Angeles spends 27 cents a day per gang member for intervention, and 24 cents a day per child (of the 300,000 children trapped in the hottest, most dangerous parts of the city, called "gang zones") for prevention. Yeah, it's like that, and I'm puttin' the city on blast. Meanwhile, of the $82 million dollars (a mere pittance for a problem called by experts as an "entrenched epidemic"), $56 million goes to suppression and $4 million goes to prevention. What has it gotten us? Well, over the past ten years, Los Angeles has had over 100,000 shootings, 12,000 homicides and locked up 450,000 youth in local jails and youth detention facilities. We should be safer, right? But consider now, there are six times as many gangs, and twice as many gang members as there were ten years ago. What does that say about suppression as the answer to violence prevention? It says that suppression is not the answer.
Suppression can serve as an appropriate supplement to a more comprehensive approach to violence prevention. Comprehensive approach is a different answer to violence in Los Angeles. Suppression hasn't even proved to be a partial answer to violence in L.A. But we know it has not proven to be the total solution.
If the city is truly in a violence crisis, and it is evident that we are, it would seem that responsible municipal government would be anxiously seeking opportunities to arrest this crisis. We must look at all the options on the table and pursue all answers that make sense. Can we truly say that this has been done? No, we cannot. In one specific instance, we know the City of Los Angeles has a study that has laid out some of the answers. Answers the city has not tried yet. When asked, why? There is just silence. Silence is detrimental to the well-being of the city. I urge city leaders to speak up on the issue of youth violence prevention, and we all suggest that the city look at options beyond suppression to address this issue.
[Author Affiliation]
Black Commentator columnist Anthony Asadullah Samad is a national columnist, managing director of the Urban Issues Forum and author of the upcoming book, Saving The Race: Empowerment Through Wisdom. His Website is www.AnthonySamad.com.
BOSTON - Paul Pierce kept Boston in the game with his scoring, but it was his pass that allowed the Celtics to earn their first victory of the season.
Delonte West made a jumper at the overtime buzzer on a pass from Pierce to lift Boston over the Charlotte Bobcats 110-108 Wednesday night.
"It was the easiest shot I had all night," said West, who had missed seven of his previous nine shots. "That was a gimme. I had to hit that."
Pierce drove the lane with time winding down and kicked the ball to a wide-open West, who knocked down a 20-footer.
"In past years I would have maybe taken a bad shot or tried to go through people," Pierce said. "Once I saw they collapsed on me, I needed to trust my teammates. That is what better players in this league do."
Pierce and Wally Szczerbiak each scored 35 points and Ryan Gomes added his first career triple-double as the Celtics kept the Bobcats winless in Boston.
Gomes had 10 points, 12 rebounds and a career-high 10 assists for the Celtics.
"I don't even think I had a triple-double at Providence, maybe in high school," Gomes said.
Brevin Knight, who finished with 22 points, missed a jumper with 7.6 seconds left in overtime for the Bobcats, who are 0-5 in Boston.
"When a great player makes the correct play, and another good player knocks down the shot, you tip your hat and move on," Knight said.
Gerald Wallace tied the game at 100 with 10.5 seconds left in regulation after Sean May's shot was blocked by Theo Ratliff and on his second rebound, Wallace laid it in.
Pierce's 18-foot jumper as regulation time expired clanked off the side of the rim.
Emeka Okafor continued his strong comeback after playing in only 26 games last season with 28 points and a career-high 18 rebounds.
"You can sing the whole song about how we fought, but we want the win," Okafor said. "We've done this enough times."
Boston overcame 25 turnovers to avoid starting the season 0-4 for the first time since 1969-70.
Pierce grabbed 13 rebounds, but had a Celtics' franchise-record 12 turnovers.
Szczerbiak scored 20 points in the third quarter on 6-of-7 shooting, including four 3-pointers, as the Celtics outscored Charlotte 37-25 to take an 82-74 lead after three. He had 33 points in Boston's last game against Washington.
Othella Harrington's fallaway jumper capped a 13-5 run and tied the game for Charlotte at 87 with 5:16 remaining.
Boston trailed 24-17 after the first quarter while shooting 7-of-23 from the field with eight turnovers.
The last four games between the Celtics and Bobcats have been decided by three points or less.
Notes:@ Celtics forward Al Jefferson is expected to miss at least two weeks after having his appendix removed. ... Raymond Felton left the game with a strained back and did not return for the Bobcats. ... Cedric Maxwell had the previous Celtics record with 11 turnovers in 1978 when they began keeping the statistic. Szczerbiak had a career-high 22 points in a quarter in 2003... Boston used its fourth starting lineup in four games.
WASHINGTON, D.C.-The Soldier's Medal pinned on his chest was not the only honor that Army Reserve Col. J. Edgar Wakayama had for his actions at the Pentagon on September 11 and the days that followed.
In a voice filled with emotion, Wakayama spoke to those who had come to see him receive the Army's highest decoration for non-combat valor.
"For nine days, I worked alongside the great soldiers of the 3rd Infantry, the Old Guard," Wakayama said. "They kept going inside the Pentagon, braving the dangers of further collapse to try to rescue survivors and then to bring out remains. For a little while, I had the honor of being part of the Old Guard."
Wakayama's "tour of duty" with the Old Guard began on the morning of Sept. 11. A Medical Service Corps officer on a one-year tour of duty with the Director for Operational Test and Evaluation (OT & E), Office of the Secretary of Defense, he evacuated the Pentagon after the hijacked airliner crashed into it.
Seeing the dense smoke coming from the crash site, he moved towards it.
What happened next is described in his Soldier's Medal citation:
"He entered the Pentagon three times to search for injured people, leading three to safety on the first trip and several more on the second attempt, before being repulsed by smoke and heat on the third entry."
Unable to get inside the Pentagon again, Wakayama started to treat the wounded on-site. He helped perform triage and administer intravenous solutions. He helped the Red Cross set up a blood draw collection point. For the rest of Sept. 11 and for the next nine days, he worked 12-hour shifts at the recovery site.
Days of being engulfed by the Pentagon tragedy can take a toll on even heroes like Wakayama. That is why the letter he was handed one day by two volunteers meant so much to him. In a way, it was his first honor.
Taking a break at the Camp Unity support site in the Pentagon parking lot, he was given a letter written in a child's hand. Dave and Shirley Hall, members of a group of volunteers from North Carolina preparing and serving food for the recovery workers, passed on the letter from Kayle Madren, a fifth grader at Altamahaw Ossipee Elementary School in Elon College, N.C. Written on Sept. 15, it read:
Dear Heroes,
Thank [you] for being our heroes. I hope you find lots of people. Please save the people that are still alive. Thank you for being our heroes.
Be Careful,
Kayle
Moved by the girl's letter, Wakayama wrote back to her that same day.
"Your kind words truly lifted my spirits during this most difficult time in our country's history," he wrote. "The mood here is sad, but the spirit of people is high because of the many letters received by students like you.
"Kayle, we are all heroes. Heroes are common people who rise during difficult times and yet somehow manage to overcome tragedy. Heroes are students like you who study hard, listen to their parents and teachers, and become good citizens."
Wakayama enclosed a diagram of the crash site, a Pentagon shoulder patch and a colonel's eagle rank insignia with the letter he sent to Kayle.
Kayle was on his mind when he received the Soldier's Medal on March 6 from Mr. Tom Christie, Director of OT &E. Also on his mind was what was taking place that week in the mountains of eastern Afghanistan on Operation ANACONDA and how this related to what he did on Sept. 11.
Wakayama said that the actions of the special operations soldiers who went back in under heavy enemy fire to retrieve a fallen comrade were also in keeping with the creed of Army medics.
"Medics don't leave wounded behind on the battlefield," he said. "For years, I've taught that to my students. When the Pentagon was attacked, I had to put my money where my mouth was."
His actions at the Pentagon clearly show that Wakayama is a teacher who can do as well as he says.
Although Wakayama now wears the Soldier's Medal ribbon on his dress uniform, he will not be able to wear any of the distinctive insignia of the Old Guard. Nor can he attach Kayle's letter to his uniform.
Those honors - serving alongside the 3rd Infantry and what Kayle wrote about Pentagon heroes - will always be present, though. They will be as much a part of the medal as its cloth.
[Author Affiliation]
By Lt. Col. Randy Pullen
[Author Affiliation]
Note: The information about Kayle's letter came from a September 25, 2001, American Forces Press Service article written by Rudi Williams. Lt. Col. Pullen is with the Public Affairs and Liaison Directorate, Office of the Chief, Army Reserve, Washington, DC.
DAILY MAIL STAFF
Silver-suited firefighters climb into the cockpit to rescue anunconscious pilot while another team sprays flames poking out of theaircraft's landing gear and then its engine. A loud boom startleseveryone, but in a few more seconds the fires are out and the pilotis lying on the tarmac.
The pilot - a mannequin - is through for the day, but for morethan two dozen firefighters from the Air National Guard and theMalden and Cottageville volunteer fire departments the day is juststarting.
A few minutes later, the aircraft is on fire again and a secondset of firefighters is learning how to combat airplane fires thanksto the $1.3 million Aircraft Firefight and Rescue Simulator purchasedby the West Virginia University Fire Extension Service.
Built by Symtron Systems Inc. of Fair Lawn, N.J., the airplane-shaped trainer can simulate fires inside and outside of the plane aswell as allow firefighters to practice rescues inside a smoke-filledplane.
Outside the plane, the extension service also has a large, metalmesh device known as "the pit" that simulates a fuel fire.
Joe French, an extension service instructor, said the training isinvaluable for firefighters who may end up fighting an airplane fire.Inside the cabin, they typically face temperatures of 400 to 500degrees.
"They'll get a real taste of what a hot fire is," he said.
French, who retired from the Air National Guard as the assistantchief of the Guard's fire station in Charleston, has 32 years offirefighting experience.
"It's the best training I've ever come across," he said.
The fire extension service has taken the trainer to six airportssince June and plans to make two more stops before storing it for thewinter. French said the trainer can't be used in cold weather becauselow temperatures interfere with some of the safety controls.
Bradley Scott of the Malden Volunteer Fire Department said theonly time he and other volunteers have received this type of trainingbefore is when they traveled to a DuPont plant in Martinsville, Va.
"This training is great. Words just can't express it," he said.
While fighting a plane fire may seem like a stretch for the Maldendepartment, in fact there have been two plane crashes in its firedistrict in the last 10 years and a third that was just outside itsdistrict.
Learning how to fight a fire in the confined space of a plane'spassenger section could well be the training that saves lives in thenext plane accident.
"It's a lot harder than it would be in a residential fire," Scottsaid.
Tech. Sgt. Taylor Daniel of the Air National Guard said thetraining is important for the Guard's firefighters and other airport-based fire crews, but it's just as important for the other firedepartments in the state.
"If we have an aircraft accident, it's probably not going to behere," he said.
Jeff Simpkins, the program coordinator for the fire extensionservice, said the trainer was purchased with a grant from the FederalAviation Administration that paid 90 percent of the cost. The WestVirginia Aeronautical Commission paid the other 10 percent.
The only other states that have similar trainers are Virginia andOregon, he said. The state decided on the purchase before the Sept.11 terrorist attack, he added.
One of its main purposes is to allow fire departments at nineairports in West Virginia to meet an annual training requirementwithout having to travel to Lexington, Ky., or Pittsburgh. Inaddition to saving travel costs, the fire departments can do thetraining with their own equipment instead of borrowed equipment, andeveryone in the department can get the training at the same time, hesaid.
"With this grant, we were able to take the training to them," hesaid.
Writer Brian Bowling can be reached at 348-4842 or by e-mail atbrianbowling@dailymail.com.
A Japanese court overturned two earlier rulings Tuesday and sentenced a man to death for a double murder he committed as a teenager, making him only the third person to be placed on death row for a crime committed as a minor since 1983.
The man, now 27, whose name is being withheld because he was a juvenile at the time of the crime, was found guilty of strangling and raping Yayoi Motomura, then 23, and killing her 11-month-old daughter, Yuka. The Hiroshima High Court ruled that he had posed as a utility company employee to enter their home, indicating the crime was premeditated.
Judge Yasuhide Narazaki said he found "no sufficient reasons to avoid the death sentence," public broadcaster NHK said.
In 2006, the Supreme Court ordered a retrial of the murders, committed in 1999 when the defendant was 18, because the life sentences handed down by two lower courts were too light, according to Tuesday's ruling. It also rejected the lower courts' finding that the killings were not premeditated.
Defense lawyers called the ruling unfair and said they would appeal to the Supreme Court.
Tuesday's closely monitored decision could set a precedent for juvenile crimes, customarily subject to a maximum sentence of life imprisonment. It comes amid a trend toward tougher penalties and a growing number of hangings.
Since the Supreme Court paved the way in 1983 for capital punishment for juvenile crimes, only two death sentences have been confirmed, each involving multiple murders, according to Japanese media reports.
Japan is one of the few industrialized countries that continues to impose capital punishment. It has executed 10 criminals in the past four months under Justice Minister Kunio Hatoyama, an outspoken supporter of the death penalty. Only one inmate was executed in 2005.
In Tuesday's case, the Yamaguchi District Court in March 2000 had sentenced the man to life in prison for the killings, a decision supported by the Hiroshima High Court two years later before the Supreme Court ordered a retrial.
Hiroshi Motomura, the murdered woman's husband, thanked the court Tuesday for "making the right decision," and added, "The punishment gave us an opportunity to think about how we can create a safe society."
A teenager was sentenced to spend 10 years in prison for her partin a robbery and assault that left a man brain damaged.
Telisa Renee McCauley of Mary Street was one of three people whoattacked Robert "Jeff" Moore, 53, of Charleston as he walked throughBrawley Walkway in May 2010. Moore was in a coma for 56 days andcontinues to require daily assistance.
McCauley, Whitney Avery and Earl Moore (no relation) werearrested for the crime.
"I want to give my apologies to Mr. Moore's family as well asmine," McCauley said in court Thursday. "I pray every day for hisforgiveness."
She told the judge she had completed her GED, and said she didn'twant to grow up in prison.
It wasn't enough to sway Kanawha Circuit Judge Paul Zakaib, whosaid he didn't see remorse in the teenager.
Zakaib said, "The defendant always had excuses for her conductand laying the blame on others. She has no meaningful remorse. Shewas asked to help rob the victim and did not hesitate and when hewas hit she relieved the victim of his wallet."
Police said the robbers took $40 from Moore's wallet after hefell to the sidewalk, striking his head. They left him bleeding andfled the scene.
But McCauley's court-appointed defense attorney, CharlesHamilton, said if it weren't for her cooperation the other twoattackers would not have been caught.
"I would like the court to consider that Telisa did not lay ahand on Mr. Moore," Hamilton said. "About a week later, she went tothe barbershop of Earl Moore wearing a wire, with her own personalsafety in jeopardy and broke this case open.
"The only reason anybody knows what happened is because of her,"he said. Hamilton asked for McCauley to be sent to the AnthonyCenter for Youthful Offenders.
Assistant Prosecutor Jennifer Meadows told the judge that thestate was willing to recommend the minimum prison sentence of 10years.
"She saw how badly he was hurt and did not call 911," Meadowssaid.
Avery was sentenced to the Anthony Center and Moore, who hit thevictim, received a 50-year prison sentence.
The victim suffered a severe brain injury and spent time in anout-of-state brain trauma rehabilitation clinic in Ohio. But hisbrother, Sam Moore, said he might need daily assistance for a longtime.
McCauley and Avery were also involved in the attack of anotherman around the same time period. Police said McCauley pulled a knifein that incident.
Before he was injured, Moore worked for West Virginia LegislativeServices for more than 24 years.
CARMARTHENSHIRE Council has received one of the best budgetsettlements in Wales.
The Provisional Local Government Revenue Settlement was publishedlast week by the Assembly Government, following the announcement ofthe draft budget.
It shows that Carmarthenshire Council will face a cut of 1.3 percent over the coming year.
This places it in the top five of the 22 Welsh authorities interms of the smallest budget cuts.
In England, local government is facing a cut of 7.1 per cent ayear for the next four years.
Cut The average cut for Wales is 1.4 per cent, with Ceredigionand Pembrokeshire facing cuts of 0.7 per cent and 1.7 per centrespectively.
Carmarthenshire Council will receive Pounds 249 million in corefunding from the Assembly Government in 2011-12 to support services,equivalent to Pounds 1,349 for every man, woman and child living inthe area.
In addition to this, Pounds 11.4 million will be available tosupport capital projects in Carmarthenshire.
The council will also receive specific grants to support a rangeof activities including early years and post-16 education, refusedisposal and recycling.
Local Government Minister Carl Sargeant pledged more cash wouldgo to schools and social services over the next three years.
Mid and West Wales Plaid AM Nerys Evans said: "It's welcoming tosee that the overall cut for councils in Wales is much lower thanpreviously anticipated.
"The Welsh Government has prioritised the needs of children, theelderly and vulnerable people, which has meant that a level ofprotection has been secured for social services and schools."
A spokeswoman for Carmarthenshire Council said: "We are pleasedthat the Welsh Assembly Government has recognised the importance offrontline local government services to the people of Wales, and hasprotected schools and social care."
Protected She warned: "There will still be very difficultdecisions to be made over the next three months or so as we striveto find many millions of pounds of savings and deal with the everincreasing pressures on adult social care, children's services andschools.
"Carmarthenshire Council will be seeking to protect the mostvulnerable members of society, support the education of our youngpeople and continue to regenerate our housing and economy."
Scoreboard at tea Thursday on the second day of the first cricket test between New Zealand and India at Seddon Park:
New Zealand, 1st Innings
Tim McIntosh c Sehwag b Sharma 12
Martin Guptill c Dravid b Zaheer 14
Daniel Flynn c Dhoni b Zaheer 0
Ross Taylor b Sharma 18
Jesse Ryder c Laxman b Sharma 102
James Franklin c Dhoni b Sharma 0
Brendon McCullum c Laxman b Patel 3
Daniel Vettori c Dhoni b Patel 118
Kyle Mills b Patel 0
Iain O'Brien std Dhoni b Harbhajan 8
Chris Martin not out 0
Extras: (1lb,3nb) 4
TOTAL: (all out) 279
Overs: 78.2. Batting time: 358 minutes.
Fall of wickets: 1-17, 2-17, 3-40, 4-51, 5-51, 6-60, 7-246, 8-246, 9-275, 10-279.
Bowling: Zaheer Khan 16-3-70-2 (2nb), Ishant Sharma 19.2-4-73-4, Munaf Patel 18-4-60-3, Harbhajan Singh 22-7-57-1, Virender Sehwag 3-0-18-0 (1w).
India, 1st Innings
Gautam Gambhir c McCullum b Martin 72
Virender Sehwag run out 24
Rahul Dravid b O'Brien 66
Sachin Tendulkar not out 25
V.V.S.Laxman not out 4
Extras (1b,2lb,3nb) 6
TOTAL (for three wickets) 197
Overs: 61. Batting time: 261 minutes.
Fall of wickets: 1-36, 2-142, 3-177.
Still to bat: M.S.Dhoni, Yuvraj Singh, Harbhajan Singh, Zaheer Khan, Ishant Sharma, Munaf Patel.
Bowling: Chris Martin 14-4-34-1, Kyle Mills 13-2-53-0 (1nb), Iain O'Brien 13-2-37-1, James Franklin 8-0-33-0, Daniel Vettori 12-1-37-0 (2nb), Jesse Ryder 1-1-0-0.
Umpires: Simon Taufel, Australia, and Ian Gould, England.
Match referee: Alan Hurst, Australia. TV umpire: Gary Baxter, New Zealand.
Toss: won by India.
YORK COUNTY
The practice of Six Sigma quality management is not novel in the busi- ness world. But the way companies and individuals receive training on the technique has begun to change.
Six Sigma can help a company refine a business process to make it more predictable, said Michael Smeltzer, executive director of the Manufacturers Association of South Central Pennsylvania. It is a tool that firms can use to improve the quality of their products by identifying problems and reducing the potential for mistakes, he said.
York College of Pennsylvania responded to the ever-increasing popularity of Six Sigma last year by beginning to offer full courses on the specialty within its Master of Business Administration (MBA) program.
"I thinkwhat's cool about what York College is doing is they're embedding it into their MBA program," Smeltzer said. "Often today, Six Sigma training occurs after someone is on the job. What York College is doing is the absolute right thing to do. It just makes perfect sense."
The topic has been touched on within graduate courses over the years, but semester- long discussions of lean operations practices is new, said Eric Hostler, director of the MBA program at the college.
The college plans to phase in a couple more classes in the practice over the next year or two so it can offer a three-course concentration in Six Sigma within the MBA program, Hoster said.
"As students are completing their MBA, if they're working in operations or supply-chain management, itwill be beneficial to them to have additional information on it," he said. "At the end, if they're interested in getting a certification, they will have most of the background and knowledge needed to sit for the exam."
Six Sigma has been evolving for several decades, becoming more specific and complex, Smeltzer said. It is not limited to manufacturing. It can be applied to any industry, including health care and banking, he said.
"It's brought more of a scientifically controlled approach to our manufacturing processes," he said. "It's all toward the goal of 100 percent quality products."
Six Sigma is more likely to be found implemented at larger companies. It is more difficult for smaller firms to use the process because they would struggle to get a return on the training investment it would require, Smeltzer said. Big companies such as HarleyDavidson Inc. probably could apply the principals to hundreds or even thousands of processes, he said. Larger firms also could afford to keep an expert on site to train employees and manage the implementation of the processes, he said.
Dentsply Caulk began using lean Six Sigma almost a decade ago, said Andrew Johnson, director of operations for Caulk, a division of Yorkbased dental-product manufacturer Dentsply International Inc. The Caulk division is based in Milford, Del., and manufactures products that dentists use to repair teeth, including cements and whiteners.
Lean Six Sigma is a way that Dentsply Caulk combines the practices of Six Sigma with that of lean manufacturing, which works to eliminate the use of resources that do not help create value.
When Caulk first began implementing Six Sigma processes, it hired private consultants and worked with state agencies to train its work force. The practices might be taught over the course of a week, with a couple of days dedicated to teaching the tools of Six Sigma and the rest of the week spent testing the process on a project, Johnson said.
In 2007, the company invested in the creation of an internal department dedicated to lean Six Sigma principals that have been customized for the dental industry, he said. The group employs five people and uses Six Sigma to improve Dentspl/s processes and to train the company's employees. The department has trained and worked with other divisions of Dentsply, including the firm's prosthetics division and Caulk's sales force, both of which are based in York, he said.
After Caulk used Six Sigma to improve its supply chain processes, including quality assurance, purchasing and warehousing, it broadened its perspective in 2006, Johnson said. The company now applies the principles to marketing research, product development and sales support, he said.
[Sidebar]
"It's brought more of a scientifically controlled approach to our manufacturing processes."
Michael Smeltzer, Manufacturers Association of South Central Pennsylvania
[Author Affiliation]
BY JESSICA BAIR
jessicab@journalpub.com
A suicide attack at a police station in northwestern Pakistan has killed an officer and wounded two civilians.
Police official Ghulam Farid said the bomber Saturday rammed an explosive laden vehicle into the wall of the station in Tank district.
He says at least two more people are believed to be buried in the rubble.
Tank is close to the Afghan border region of South Waziristan, a Taliban stronghold where the Pakistan army began a major offensive in mid-October.
THIS IS A BREAKING NEWS UPDATE. Check back soon for further information. AP's earlier story is below.
PARACHINAR, Pakistan (AP) _ Militants ambushed Pakistani security forces at checkpoints near the Afghan border Saturday, sparking gunbattles that left 22 insurgents and two troops dead, officials said.
Government officials Mohammad Yasin and Mohammad Naseem said two more troops were wounded in the clashes in the Orakzai and Kurram tribal regions. They said a search and clearance operation launched after the clashes also seized 25 suspected insurgents.
The force commander in Kurram, Col. Tausif Akhtar, told reporters the troops had cleared six villages of Taliban fighters. "We have also cut a main route the militants would use to enter the region," he said.
Many militants fleeing a Pakistani military offensive in the Taliban stronghold of South Waziristan have ended up in the two regions, where they often target government forces.
Washington has welcomed the military campaign but is pushing the Pakistani army to do more to target the Taliban blamed for violence across the border in Afghanistan. The Pakistani army has said it is too taxed to launch another operation right now.
"We have gone in Orakzai and Kurram because they were affecting our operations in South Waziristan," Pakistani army spokesman Maj. Gen. Athar Abbas told DawnNews TV on Friday night. "We are too thin on the ground. We are too over stretched. It is not possible to get into any other area for operations."
The army deployed some 30,000 troops against the Pakistani Taliban in South Waziristan in mid-October and has retaken many towns in the region. But many fear the militants have just set up in other parts of the vast, lawless border regions and will continue to threaten the Pakistani government and U.S. troops in Afghanistan.
Meanwhile, Interior Minister Rehman Malik said Saturday that a paramilitary soldier had been arrested for involvement in the Oct. 5 suicide attack the U.N. food agency's office in Islamabad that killed five staffers.
Pakistani Taliban at the time had claimed responsibility for targeting the World Food Program, saying the agency's work was not in "the interest of Muslims."
Maik didn't reveal identity of the man, but said he was also involved in the Dec. 2 suicide attack outside the entrance of Pakistan navy's headquarters in Islamabad that killed one guard and wounded 11 other people.
Pennsylvania's 10 casinos collected $211.3 million in revenue last month, up more than 8 percent over the same month last year, the state gaming Control Board said.
The state received $115.4 million in tax revenue from slots last month, the board said.
The nine casinos that were open in April 2010 produced $196.8 million in revenue last month, compared with $195.3 million during the same month last year.
Dauphin County's Hollywood Casino at Penn National Racecourse saw a small loss in April comparative slots revenue. Hollywood collected $22.4 million in revenue last month, down from $22.5 million it generated during April 2010, the board said.
Table game revenue is separately reported.
-Eric Veronikis
Manchester United goes into the second leg of the League Cup semifinals against Manchester City with Wayne Rooney in the sort of form that helped the team decide to let Carlos Tevez leave.
Tevez has rediscovered the touch that led United to sign him in 2007 with 17 goals in 26 matches for City this season, including two in last week's 2-1 first-leg win over his old side.
But Rooney is on course for his most productive season ever, with his four goals in Saturday's 4-0 win over Hull taking United back to the top of the Premier League and his total to 20 in 28 matches.
With defending League Cup champion United having beaten City 4-3 at Old Trafford already this season, City and Tevez may not have everything their own way on Wednesday.
"The game against City will be very hard, but it's great that we have someone like Wayne who is in good form and feeling confident," United winger Nani said. "It's great to see one of your strikers score four goals and it gives everyone confidence because we know we have someone who can score goals in every match."
The first leg was defined by Tevez's two goals and the reaction to his celebration in front of the team benches.
Gary Neville reacted with a middle-finger gesture at his former teammate and Tevez subsequently insulted the United captain in an Argentine radio interview.
The Football Association warned both players over their conduct and told the clubs to show restraint in their behavior on Wednesday, while Tevez's agent, Kia Joorabchian, tried to downplay the rift.
"If you translate a slang terminology into an actual word, it may turn out to look like a very bad word," Joorabchian said. "In reality, it might not be such a bad word."
United was buoyed at the weekend by an unchallenging return to action after three months out for Rio Ferdinand and the defender should be there to help monitor Tevez.
But even if United reaches the Feb. 28 final, it will almost certainly have to deal with the loss of the league lead.
Second-place Arsenal is at Aston Villa on Wednesday, when third-place Chelsea hosts Birmingham.
The London pair are just two points behind the defending Premier League champions, but Arsenal has played one game fewer and Chelsea has two games in hand.
Arsenal manager Arsene Wenger will revert to his regular lineup for the first of four tough games after fielding largely a second-string side in Sunday's 3-1 FA Cup loss at Stoke.
"I do not regret the side I put out," said Wenger, whose team plays Villa, United, Chelsea and Liverpool in consecutive league fixtures over the next two weeks. "I did not have much choice. We had 10 injuries and a very difficult program coming up."
Also Wednesday, Wigan is at Blackburn and Sunderland is at Everton.
In Tuesday's games, Burnley visits Bolton, West Ham is at Portsmouth, Tottenham hosts Fulham and Liverpool is at Wolverhampton.
Tottenham may need victory to keep hold of fourth place, with Liverpool just a point back after beating Spurs 2-0 last week.
I Am writing about parking on the pavement in the car park of Ikeaon Sunday.
The car park was full; a car was even parked on the footpath byNapier Road, so that even mothers with prams could not get out.
I got in touch with Ikea and we had to wait for someone to move acar so we could get out.
I Turner (Mrs), Eastville.
ABSTRACT
We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.
INTRODUCTION
Fluorescence correlation spectroscopy (FCS) (1) probes dynamical processes in fluorescent species over the large range of timescales from nanoseconds to seconds. By introducing a sufficiently small confocal volume to FCS, single molecules can be detected (2), and the applications of FCS to analysis of biological processes have thereby multiplied (3). FCS has been proposed as a way to analyze rare species (4,5). Unfortunately, its usefulness can be limited in cases where multiple fluorescent species contribute to the same detection channel, contaminating the signal from a species of interest. If the dynamical processes of the contaminating species occur on similar timescales with the species of interest, it is very difficult and sometimes impossible to distinguish between contributions from different species. The correlation function for any minor species is obscured by contributions from other, more abundant species.
For example, we are performing solution-based single molecule experiments that monitor a DNA sliding clamp protein as it moves on DNA (β-clamp of Escherichia coli), by monitoring fluorescence energy transfer (FRET) between a donor (D) fluorophore on the β-clamp and an acceptor (A) at a specific location on a DNA plasmid. FRET is caused by the nonradiative transfer of excitations from D to A when they are in close proximity (within ~5 nm). We perform these single-molecule FRET measurements of the dynamic complex of the β-clamp on DNA using alternating laser excitation (ALEX) (6) in the presence of free plasmids and free β-clamp proteins. With ALEX, three photon streams or channels are available: photons detected from donor fluorescence resulting from excitation by the donor excitation laser (hereafter, "donor channel"); acceptor-emitted photons detected in the acceptor channel that are the result of FRET excited by the donor excitation laser ("FRET channel"); and photons detected in the acceptor channel, that are the result of acceptor fluorescence, excited by the acceptor excitation laser ("acceptor channel"). In addition to signals from complexes undergoing FRET, the FRET channel contains contaminating signals caused by leakage of the donor emission into the acceptor channel and by direct excitation of the acceptor by the donor excitation laser (Fig. 1), Although both of these problems must be considered, the former is amplified in our experiments by aggregates of the β-clamp protein causing bright fluorescence bursts that leak into the acceptor channel. These bright bursts can appear indistinguishable from bursts caused by actual FRET. Autocorrelations performed on the FRET channel, therefore, have contributions from FRET and these contaminating sources, calling into question any conclusions drawn from correlation analysis, especially in the case where complexes are observed less frequently than the free components.
The cross-correlation (7) between the FRET channel and the acceptor channel obtained from the acceptor laser excitation (8) eliminates contributions from the free protein and aggregates since those species are not excited by the acceptor excitation laser. However, fluctuations in FRET efficiency, which should reveal the protein-DNA intermolecular movement sought in these experiments, are unobservable by a cross-correlation between the FRET channel and acceptor channel. This is because the acceptor signal excited by the acceptor laser is only correlated with diffusion in and out of the detection volume, not fluctuations in the FRET efficiency. Hence, this cross-correlation reflects only the fluctuations caused by the translational diffusion of the complexes in and out of the detection volume.
Solution-based single-molecule fluorescence spectroscopy (SMFS) uses ratiometric variables and fluorescence lifetime measurements to allow for the identification and sorting of many species in complex mixtures (9-12). Signals from single molecules are detected by searching for "bursts" of photons with signal intensities above a preset threshold level, determined by the background levels and expected signal intensities. The recently introduced alternating laser excitation (ALEX) of single molecules allows sorting of species based on distance and association (6,13). Using ALEX, β-clamp-DNA complexes are easily distinguished from free components by searching for fluorescence bursts in the FRET channel. Only those bursts that have coincident bursts in the FRET channel and in the acceptor channel are due to complexes. Any burst with a coincident large burst in the donor channel is due to a β-clamp aggregate.
Although SMFS and FCS often use the same experimental setups and samples, techniques that take advantage of the power of SMFS to sort species while simultaneously using the ability of FCS to probe temporal dynamics remain underdeveloped. Selective fluorescence spectroscopy (10), which selects single molecule bursts for further correlation analysis, is the most advanced technique in this direction. The region over which the correlation is calculated is truncated at the beginning and end of the burst, allowing for analysis of fluctuations within the timescale of the bursts. To maximize the timescales monitored using the correlation analysis, only the brightest (>200 kHz) and longest single molecule bursts (>70 ms) are selected. These exceptional bursts correspond to fluorescent molecules that remain in the detection volume the longest. Nevertheless, the truncation used in the analysis prevents correlation analysis of fluctuations on the same timescale of the burst, including, for example, translational diffusion into and out of the optical detection volume.
The approach taken here also uses a selection of bursts, but it differs from selective fluorescence spectroscopy in two ways. First, the burst selection criteria are not as restrictive; we use much lower thresholds (5-15 kHz thresholds are typical), and allow much shorter bursts, only requiring sufficient signal over a 10 ms time bin. More importantly, the correlation calculations are not truncated at the burst edges. By expanding the region of the correlation around detected bursts, we introduce a way to use SMFS sorting to analyze temporal dynamics of specifie species, including translational diffusion into and out of the optical detection volume, using standard FCS fitting equations (14,15). Truncation of the signal is moved to regions uncorrelated with the signal from the selected burst, allowing the functional forms of FCS fitting functions to remain unchanged except for a multiplicative factor.
Thus, by selecting only those bursts that are due to the species of interest and averaging the resulting correlations over all selected bursts, we can "purify" the signal of interest. This purification eliminates contributions both from leakage of the donor emission into the acceptor channel and direct excitation of the acceptor (Fig. 1 ). Due to the exclusion of contaminating signals, autocorrelations of the FRET channel calculated after signal purification may be used to study the fluctuations of an individual species. Signal purification may also be used for photon arrival-time interval distribution (PAID) functions (16) in the same way as for FCS. We call our method of purifying correlations signals by performing correlations over selected bursts "purified FCS", or PFCS. We will refer to performing correlations only over the photons in the truncated single molecule bursts without correlation region expansion as "selective FCS", or SFCS.
Here we demonstrate the use of PFCS using our /3-clampDNA experiments. We also investigate how precisely a diffusion time can be extracted from the correlation calculated for a single burst. We call the method of analyzing FCS for single molecule bursts "single-molecule FCS". Here, single-molecule FCS will be applied to experiments containing two species-one of free labeled DNA oligomers, the other of those oligomers hybridized to ssDNA plasmids. In this application of our method we show that many DNA oligomers weakly bound to plasmids during hybridization reactions with excess DNA oligomer are removed by gentle heating (at 37�C) of diluted solutions of the hybridized DNA.
THEORY
Purified FCS with correlation region expansion
We illustrate our new method through a simulation of an experiment of two interacting proteins, as shown in Fig. 2. In the simulated experiment, we monitor the fluctuations of the emission in the FRET channel from the intermolecular interaction between a protein labeled with a donor fluorophore D and a second protein labeled with an acceptor fluorophore A (Species 1 in Fig. 2). The emission in the FRET channel is contaminated by the presence of aggregates of the D-labeled species (Species 2 in Fig. 2). The D emission from Species 2 leaks into the FRET detection channel, leading to bursts that appear similar to those from Species 1 (the acceptor detection channel excited by the acceptor excitation laser is not simulated). Using values chosen to correspond roughly to the values found in our β-clamp experiments, Species 1 and 2 are both present with a molecular occupancy of c^sub 1^ = c^sub 2^ = 0.05 in the detection volume, and a molecular brightness of q^sub 1,FRET^ = q^sub 2,FRET^ = 35 kHz in the FRET channel. The molecular brightness is the number of photon counts per second received from a single fluorescent molecular species, averaged over the confocal detection volume. In the donor channel, Species 2 has a brightness of 172,0 = 141 kHz and Species 1 has a brightness of q^sub 1,b^ = 0 kHz (the leakage of A into the donor channel is negligible, and will not be considered further). Due to translational diffusion through the optical detection volume, each molecular species is associated with a characteristic "diffusion time", i.e., the average time a molecule remains in the detection volume. The diffusion times are τ^sub D,1^ = 3 ms and τ^sub D,2^ = 6 ms for Species 1 and 2, respectively. Species 2 is distinguished from Species 1 by the presence or absence of a coincident burst in the donor channel.
Bursts from Species 1 and 2 are distinguished using single molecule fluorescence analysis. Single-molecule fluorescence bursts are identified using the burst search method described in Kapanidis et al. (6), with the addition of a medianbased background subtraction (Materials and Methods). A histogram of FRET efficiency ratio E (proximity ratio) for all bursts (17) clearly shows two subpopulations (Fig. 2 b).
The data recorded for photon-timing SMFS/FCS experiments are series of photon time stamps with time-resolution Δt. The value t^sub i^ is the arrival time of the i^sup th^ photon from channel A, and u^sub j^ is the arrival time of the j^sup th^ photon from channel B. Assuming stationarity, the ensemble averages in the expression for C^sub AB^(T) are converted to averages over all time. Averaging over a finite experimental time T with N^sub A^ and N^sub B^ photons detected in the respective channels gives a correlogram C^sub AB^(τ), an estimate of the actual correlation function.
If we combine correlations performed only on photons within bursts, the functional forms for the correlation do not match the FCS fitting equation in Eq. 2 on timescales near the burst width (Fig. 2 c). The autocorrelation of the FRET channel for the whole experiment fits well to the singlecomponent FCS model in Eq. 2 with a diffusion time of 4.2 � 0.1 ms (solid squares, simulation data; solid line, fit). FCS was not able to distinguish the two diffusing species present with a factor-of-2 difference in diffusion times. Using selective FCS (truncating correlations at burst edges) for all of the bursts from Species 1 or Species 2, we find a difference in the diffusion time between the two species (light shaded circles and dark shaded triangles in Fig. 2 c). The autocorrelation of the FRET channel for bursts from Species 1 (dark shaded region in Fig. 2 b) is fit by Eq. 2 with a diffusion time of 1.7 � 0.1 ms (dark shaded triangles and curve in Fig. 2 c). The autocorrelation of the FRET channel for bursts from Species 2 (tight shaded region in Fig, 2 b) is fit by Eq. 2 with a diffusion time of 3.3 � 0.1 ms (light shaded circles and curve in Fig. 2 c). Unfortunately, both fits are poor, and the extracted values do not match the simulation values put in.
The primary problem encountered in Fig. 2 c is that burst searching routines select only those parts of the signal that are bright. The selected time regions have widths on the same timescale as the diffusion time, truncating a significant amount of correlated signal. To properly characterize the signal fluctuations, the timescale over which the correlation function is performed must be longer than the timescale of the fluctuations themselves. We introduce a simple way to do this: expand the region of the correlation function around the burst so that the region has a time-width much longer than the diffusion time (see Fig. 2 d). We expand it here by 10-fold (i.e., 100 ms) on either side of each burst. We expand enough to allow the correlation functions calculated to have the same functional form as the standard FCS fitting model in Eq. 2. We do not expand too much, so that we can exclude unwanted single molecule bursts from other species. We also want to exclude contributions from more persistent fluctuating signals, such as leakage from low-intensity, higher concentration signals. The correlations are calculated as in Eq. 5, except that now k is an index for expanded correlation regions rather than just the time of the bursts.
There is one change in the functional form in Eq. 2 for purified FCS due to the selection only of regions with bursts. FCS detects the molecular occupancy by comparing the variance and mean of the signal intensity. We are selecting regions that contain single molecule bursts, so the mean and variance of the signal intensity of the selected regions are different from the mean and variance for the entire experiment. Hence, the normalization as shown in Eq. 5 does not work properly. We use a multiplicative correction factor � as a parameter in all of our fits, accounting for this problem.
There are two uses for the expanded correlation regions for selected bursts. First, one may select only those correlation regions containing bursts of a specific species, excluding bursts from other species as well as leakage of higher concentration species into the channel of interest. The correlations for all regions can be averaged according to Eq. 5, obtaining the purified correlation function for a selected species. This methodology is an example of the use of singlemolecule fluorescence to sort molecules for later subensemble analysis (13). Second, one may fit the correlations for individual regions to an FCS model, and the distribution of fitted diffusion times may be used to directly observe the distribution of diffusion times in the sample. This "singlemolecule FCS" is described later.
There are two clear limitations to this method. First, PFCS is limited to cases where the methods of single molecule spectroscopy can distinguish the species involved; there must be some distinguishing parameter such as E that clearly reveals two or more subpopulations. second, the concentration of fluorescent molecules monitored must be low so that bursts from multiple species are not included in the correlation region. In the correlation function example, handling cases where only one or two additional bursts are within the expanded correlation region is not difficult. In Fig. 2 d, the correlation region was expanded around the central burst from Species 1, and includes an additional, earlier burst. Since that burst is also from Species 1, that region is included in the analysis. However, in Fig. 2 e the expanded correlation region included a burst from Species 2, and that region is excluded from the analysis. Similar rules can be developed for a specific experimental situation.
Fig. 2 f shows that the purified correlations calculated for Species 1 and 2 match the correlations expected for those species if they were alone in solution. The correlation function for regions of interest containing bursts from Species 1 (dark shaded region in Fig. 2 b) is well fit by a singlecomponent model with a diffusion time of 3.0 � 0.1 ms (dark shaded triangles and curve in Fig. 2f). The correlation function for regions of interest containing bursts from Species 2 (light shaded region in Fig. 2 b) is well fit by a singlecomponent model with a diffusion time of 5.6 � 0.1 ms (light shaded circles and curve in Fig. 2f). The fitted values for the diffusion times are within 10% of the simulation input values.
Another situation in which PFCS may be used is the case in which Species 1 is again a complex undergoing FRET at single molecule concentrations, but Species 2' is a nonaggregated donor-labeled protein present at higher concentrations. The donor emission from Species 2' again leaks into the FRET channel, but now presents a low-intensity, fluctuating background that contributes to the FRET autocorrelation function. For PFCS to work in this situation, the species of interest must be significantly brighter than the fluctuating background. For these simulations, we used the same parameters for Species 1 as before, but with a lower molecular occupancy of c^sub 1^ = 0.02. We replace Species 2 with Species 2', the molecular occupancy is c^sub 2^ = 2.5, the brightness in the FRET Channel is q^sub 1-FRET^ = 2.3 kHz (15-times smaller than for Species 1), and the diffusion time is τ^sub D.2'^ = 600 �s. By selecting correlation regions around large fluorescence bursts in the FRET channel (as shown in Fig. 2), we effectively concentrate the signal of interest, excluding most of the experimental time where only the leakage signal from Species 2' is present. The relative contribution of each species to the amplitude of the autocorrelation functions of the FRET channel can be calculated as A^sub i^ = c^sub i^q^sup 2^^sub i,FRET^, the contribution to the numerator in Eq. 3. For the autocorrelation of the FRET channel for the whole experiment, the amplitudes are A, = 24.5 kHz^sup 2^ and A'^sub 2^ = 13.6 kHz^sup 2^. After using PFCS, the amplitudes are A^sub 1^ = 183 kHz^sup 2^ and A'^sub 2^ = 13.6 kHz^sup 2^, increasing the contribution of Species 1 to the correlation amplitude from 64% to 93% of the total correlation amplitude. For the autocorrelation of the FRET channel over the entire simulated experiment, we obtain a diffusion time of 2.1 � 0.1 ms with a poor fit to Eq, 2. Using PFCS to exclude most of the fluctuating background, we obtain a good fit to the autocorrelation of the FRET channel with a diffusion time of 2.9 � 0.2 ms, matching the simulation value.
One important feature of PFCS is that the concentration of the species of interest does not affect the correlation obtained, except for the total experimental time h takes to obtain the correlation. The purity obtained (93%) for the correlation amplitude of Species 1 is lower than 100% since the contaminating, fluctuating background is always present. This upper limit on purity depends on the concentration and brightness of Species 2' and on the brightness of Species 1, but not on the concentration of Species 1. As long as bursts can be identified, purified correlations may be obtained. For example, if we reduce the molecular occupancy of Species 1 from 0.02 to 0.005, the amplitude of the autocorrelation of the FRET channel decreases from A^sub 1^ = 24.5 kHz^sup 2^ to A^sub i^ = 6.3 kHz^sup 2^, Under these conditions, only 20% of the correlation amplitude comes from Species 1, and the measured diffusion time is 1.1 �0.1 ms, close to the diffusion time of Species 2'. Using PFCS, the amplitude increases to A^sub 1^ = 172 kHz^sup 2^. Hence, Species 1 comprises 92% of the PFCS correlation amplitude (nearly identical to the 93% obtained above), and the diffusion time extracted is 2.8 � 0.2 ms, close to the simulation value for Species 1.
We have demonstrated that PFCS can purify correlations for species present at single molecule concentrations with a distinguishing parameter. PFCS can also purify correlations when a low-intensity fluctuating background (caused by leakage of other fluorescence signals) is present. Unfortunately, there is currently no elegant, general theory for analyzing the effects of burst analysis on calculated correlations. This makes a quantitative theory of PFCS difficult to obtain. In place of such a general theory, we recreate experimental situations in simulations, and test for the accuracy of the PFCS methodology. For new experimental situations that differ significantly from the above simulations, it will be necessary to perform new simulations that match those conditions. One example is applying PFCS to species that are not as well separated by the E histogram, such as the folded and unfolded states of proteins (20).
Biases in fitted parameters using purified PCS
We now show that, for reasonable burst search thresholds, there are no large biases in the extracted fitting parameters. A previous work (21) describes how the detected diffusion time for a single burst depends on the threshold. High thresholds tend to increase the detected diffusion time, since bursts with larger numbers of photons tend to be those events that stayed in the detection volume longer. This implies that, for PFCS, there is a balance between selectivity from a higher threshold and lower bias obtained with a lower threshold. We investigate here the effects of the burst search threshold on PFCS. We also investigate the use of our expanded burst selection regions for the photon-arrival interval distribution (PAID) function (16), which adds an additional dimension of photon counts to the correlation function.
In Fig. 3, we illustrate the biasing effects of burst selection on fitted parameters. To quantitatively analyze bias, the simulation in this example contains only a single species. Ten simulations of 60 s each were performed with molecular occupancy c = 0.1, diffusion time τ^sub D^ = 3 ms, brightness q = 35 kHz, and background Abkgd = 1 kHz. We plot the fitted parameters as a function of burst search threshold, ranging from 5 kHz to 45 kHz over 10 ms bins (Fig. 3, a-d). For fitted diffusion time τ^sub D^ and brightness q, an upward bias is seen as the threshold is raised, both in FCS and PAID fits (Fig. 3 b). However, this bias is small (within 5%) even for a significant threshold (up to 15 kHz). Hence, purified FCS and PAID do not introduce unreasonable bias in the fitted parameters as long as the burst search threshold is below the average burst intensity.
In Fig. 3 c, we plot the fitted occupancy c from PFCS and purified PAID, and fitted background level k^sub bkgd^ from purified PAID. FCS values for c are higher since FCS cannot distinguish between increases in k^sub bkgd^ and increases in c. The fitted occupancy values are less consistent than the values for τ^sub D^ and q. This is not surprising, since the mean and variance of the signal intensities are affected by the correlation region selection process, and FCS detects the molecular occupancy by comparing the variance of the signal intensity with the signal mean. The fitted background from PAID drops off nearly linearly, vanishing at high thresholds. Since we are excluding regions that contain only background, this is not surprising. The fitted correction factor a decreases as the threshold is raised (Fig. 3 d). The χ^sup 2^ values for the fits are near 1 for all of the FCS fits. However, the χ^sup 2^ for PAID increases to high values for higher thresholds. The burst selection changes the shape of the PAID function (see Fig. 3, e and f). The largest change is a decrease in the correlation amplitude to the lower right of the main peak, accounting for the lower fitted value for k^sub bkgd^. The main peak is largely unchanged, accounting for the slow change in q with threshold. The changes are due to the exclusion of regions with only background. Although Fig. 3 b shows that purified PAID may be used to extract accurate values of τ^sub D^ and brightness g for a single species, analysis of multiple subspecies with different q cannot be performed unless the PAID function model is changed to account for the burst selection.
Cross-correlations are often used in FCS to determine binding of two labeled, interacting molecules (7). In SMFS, ratios of fluorescence intensities from single bursts have also been used to determine the extent of binding (22). It is possible to use PFCS to select a species using ratios from SMFS, and to calculate cross-correlations of that species. We find that the selection of bursts with a specific ratio does not introduce spurious cross-correlations for timescales below the burst search timescale, allowing PFCS to distinguish bound molecules from random coincidence.
Fig. 4 shows the effects of burst selection on crosscorrelation experiments. Two sets of 10 simulations of 60 s each with three species were performed. There are two detection channels A and B, with background levels k^sub A,bkgd^ = k^sub B,bkgd^ = 1 kHz . In both sets of simulations. Species 1 is present with molecular occupancy c^sub 1^ = 0.05, diffusion time τ^sub D,1^ = 3 ms, brightness q^sub A,1^ = 35 kHz in Channel 1, and qB1 = 0 kHz in Channel 2. For Species 2, C^sub 2^ = 0.05, q^sub A,2^ = 0 kHz, q^sub B,2^ = 35 kHz, and τ^sub D,2^ = 3 ms. Species 3 simulates binding of Species 1 with Species 2, with q^sub A,3^ = 35 kHz and q^sub B,3^ = 35 kHz, and τ^sub D,3^ = 3 ms. In the first set of simulations, C^sub 3^ = 0; in the second set, C^sub 3^ = 0.005. The burst search routine searched for consecutive 10-ms time bins where the sum of counts for both channels is above 5 kHz. In the first set of simulations, there are two species that emit only in one channel each, with no crosstalk. The ratio of the intensity in one channel over the sum of both channels, r = I^sub A^/(I^sub A^ + I^sub B^) is a bimodal distribution (shaded line, Fig. 4 b), The events with 0.3 < r < 0.7 are caused by random coincidence. In the second set of experiments, a third, minor species depicting bound molecules of Species 1 and 2 was added that emits in both channels equally (solid line. Fig. 4 b). A small peak in r near 0.5 is observed.
A cross-correlation of the whole experiment produces a flat line for the first set of simulations (i.e., no crosscorrelation, the dashed shaded line in Fig. 4 b), and a positive correlation for the second set of simulations (dashed solid line in Fig. 4 b). We first select only those bursts with 0.3 < r < 0.7, but do not further exclude any regions that contain other bursts outside this range in r. The first set of simulations produces a flat line (shaded line), and the second set of simulations produces a positive correlation (solid line), The shaded line is above 1.0 because of the modified normalization as discussed earlier. There is a drop in the crosscorrelation at long timescales (> 10 ms) that is introduced by the burst selection. Even if the coincidence of bursts in both channels is only due to random coincidence, the crosscorrelation can detect this as revealed by the drop at long timescales (shaded line). Although this must be accounted for in any experiments, it is easily distinguished from an actual cross-correlation signal; a real cross-correlation caused by molecular binding also contains the correlation with the diffusion timescale (solid line). If we now selected the bursts with 0.3 < r < 0.7, and further exclude regions with other bursts with r > 0.7 or r < 0.3, we obtain the light shaded line. This line is further above 1.0 than the shaded line, and also shows a larger drop in the cross-correlation beyond 10 ms. However, in all cases, the cross-correlations of the first set of simulations are clearly distinguished from those of the second set of simulations. No spurious crosscorrelations are introduced by purified analysis of crosscorrelations at or below the diffusion time. However, the increase in the constant level as well as a drop in correlation at long times (beyond the burst width time) must be accounted for.
Although it is necessary to consider the biases in any use of this methodology, the results in this section indicate that these issues will not change the extracted results >10%, as long as the burst search threshold is below the average burst intensity.
Single-molecule FCS
Is it possible to get meaningful fits of correlation functions for regions containing only a single burst? It is not possible to get arbitrarily precise estimates of diffusion times for one single-molecule transit across the optical detection volume. Even arbitrarily strong signals will not help: FCS is a statistical method, and requires averaging over many such single-molecule transits to obtain a precise estimate of the diffusion time (21). However, as shown in Fig. 5, it is possible to obtain meaningful estimates of the diffusion by fitting correlation functions for single-molecule bursts when we expand the correlation region as described in Fig. 2.
The means of the distributions match the diffusion times of the simulation parameters. Fitting the distributions with a log-normal distribution, the standard deviation of distributions is 0.52 � 0.05 in units of 1n(τ^sub D^). The full-width half maximum values for the distributions are ~0.5 in units of log10(τ^sub D^)- For Species 1, the log-normal fit results in a central value of 3.2 � 0.2 ms. For Species 2 in the donor channel, the log-normal fit results in a central value of 5.4 � 0.3 ms. This is the same as for the FRET channel (fivefold dimer), where the log-normal fit results in a central value of 5.7 � 0.3 ms. Hence, the width of the distribution is not limited by signal/noise, but by having only one transit through the detection volume.
Such single-molecule FCS analysis is useful for detecting subpopulations with large differences in diffusion time. Standard FCS analysis can do similar analysis using multicomponent fits, but it is often difficult to determine if the multiple timescales seen are really due to multiple species or are due to photophysical dynamics of a single species. The singlemolecule FCS analysis introduced here allows these two cases to be distinguished.
The correlation function does not use the full information available in the photon stream. One way to improve on single-molecule FCS is to take advantage of more of this information. For example, analysis with a recursive Bayesian estimator would likely produce improved measurements of τ^sub D^ for a single molecule event (23).
MATERIALS AND METHODS
Simulations
The simulations are performed as described previously (16). A Gaussian detection volume was used in all cases, with transverse width of 0.35 �m, and longitudinal width of 1.75 �m. The three-dimensional simulation box is of size 3.5 � 3.5 � 17.5 �m^sup 3^, with periodic boundary conditions (a molecule that leaves one side reappears at the opposite side with the same lateral position).
Single-molecule confocal fluorescence microscopy
Solution-based single molecule measurements are performed as in Kapanidis et al. (6). The alternating-laser excitation experiments were performed using the 488 nm line of an Argon ion laser (Innova 90C, Coherent, Santa Clara, CA) and the 633 nm line of a Helium-Neon laser ( 1163P, Uniphase, Milpitas, CA). The lasers are turned on and off using TTL timing pulses and an acoustooptic modulator (AOTF 48062 2.5-0.55, NEOS Technologies, West Melbourne, FL) rather than electro-optic modulators as used previously. The alternation period is set at 25 �s.
The excitation light is reflected using a custom dichroic mirror (488-633 DBDR, Omega Optical, Brattleboro, VT). A 1.4 NA oil-immersion objective (60 � 1.4 NA oil immersion Plan Apochromat, Nikon, Tokyo, Japan) mounted on a Nikon TE300 inverted confocal microscope is used for the excitation; a 100 �m pinhole is used on the emission detection path. The emission is split using a second dichroic mirror (580 DRLP, Omega Optical). The donor channel (for Alexa 488; Molecular Probes, Eugene, OR) is filtered using a bandpass filter (535DF45, Omega Optical), and the acceptor channel (for Alexa 647; Molecular Probes) is filtered using a longpass filter (665AGLP, Omega Optical). The photodetectors, timing electronics, and software are as described previously (6). A neutral density filter (OD 1.2) is placed in front of the detector for the donor channel to reduce the signal intensity from the donor-labeled β-clamp. We time every photon, and without the neutral density filter our data acquisition was producing enormous files with uninteresting data produced by the donor channel. We still needed to monitor the donor channel to watch for β-clamp aggregates (see Fig. 6), but we did so with a much reduced count rate.
Median-based background subtraction and burst searches
In processing the single-molecule signals and performing burst searches, we use a median-based background subtraction. A time trace with 10-ms time resolution is formed from the photon streams obtained from the single molecule microscope. At each time point, the background is determined by calculating the median of the previous 100 time bins. The median Js used to avoid weighting the bursts in the signal too much in the calculation of the background. This background estimate is subtracted from each time point.
Obtaining error estimates using the bootstrap
We use a bootstrapping methodology to obtain error estimates for our fits of the purified correlation functions (16,24). In calculating the correlation, we average the correlation for all N^sub burst^ regions of interest surrounding selected bursts according to Eq. 5. For each correlation, we calculate 50 bootstrap instances of the correlations using the following procedure. We randomly select with replacement N^sub burst^ regions of interest from all N^sub burst^, regions of interest. Because we randomly select with replacement, a particular region of interest may be selected multiple times, or not at all. Using Eq. 5, we average the N^sub burst^ randomly selected regions of interest and obtain a bootstrap instance. Each bootstrap instance will have some regions missing, and some present twice or more. This allows the resulting bootstrap correlations to mimic additional experiments with similar noise characteristics. Using all 50 bootstrap instances, we calculate the variance for correlation time bin, and use this in weighting the fits for the correlation functions. Also, we fit each of the bootstrap instances to provide error bars for the fitted values.
DNA substrates
Analytical HPLC was performed on a HPl 100 series instrument with 220 and 280 nm detection using a Vydac C18 column (5 �m, 4.6 � 150 mm; Grace Vydac, Hesperia, CA) at a flow rate of 1 mL/min. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (solvent A) versus 0.1% trifluoroacetic acid, 90% acetonitrite in H2O (solvent B). Ultraviolet-visible spectroscopy was carried out on an Agilent 8453 diode array spectropbotometer (Agilent, Palo Alto, CA). Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all compounds and components of reaction mixtures. ES-MS was performed on a Sciex API-150EX single-quadrupole electrospray mass spectrometer (MDS Sciex, Foster City, CA). Calculated masses were obtained by using ChemDraw 7.0.1 (CambridgeSoft, Cambridge, MA) or ProMac Ver. 1.5.3 (Sunil Vemuri and Terry Lee, City of Hope, Duarte, CA). Fluorescently labeled oligonucleotides were purchased from Invitrogen (Carlsbad, CA) or IDT (Skokie, IL) and purified by reverse phase HPLC. Fluorescent dyes were purchased from Invitrogen. Bio-Gel A-15m agarose was purchased from BioRad (Hercules, CA). All other chemicals were obtained from Aldrich (Milwaukee, WI) unless otherwise indicated.
The M13mp18 phage was prepared by two consecutive bandings in cesium chloride, as described in Turner and O'Donnell (25). The ssDNA M13 plasmids with single hybridized DNA oligomers were prepared by annealing the synthetic DNA oligomers to purified single-stranded M13mp18 DNA, as described in Yao et al. (26). Briefly, 9 pmol of DNA oligomer(s) were added to 45 pmol of ssDNA template in buffer A (10 mM Tris-HCI, 300 mM NaCl, pH 8) and the final volume was adjusted to 500 ml. The reaction mixture was heated to 100�C for 5 min and slowly cooled to room temperature for 1 h. The reaction mixture was applied to a 5-ml column on Bio-Get A-15m equilibrated in buffer B (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). Fractions of 100 �l were collected and analyzed for UV absorption using the Agilent spectrophotometer. The molar concentration of M13 ssDNA with annealed DNA oligomer was calculated using known molar absorption coefficient
DNA hybridization experiments
Single-molecule samples are prepared by diluting DNA oligomers hybridized to ssDNA plasmids to ~100 pM concentration in a 20 mM 7.5 pH Tris buffer with 0.1 mM ethylene diamine tetraacetic acid (EDTA), 4% glycerol, 40 �g/ml bovine serum albumin, 8 mM MgCl^sub 2^, and 50 mM NaCl. A well is formed by using silicone well (Grace Biolabs, Bend, OR) on a coverslip. Ten microliters of sample is placed in the well, and a second coverslip is placed on top. The solutions are monitored using the single-molecule fluorescence microscope for 5 min with 70 �W excitation from the 633-nm laser.
In the experiments of Fig, 7 a, three sample solutions were prepared: one with ~100 pM of labeled, DNA oligomer hybridized to ssDNA plasmid (excess plasmid); a second with ~100 pM of labeled DNA oligomer without plasmid; and a third mixture sample prepared as a 1:1 mixture of the previous two samples. The samples were observed before and after heating at 37�C for 10 min. In the experiments of Fig. 7 c, the sample with ~100 pM DNA oligomer hybridized to ssDNA plasmid (excess DNA oligomer) were observed before and after heating at 37�C for 10 min.
Cloning and bacterial expression of β-clamp
The gene fragment encoding DNA β-clamp was amplified by polymerase chain reaction using E. colt K12 genomic DNA as template. The 5'-DNA primer (5'-GGT GGT CAT ATG AAA TTT ACC GTA GAA CGT GAG CAT TTA TTA AAA-3') and the 3'-primer (5'-GGT GCT TGC TCT TCC GCA GCC CAG TCT CAT TGG CAT GAC AAC ATA-3') introduced NdeI and SapI restriction sites, respectively. The polymerase chain reaction-amplified DNA was purified, digested simultaneously with NdeI and SapI and then ligated into a NdeI, SapI-treated pTXB1 plasmid (New England Biolabs, Ipswich, MA). The resulting pEY10 plasmid was shown to be free of mutations in the β-clamp-coding region by DNA sequencing.
Bacterial expression was carried out as follows. E. coli BL21 (DE3) pLysS cells (Novagen, Madison, WI) were transformed with pEY10. Cells were grown at 37�C to midlog phase (OD^sub 600^ [asymptotically =] 0.6) in Luria-Bertani medium and induced with 0.2 mM isopropyl β-D-thiogalactopyranoside at 30�C for 6 h. Cells were collected by centrifugation at 5000 rpm in a GS3 rotor for 10 mtn. The cell pellet from 1 L of bacterial culture was resuspended in 20 mL of lysis buffer (0.1 mM EDTA, 1 mM phenylmethanesulphonyl fluoride, 25 mM sodium phosphate, 150 mM NaCl buffer at pH 7.4 containing 10% glycerol) and lysed by sonication. The iysate was clarified by centrifugation at 14,000 rpm in a SS-34 rotor for 30 min. The clarified supernatant (~20 mL) containing the β-clamp-Gyrase intein fusion protein was incubated with 5 mL of chitin beads (New England Biolabs) at 4�C for I h with gently shaking. The chitin beads were washed with 50 mL column buffer (0.1 mM EDTA, 50 mM sodium phosphate. 250 mM NaCl buffer at pH 7.2) containing 0.1% Triton X-100 then equilibrated with column buffer. The fusion protein adsorbed on the beads was subsequently cleaved with 100 mM NH^sub 2^OH in PBS at pH 7.0 ([approximate]6 mL) overnight at 18�C to yield free β-clamp. The protein was further purified by fast protein liquid chromatography on a MonoQ 5/50 GL column (Pharmacia, Peapack, NJ) using a flow rate of 0.5 mL/min and a linear gradient from 0 to 500 mM NaCl in 50 mM Tris-HCl buffer at pH 8. The purified β-clamp was characterized as the desired product by ES-MS (Expected mass (average isotopic composition) = 40,642 Da; measured MW: 40,659 � 15 Da). The isolated yield for purified β-clamp was at ~20 mg/L.
Fluorescence labeling reaction
Fifteen microliters of Alexa488-maleimide in dimethylformamide (1 mg/100 �l) was added to 500 �l of 50 �M β-clamp in phosphate-buffered saline (50 mM phosphate, 150 mM NaCl, 2 mM Tris(2-carboxyelhyl)phosphine hydrochloride, pH 7). The reaction was allowed to proceed for 2 h in the dark at room temperature. Excess glutathione was added to terminate the reaction and the reaction mixture was applied to a Sephadex G-25 gel filtration column ((Amersham Biosciences, Uppsala, Sweden)) and then eluted with buffer B to separate β-clamp from the small molecular weight reactant. The reaction resulted in ~10% of fluorescenlly labeled β-clamp and, under this condition, no multiple labeling was observed.
Loading of β-clamp on DNA
The loading of the β-clamp is performed as in Yao et al. (27), but with a lower concentration of DNA and β-clamp. Briefly, a 100-�l reaction mixture was formed in a 20 mM 7.5 pH Tris buffer with 0.1 mM EDTA, 4% glycerol, 40 �g/ml bovine serum albumin, 8 mM MgCl^sub 2^, and 50 mM NaCl. Forty femtomoles of M13 plasmid with annealed DNA oligomer (described above) are added with 220 pmol of single-stranded binding protein. A quantity of 0.4 pmol of γ-clamp loading complex and 1 pmol of β-clamp (labeled monomer) are then added. Finally, adenosine triphosphate is added to a final concentration of 1 mM. Fifty microliters of this reaction mixture are placed in a well formed in a cell incubation chamber (WillCo-dish GWSt-3522, WillCo Wells, Amsterdam, The Netherlands) with a silicone gasket (Grace Biolabs). The solution is covered with a coverslip and is heated from room temperature to 35 � 2�C over a period of 2 min using a microscope-based heater (Warner Instruments, Hamden, CT). ALEX-based single-molecule spectroscopy is then performed for 20 min at 35 � 2�C.
RESULTS AND DISCUSSION
Purified FCS and the analysis of β-clamp-DNA complexes
We now apply purified FCS (PFCS) to the β-clamp-DNA interaction experiments mentioned in the Introduction. As seen in the time traces in Fig. 6, we use a relatively high concentration (10 nM) of donor-labeled β-clamp protein (from an even higher, 50 nM concentration of protein, 80% of which is unlabeled), and a low concentration (SOO pM) of acceptor-labeled DNA oligomers hybridized to ssDNA plasmids. The β-clamp protein forms a dimer, so that we have 10 nM of donor labeled β-clamp dimer, implying that 40% of the dimers are labeled with at least one donor. There is a neutral density filter on the donor channel to reduce the signal intensity on the donor channel. Thus, there are four potential fluorescent species: labeled DNA oligomers free in solution; labeled DNA oligomers annealed/hybridized to ssDNA plasmids; labeled β-clamp free in solution (in dimeric form and occasional aggregates); and labeled β-clamp on DNA. Individual events are easily distinguished in time traces of the emission in the FRET (black) and acceptor (red) channels, but individual events corresponding to single β-clamp molecules are not identifiable since their concentration is too high. Bursts from complexes undergoing FRET (as in Fig. 6 a) are easily distinguished from β-clamp aggregate events leaking into the FRET channel (as in Fig. 6 b) by looking for coincident bursts in the donor channel (implies aggregates) or in the acceptor channel (implies complexes with FRET). Then, based on ratiometric expressions calculated using these signals, we select correlation regions containing only bursts from complexes undergoing FRET. For each selected burst, the correlation regions are expanded to include 100 ms before and after the burst. Correlations are calculated for each correlation region, and summed over all selected bursts.
The autocorrelation of the FRET channel (black line in Fig, 6 c) found using PFCS is significantly different from the autocorrelation of the FRET channel calculated for the entire experiment (green line in Fig. 6 c). The purified FCS FRET autocorrelation is well fit by Eq. 2 with a diffusion time of 4.0 � 0.6 ms (red line). In contrast, the FRET autocorrelation for the whole experiment is poorly fit by Eq. 2 with a diffusion time of 2.0 � 0.2 ms (blue line). The shorter diffusion time is due to contributions from leakage of the donor signal into the FRET channel (the β-clamp diffuses more quickly than the plasmid). Out of the 159 bursts in the FRET channel detected in this experiment, 21 were excluded because there was a coincident large burst in the D channel, indicating that the burst was likely a β-clamp aggregate. The measured diffusion time for the excluded bursts was 3 � 1 ms. They were typically dimmer than the selected bursts, so these aggregates do not account for the difference in the above-measured diffusion times. It appears that the primary benefit of PFCS was to exclude the dimmer, but more consistent leakage signal from free, nonaggregated β-clamp, which accounts for a consistent, though fluctuating source of background photons collected in the FRET channel throughout the experiment.
Tables 1 and 2 show the effects of PFCS on the various auto- and cross-correlations calculated for this data set Table 1 shows values for molecular occupancy, diffusion time, and brightness in three channels as extracted using photon arrival-time interval distribution (PAID) (16). Using PAID, we were able to extract diffusion times for each species, but the full autocorrelation was not able to be viewed independently from the other species. This ability, provided by PFCS, will be necessary for our application monitoring the movement of the DNA sliding clamp on DNA. Table 2 shows the contribution to the correlation amplitudes, or the numerator in Eq. 1, of each species in Table 1. First, the contributions are shown for the entire experiment. Second, the contributions are shown after PFCS is used to select for Species 4, the DNA sliding clamp-DNA complexes undergoing FRET. The contribution of these complexes to the autocorrelation of the FRET channel, which is of primary interest to us, is seen to increase from 60% to 91%.
Could the difference in diffusion times measured be an artifact of the burst selection? In addition to the simulation results above, several lines of reasoning indicate that it is not.
First, the diffusion of the β-clamp-DNA complexes is expected to be characterized by a single diffusion time, and the PFCS autocorrelation of the FRET channel fits better to a single-component FCS model of Eq. 2 than the autocorrelation of the FRET channel for the whole experiment.
Second, the PFCS cross-correlation is much more consistent with the total cross-correlations between the FRET channel and acceptor channel from the DNA oligomers hybridized to ssDNA plasmids (Fig. 6 d). The PFCS cross-correlation fits to Eq. 2 with a diffusion time of 6.4 � 1,1 ms, and the cross-correlation for the whole file fits well to Eq. 2 with a diffusion time of 7.4 � 0.4 ms, equivalent to within error. In this cross-correlation case (as mentioned in the Introduction), there are only two contributing signals: complexes undergoing FRET and direct excitation of lone DNA oligomers hybridized to ssDNA plasmids by the donor excitation laser. Both have similar diffusion timescales, and we expect cross-correlations for the whole experiment to match cross-correlations obtained with PFCS.
Third, the timescale for the PFCS autocorrelation of the FRET channel makes more sense compared to the diffusion time measured for the plasmid using the acceptor excitation laser (9.6 � 0.4 ms). The detection volumes for the donor and acceptor excitation lasers are not the same, so we do not expect identical diffusion time values to be found in the two results. However, the diffusion times for the cross-correlation between FRET and acceptor channels should be halfway in-between the diffusion times for the autocorrelations of the FRET and acceptor channels (7). If we use the value of 9.6 � 0.4 ms for the acceptor autocorrelation, and 7.4 � 0.4 ms for the cross-correlation, we expect an autocorrelation diffusion time of 5.2 � 0.6 ms. Within error, this matches the value of 4.0 � 0.6 ms from the purified correlation analysis much better than the 2.0 � 0.2 ms value extracted from the entire experiment.
As shown in this example, purified FCS with the correlation region expansion allows us to monitor the temporal dynamics of individual species even in the presence of other species. Using this methodology, we are studying the motion of the β-clamp protein on DNA (unpublished).
Single-molecule FCS and DNA hybridization
In our initial β-clamp experiments, we incubated the reaction mixture for 10 min at 37�C before spectroscopy (in later measurements the sample was heated on the microscope). In these first experiments, we noticed that the diffusion time measured in the acceptor channel decreased significantly after incubation. This was an apparent paradox since the signal in the acceptor channel should be exclusively from the labeled DNA oligomers annealed to ssDNA plasmids, which should not be affected by gentle heating or by loading of the β-clamp. A clue was that we found the change in diffusion time after heating occurred even without the β-clamp or clamp loading complex. FCS measurements (not shown) suggested two components, one with a long diffusion time (3.5 ms) and one with a short diffusion time (0.7 ms). The strength of the component with the shorter diffusion time increased after heating. By standard FCS, however, there is an ambiguity as to how much of the deviation from a one-component fit is due to a second diffusing species and how much is due to internal dynamics of the large DNA (28,29). Single-molecule FCS provides a way to show that there are indeed two diffusing components with different diffusion times.
Using single-molecule FCS, we found that the change in diffusion time was due to unbinding of labeled 30-mer DNA oligomers weakly bound to the ssDNA plasmid. We annealed the short DNA oligomers to the ssDNA plasmid using a 10:1 excess of DNA oligomers to be sure that each ssDNA plasmid was hybridized by a DNA oligomer. Depending on the sequence specificity of the short DNA oligomers, multiple DNA oligomers can be attached to a DNA plasmid at 40�C, as seen previously in FCS experiments under similar conditions (30). We hypothesize, therefore, that additional DNA oligomers are likely attached to the ssDNA plasmid at the lower temperature we used for purification, and then detach from the ssDNA plasmid upon heating of the DNA oligomer-plasmid complexes to 37�C.
In Fig. 7 a, we show that single-molecule FCS distinguishes between free 30-mer DNA oligomers and 30-mer DNA oligomers attached to ssDNA plasmids. The 30-mer is labeled with Alexa647 dye at the 5' end. The green line in Fig. 7 a is the histogram for a solution with free 30-mer DNA oligomers. The red line is the histogram for DNA oligomers attached to plasmid, prepared using a 5:1 excess of plasmid to prevent attachment of weakly bound DNA oligomers. The black line is the histogram for a solution formed as a 1:1 mixture of the pure samples. The histogram for the mixture can be accurately predicted simply by averaging the results for the two pure samples (blue line). All of these histograms remain the same after heating to 37�C (not shown).
As can be seen by comparing results from Fig. 7, a and b, single-molecule FCS produces a better separation of species than can be obtained by using histograms of single-molecule burst widths. In comparing the histograms, one must keep in mind that the faster diffusing species produces more bursts than the slower diffusing species for the same concentrations. In Fig. 7 a, even though more bursts are detected from the faster diffusing species, the slower diffusing DNA oligomers attached to ssDNA plasmids are at a higher concentration. (Refer to Fig. S1 in the Supplementary Material to see histograms corrected for this effect.)
Since we measured the histograms produced by the free DNA oligomers alone and the DNA oligomers bound to the ssDNA plasmids alone, we can calculate the probability that the burst identification is correct in the 1:1 mixture sample. If, for the extracted diffusion time of a given burst, there were more bursts in the free DNA oligomer histogram, then most likely the burst was from that species. If there were more bursts in the histogram for DNA oligomers bound to the ssDNA plasmid, men most likely the burst was from that species. For a burst with a specific diffusion time, the probability that the species identification is correct is the ratio of the maximal number of bursts in one histogram with that diffusion time divided by the sum of the number of bursts in the histograms for both species with that diffusion time. This probability is plotted in Fig. 7 a as the cyan curve. A corresponding plot is shown in Fig. 7 b using burst widths. As can be seen, the probability of correct identification is significantly larger using single-molecule FCS than by using burst widths. Summing over all bursts, the probability of correct identification using single-molecule FCS is calculated to be 92%. For burst widths, the probability is 72%.
Now, suppose we did not use any of this information, and only used the total number of bursts in the individual species histograms. There were 2101 bursts in the histogram for DNA oligomers bound to ssDNA plasmids, and 4900 bursts in the histogram for free DNA oligomers. Using this, we could guess, based on no other information, that any burst has a 70% chance of coming from a free DNA oligomer. Hence, we see that the information gain in the case of burst widths is minimal, at only 2%. For longer burst widths, there is a >95% chance that the burst comes from the slower diffusing species, but there is only minimal discrimination for shorter burst widths. However, single-molecule FCS produces significant gains in information for both long and short bursts. There are single molecule diffusion times that provide a >95% chance of correct species identification both for the slower and faster diffusing species.
In Fig. 7 c, we show the results of experiments with a 10:1 excess of labeled DNA oligomer. In this case, there is a large increase in the amount of free DNA oligomer after the 10-min incubation at 37�C. There is some decrease in the number of long-diffusion time bursts, likely because these bursts are dimmer after losing weakly bound DNA oligomers. In these histograms, there are many more short diffusion-time bursts than long diffusion-time bursts. Here, there is approximately a factor-of-5 difference in the diffusion times of the labeled DNA oligomer and DNA oligomers hybridized to ssDNA plasmids, leading to a factor-of-5 more bursts from the labeled DNA oligomer even with the same concentration.
CONCLUSION
We have shown that purified FCS allows correlation analysis on individual subpopulations selected using single molecule measurements. We can use standard FCS models by expanding the region of interest around the detected bursts. This methodology will be useful in purifying correlations for species of interest. This will help improve our ability to apply FCS and single-molecule analysis to questions involving fast fluctuations in rare species.
Additionally, we have demonstrated single-molecule FCS analysis that may be used to distinguish between bursts from species with at least a fivefold difference in diffusion times. Here we showed that weakly bound DNA oligomers can fall off ssDNA plasmids even with gentle heating at 37�C. This methodology can be further used for measuring binding kinetics of large proteins/DNA with smaller proteins, DNA, or small molecules. These results indicate that, although the amount of information from single molecules bursts is finite (31), we have not yet taken full advantage of the information that is there.
SUPPLEMENTARY MATERIAL
An online supplement to this article can be found by visiting BJ Online at http://www.biophysj.org.
We thank the laboratory of Professor Mike O'Donnell of Rockefeller University for supplying us with β-clamp, ssDNA plasmids, single-stranded binding protein, and clamp loader γ. We thank Professor Thomas Huser of University of Califomia-Davis Medical Center for critically reading the manuscript.
This work was performed tinder the auspices of the U.S. Dept of Energy by the University of California, Lawrence Livermore National Laboratory, under contract No. W-7405-Eng-48. This work was supported by the Laboratory Directed Research and Development program at Lawrence Livermore National Laboratory.
[Reference]
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[Author Affiliation]
Ted A. Laurence, Youngeun Kwon, Eric Yin, Christopher W. Hollars, Julio A. Camarero, and Daniel Barsky
Chemistry, Materials, and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California
[Author Affiliation]
Submitted July 17, 2006, and accepted for publication November 29, 2006.
Address reprint requests to T. A. Laurence, E-mail: laurence2@llnl.gov.
