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The Detroit Red Wings have called up goaltender Jimmy Howard from the AHL's Grand Rapids Griffins and will start him in Minnesota against the Wild on Saturday.
Chris Osgood, who had been expected to start, will remain in Detroit to work with goalie coach Jim Bedard. Osgood will also be a healthy scratch for Friday night's match against the Anaheim Ducks, with Howard backing up Ty Conklin.
"Jimmy is playing as well as he has at any point in his four-year (professional) career and it's an opportunity for Chris to have 10 days (off), get a mental breather and clear his mind," Detroit general manager Ken Holland said. "We need him to be at his best. Sometimes when you get bad bounces, it just snowballs. The hope is he gets to where he was last year."
Both the Chief and Malik have commented multiple times on the Jimmy Howard call-up and Chris Osgood's removal from the Red Wings' lineup so I'll throw my two cents in, too.I've said before that I'm an Osgood fan. I've liked the guy since 1994, when he was crying in the locker room after the Game Seven loss to San Jose, the first time it actually looked like a Red Wings gave a damn about all of those early playoff exits from the early 1990s.Osgood's stop on Peter Zezel en route to a double-OT shutout in Game Seven of the 1996 Western Conference Semifinals is still one of my favorite saves of all time.Look at his eyes after that stop, he's unfazed. Osgood's mental toughness has always been his strong point.And that's the problem this season. That toughness isn't there, it's been replaced with frustration at himself and with his team. So as much as I like Osgood I won't deny that he's very off his game.If the playoffs started today, who would I start? I want to give Osgood the benefit of the doubt with the expectation that he's out as soon as he falters, much like Hasek last season. That said, if he doesn't snap out of it, I can't say I'd blame Mike Babcock for going with Ty Conklin for Game One.The one thing I have trouble with is the idea that the Wings are playing poorly in front of Osgood and better in front of Conklin this season. The Wings sit second in the Western Conference and have won a ton of games again this year, the idea that they're playing poorly - no matter who the goalie is - seems ridiculous. It's true, of course, but more than playing poorly the Wings are playing differently.For years, Detroit has been a puck-possession team. Hold the puck, move the puck, keep the puck. If you have it, the other team doesn't. And part of that kind of play is that the goalie is expected to keep play alive.Osgood thrived in that system last year, controlling rebounds and putting them to places where his defenders could get to them. This year the Wings don't get to the loose pucks as well and those formerly-controlled rebounds become rebound chances for the opposition. Conklin freezes the puck more than Osgood, saving himself from those rebound chances and relying on the Wings' superb faceoff skills to get the puck back.So do you blame Osgood for failing to adapt to his defense's new laziness or do you blame the defense for not getting to the puck? As a goalie of a different kind, I understand Osgood's frustration, preparing for a season expecting one kind of play from his defense only for them to play another style entirely.All of that said, Osgood does need to get focused and this time off should serve him well. While he's refocusing, though, the rest of his team needs to tidy up their game as well.
I hate it when people always ask the same question, but is there somewhere I can watch the game online besides NHL.com? Or is that my best bet, cause I heard it sucked.
If you can even watch it on NHL.com? XD
@&*%^@&*&*$* Nope, I'd have to pay, screw that.
ATDHE feed for tonight's game: http://atdhe.net/live-tv-2.php?i=884
Backstrom gets the start for the Wild. Andrew Brunette is questionable for tonight but took the morning skate.
I'll be damned, Draper is the healthy scratch tonight. I thought for sure it'd be Kopy.
There was a healthy scratch? I missed the pregame show.Wild scores first. Bad sign.
I think Howie will want that one back. He got a piece of it but not enough.
Kopecky should be scratched, he's done nothing this year.
Kopecky should be scratched, he's done nothing this year.
I strongly disagree! I think he's done a lot and in fact I like him quite well. It probably just doesn't seem like much because I know he had to deal with a lot of injuries in previous years?Maltby, on the other hand...
Kopecky should be scratched, he's done nothing this year.
I'd say the whole fourth line has done nothing. Kopy was the only one using his body at the start of the season so I give him credit for that.
I can tell more 'fascinating' stories
Sorry for bailing out last night, but it was my cousin from Hawaii's last night here, so I was paying attention to him.
How dare you pay attention to family over hockey. And posting about hockey on the Internet. Where are your priorities?!
Ok, I'm at a loss for words. My inner ghetto self took over to step into the spotlight. I have nothing sarcastic to say to your sarcastic remarks
Not so much poor me, I'll take goals from anyone whether I think they should be in the lineup or not.
You know, that was a really nice move by Leino to get it out to the front of the net. Kopy kind of lucked out (though at least he went to the net, that's more than a lot of Wings have been doing).
Not so much poor me, I'll take goals from anyone whether I think they should be in the lineup or not.
That is a great thing that all sports fans should learn to do
Helm goddamned near had one there. He's gonna get one tonight the way he's playing. Wings finally have some jump.
I might need to turn this off, I'm dog-sitting and she's looking at me funny, wondering why I'm growling at the TV. All that jump early in the period and the Wings blow it late.
Crisis, huh?
Worse then the Great Fire of London.I hate to be pessimistic, but the game is most likely lost. But the Wings will hopefully get another goal
The Wings can score three in five minutes, I just don't expect them to.
Oooh, there's one. Two in five minutes then.
The Wings can score three in five minutes, I just don't expect them to.
Edited it for you? :c Mr. Logic.Two more to go!
The way I see it, they need another before the two-minute mark. Then they can pull Howard and go for the tie. Down by two with 2:00 left isn't enough time. Yeah, it might work out, but it's pushing it.
Ooh, I thought they were going to bat it in there.
Exciting last minute, with no success.
As I typed, empty netter.
All that and the Wild get the ENG. I swear they just stacked five guys in the crease at the end there.
Well, hey, good thing Howard played and not that bum Osgood. Or something.
AS:LDJAKJS:KLSAKLSAJ YOU JUST SAID OR SOMETHING? D: That is awesome. I say that all the time on purpose :c Hmmm. I don't know. I think all the 'Nogood' haters should remain quiet the rest of the night. No, he's definitely not my favourite goaltender, but he has done a lot But then again...that was probably more sarcasm, wasn't it? x]
Jimmy Howard
Credit: Clark Rasmussen
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"I never doubted what Peter told us," Hornbuckle said.
Former Councilman Jay Humphrey, who began his term in 1994, said Naghavi was patient when explaining the issues.
"I can't say enough positive things about Peter," Humphrey said. "The moment he does physically retire, the city will have lost an important piece. No doubt about it. They will have lost something that was irreplaceable."
Hornbuckle recalled how Naghavi reacted on one of the most momentous days in city history: March 17, 2011, when 29-year-old city maintenance worker Huy Pham committed suicide by jumping off the roof of City Hall.
"Peter was right there," Hornbuckle said. "He showed up, he held hands, he talked with people, he shed a tear with people. He's a very caring and very human person.
"In just being there, he showed his support for the city employees and for the family and everything that was going on."
Naghavi said it was his toughest time at the city.
"While I myself was very sad and could hardly hold my tears back, I felt the weight of the entire City Hall on my shoulders, as I felt it was up to me to provide the soft, yet stable and strong presence for the rest of the staff who were confused and in disbelief," he wrote in an email. "I always have been good in talking to people, but those days, I didn't know what to say … this kept me up many hours at night."
'The perfect public servant'
Some years ago, Costa Mesa resident and blogger Geoff West had made an appointment to speak with Naghavi over lunch. The two were to meet at Z'Tejas Southwestern Grill in South Coast Plaza.
A small earthquake, however, had shaken things up — literally and figuratively — that afternoon.
Naghavi was 30 minutes late. West tried calling Naghavi at City Hall. No luck.
Eventually, he did see Naghavi coming — as quickly as he could.
"I saw him sprinting down the parking lot," West said. "I shook his hand, and he said, 'Sorry, I have to go back to City Hall. The building is in chaos. The phone system is out.'"
Naghavi wanted to keep his appointment somehow, West said, even though an emergency had impeded communication.
"He's probably the perfect public servant," West added, the real "go-to" guy who's service-oriented.
Helen Nenadal, president of the Costa Mesa City Employees Assn. and a nearly 30-year Costa Mesa employee, said for Naghavi, "It didn't matter what job you had or what level you were at, he was always there for us employees."
He respected the staff, and they respected him in return, she said.
"The employees felt they could go to him for anything," Nenadal said.


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If you're really observant or just naturally curious, (you have to be one of the two to be on this page), you'll notice that I'm sandwiched between two very energetic and passionate personalities in the About Us dropdown menu – Marcus and George. A short and succinct way to describe my role here at The Sales Lion is – I'm the peanut butter holding the bread together!
For the majority of my working life, I've been in marketing. The common thread running throughout my entire career is and always has been: helping businesses earn new customers. The word earn is crucial to me – for two reasons – it's not as cold and impersonal as the industry buzzwords – acquire, win, generate and attain but more importantly, it signifies that it takes planning, patience, perseverance and desire to achieve successful results. That applies as much to us as service providers as it does to our clients. You gotta have skin in the game to make things work!
That's what attracted me to The Sales Lion in the first place – I observed Marcus Sheridan doing more transformative work on mindsets and company cultures than with the rote aspects of marketing execution. The lasting, meaningful change occurs when hearts and minds are aligned and I'm excited to be deeply involved in cultivating that alignment.
I've founded and run two businesses – both direct mail agencies that earned new customers for Big Box retailers (Home Depot, CVS Pharmacy, Costco, Bed Bath & Beyond…) by targeting consumers entering specific life stages (new homeowners, new parents…). The second agency was an Inc. 500 company (#363). I became a certified HubSpot reseller in 2009 when I saw the direction HubSpot was taking with their marketing automation platform.
On the personal side:
I was born and raised in Boston (Needham, MA) and am a die-hard Red Sox, Patriots, Bruins fan (Sorry Celts – the NCAA is more interesting than the NBA!) I graduated from Bentley University with a BS in Management.
My wife Karen and I have been married for 26 years.
We have two boys (men, sorry guys). Chris is a University of Virginia grad living his snowboarding dream by working at Big Sky Resort in Montana. Tim is a third year student student at the University of Michigan majoring in psychology.
Benny the dog is a 12 year old English Lab who thinks he is a puppy.
I love to ski, snowboard, water ski, rock climb and play golf .
Random Facts about Rich:
- Jumped out of a perfectly good airplane and lived to tell about it
- Crashed a motorcycle and lived to tell about it
- Lived in a condemned building in San Diego for six months and lived to tell about it
- Hit by a red-light runner who spun and flipped my car upside down and lived to tell about it
- Attended a Patriots-Ravens (Patriots won!) game in Baltimore in full Patriots gear and lived to tell about it
- Five lives down with four to go – I'm picking my battles wisely now!


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Professor Iris Krasnow
Program Director
Professor Iris Krasnow
Program Director
American University's Lifelong Empowerment And Professional Development (LEAP) Program provides women with the impetus and skills to help guide them through significant transitions in their personal and professional lives. These courses are offered through AU's School of Professional and Extended Studies both in Washington, D.C. and in Annapolis, Maryland, where we have launched AU on the Bay.
Read "The ticket to longevity," a blog post by LEAP Professor Iris Krasnow
Founded on the idea that substantial change offers opportunities for personal growth, LEAP helps a woman navigate the inevitable and important changes that arise in family structures, with the growing independence of children, a return to the workplace and/or a desire to shift careers.The holistic, multi-disciplinary curriculum in the four-course LEAP program addresses gender-based needs and experiences in both professional and personal development from one's thirties through midlife, retirement and beyond. Courses can be taken individually for those students that cannot enroll in the full-load program at this time.
The program courses enable self-reflection, intellectual challenge, peer-to-peer knowledge sharing, and development of concrete skills. LEAP is designed to help women advance as leaders and prepares them to accomplish short- and long-term personal and professional goals. Upon completing the program, participants will have increased self-awareness and self-confidence; strengthened professional and business skills, and developed practical approaches to ownership/leadership that they can draw upon and apply at every point of life's transitions. To earn certification of completion in the Lifelong Empowerment and Professional Development program, students must complete these four LEAP courses, which can be taken individually both in Washington, D.C. and in Annapolis:
Adult learners in the LEAP will have access to the intellectual resources and networks available at American University, an internationally recognized university in the nation's capital.


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Angry Birds and Doodle Jump to add to Windows Phone 7's 18,000 apps
Microsoft's Windows Phone 7 mobile operating system now has more than 18,000 applications in its App Market Place the Redmond based tech giant has revealed.
Speaking at the official preview of the operating system's next major update, Microsoft confirmed that its App outlet has surpassed the latest content milestone just seven months after the system was made available to both developers and consumers.
"We stayed focussed on giving consumers the apps they wanted most," declared Corporate Vice President of Windows Phone 7 marketing, Achim Berg on the services rapid rise in available app content.
Apps are set to play are major role in the development of the service with Microsoft outlining the downloadable content as one of its key areas of development ahead of the Mango update that will hit devices in autumn this year. Apps that will hit Windows Phone 7 in the coming weeks include mobile gaming powerhouses Doodle Jump and Angry Birds.
"Seven months ago we started our mission to make smartphones smarter and easier for people to do more," said Andy Lees, President of the Mobile Communications Business at Microsoft. "With Mango, Windows Phone takes a major step forward in redefining how people communicate and use apps and the Internet, giving you better results with less effort."
Can Microsoft's Windows Phone 7 OS challenge the likes of Apple and Google in the App stakes? Let us know what you think via the T3 Twitter and Facebook feeds.
Windows Phone 7 Mango video
Source: Windows Phone 7 Mango | T3 Tech Videos


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COLUMBIA, S.C. -- An attorney representing state NAACP President Lonnie Randolph on charges of refusing to pay his dry cleaning bill and fighting with officers says he wants a new prosecutor handling the case.
Joe McCulloch says in documents filed Tuesday that city prosecutor David Fernandez is compromised because of pressure from publicity and local politicians.
McCulloch says the trespassing, disorderly conduct and resisting arrest charges should have been dismissed and that his client was having a diabetic episode.
Randolph was arrested July 12 at a Columbia dry cleaners after employees said he would not pay for his clothes or leave. City Manager Teresa Wilson went to the scene, which Mayor Steve Benjamin later criticized.
Last week, Fernandez told a judge he wanted more time to evaluate whether to prosecute.
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The purpose of automobile bumpers is to protect functional and cosmetic areas of the car from damage during low-speed impacts. For many decades, bumpers were heavy chrome-plated steel bars bolted onto a vehicle's front and rear. Today's vehicles still have an actual metal bumper beam, but it's structural only and lighter in weight. A plastic piece known as a bumper...
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Need more explanation of mocks
21 May 2011, 12:36
Michael Blyth (4 posts)
I've only just gotten to the section Introducing IO under Making Your Tests Fast. The discussion jumps into using RSpec mocks without any explanation as far as I can see. I am a beginner with RSpec and am trying to make my way through The RSpec book, and the various ins and outs of mocks and related topics are still fuzzy to me. I think that the book needs a quick intro to the meaning and syntax of mocks, maybe in an appendix if you think it's too basic for most of the readers.
An example of what is confusing to me is the line
I think I understand @should_receive@ as setting up a message expectation, but here is it replacing the actual Twitter::Search with a mock?
30 May 2011, 16:25
Andrés N. Robalino (4 posts)
RSpec mocks Twitter::Search's new method and returns mock_client. Yes, it is basically disconnecting the real "new" method.
This is done after you test it works properly, once this happens… you mock it such that your test runs fast.
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Your child's bedroom is her comfort zone, so you'll want to make it as safe and cozy as possible. Here are a few of the most important bedroom safety concerns:
Crib safety basics
If your child hasn't yet made the transition to a bed, make sure her crib meets current safety standards. New cribs have to meet safety regulations to be sold in the United States, but be careful with older or borrowed cribs: Most crib-related accidents and deaths happen in previously used cribs that don't meet current safety standards.
Check BabyCenter's Product Recall Database to see whether the crib you're using has been recalled.
- Make sure crib slats are no more than 2 3/8 inches apart (you shouldn't be able to fit a soda can between the slats) to prevent your child's head from getting caught between the bars. The crib shouldn't have any missing or broken slats. And be sure the mattress fits snugly — no more than two fingers should fit between the mattress and the side of the crib.
- Remember to lower the crib mattress and remove the bumper pads as soon as your child can pull herself up. In fact, if she moves around a lot in her sleep, you may want to remove the bumpers even earlier. Some parents warn that their child's head has gotten trapped under the bumper.
- Corner posts should be no higher than 1/16 of an inch and no shorter than 16 inches if there's a canopy. And make sure the headboard and footboard don't have any decorative cutouts that could trap your child's head or limbs.
Window blinds pose a particular hazard because your child's neck could become entangled in the cords that raise the blinds or run through the slats. If the crib must be near a window, either cut off the pull cords or use cord shorteners, safety tassles, or wind-ups to keep them out of reach.
Window blinds sold since November 2000 have attachments on the pull cords to prevent a loop from forming between the slats if a child pulls on them. If you bought your blinds before November 2000, visit the Window Covering Safety Council Web site or call (800) 506-4636 to order a free repair kit.
To prevent falls, keep the drop side of the crib up and locked when you're not with your child.


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Loren Steffy
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How to Tell the Difference Between a Kayak and Canoe
There are many differences between a kayak and canoe and it is helpful for those who wish to start these water-sports to understand which is which.It is better if you are experienced to go kayaking or canoeing...make sure you know what you are doing!
1Learn the terminology. For example: K1, C1, C2 - the letter refers to what the boat type is:
- Either a kayak (K); or
- A canoe (C).
- The number refers to how many people can be seated in the boat.
2Note which paddles are used:
- In a kayak, known as a K1, the paddler uses a double bladed paddle.
- In a canoe, also known as C1 or C2 depending on how many paddlers are in the boat, individuals only use a one bladed paddle.
3Note the seating position differences:
- In a kayak, the paddler sits with their legs out in front of them, sometimes tucked under a covered kayak, or in thigh braces.
- In a canoe, your legs are in a sitting position, or kneeling position. Instead of braces (if used), a canoe can have straps over the thighs.
4Be aware that it is not unusual to have mixed parties of kayaks and canoes paddling together.Ad
We could really use your help!
- Don't be afraid to ask an experienced paddler to show you the differences. They might even let you have a go yourself.
- Before deciding which one you want, have a try in both kayak and canoe and then decide.
- Always wear a life jacket or buoyancy aid for protection in case you fall in the water. It does happen, even on the calmest of days. Helmets are also a must for whitewater or any kind of moving water in the proximity of rocks.
- Before undertaking any paddle sports, make sure you are aware of the risks and take adequate precautions, or ask your instructor to explain the risks, and safety precautions being taken. Whitewater kayaking is an "assumed risk" sport, but sea kayaking/canoeing can also present life-threatening situations.
Article Info
Categories: Canoes, Kayaks, and Rowboats
In other languages:
Español: saber la diferencia entre un kayak y una canoa, Italiano: Sapere la Differenza Tra Canoa e Kayak, Português: Diferenciar um Caiaque e uma Canoa, Русский: отличить каяк от каноэ
Thanks to all authors for creating a page that has been read 185,078 times.


text:
A method for high-resolution image recording of at least one object with a microscope, includes the steps of: (a) positioning the object in a receptacle being arranged in the optical axis of the microscope, (b) generating at least two first data sets per object which represent intermediate images of the object with at least two different orientations relative to the optical axis of the microscope, wherein the different orientations of the object are provided by moving the object relative to the receptacle, and (c) evaluating the data sets for obtaining quantitative three dimensional information.
1. A method for high-resolution image recording of at least one object with a microscope, comprising the steps of:
positioning the at least one object in a receptacle arranged in an optical axis of the microscope,
generating at least two first data sets per object, wherein: (a) the at least two first data sets represent intermediate images of the at least one object with at least two different predetermined orientations relative to the optical axis of the microscope, (b) the at least two different predetermined orientations of the object are provided by controlled movement of the at least one object relative to the receptacle, and (c) the controlled movement comprises a rotation of the at least one object by an influence of electrical field forces, said object being rotated around at least one of a predetermined axis and a predetermined rotation angle, and
evaluating the data sets to obtain quantitative three-dimensional information of the at least one object, wherein said quantitative three-dimensional information represents a three-dimensional shape of the at least one object.
2. The method according to
3. The method according to
4. The method according to
5. The method according to
6. The method according to
a rotation in a continuous mode or for predetermined time periods and angles, and/or
a rotation with changing rotational axes.
7. The method according to
8. The method according to
9. The method according to
10. The method according to
11. The method according to
12. The method according to
13. The method according to
14. An imaging device for high-resolution image recording of at least one object, comprising:
a microscope imaging system with an optical axis,
a receptacle for accommodating said at least one object, said receptacle being arranged in said optical axis, and
a control circuit being arranged for: (a) generating at least two first data sets per object, wherein said at least two first data sets represent intermediate images of the at least one object with at least two different predetermined orientations relative to the optical axis, (b) providing controlled movement of the at least one object relative to the receptacle to generate the at least two different predetermined orientations, wherein the controlled movement comprises a rotation of the at least one object by an influence of electrical field forces, said object being rotated around at least one of a predetermined axis and a predetermined rotation angle, and (c) evaluating the data sets to obtain an object image representing a three-dimensional shape of the at least one object, and
a driving device adapted to controllably move the at least one object relative to the receptacle.
15. The imaging device according to
16. The imaging device according to
17. The imaging device according to
18. The imaging device according to
19. A method for high-resolution image recording of at least one object with a measuring device with a predetermined measurement field, comprising the steps of:
positioning the at least one object in a receptacle arranged in the measurement field of the measuring device,
generating at least two first data sets per object, wherein: (a) the at least two first data sets represent intermediate data of the at least one object with at least two different predetermined orientations relative to the measurement field of the measuring device, (b) the at least two different predetermined orientations of the object are provided by controlled movement of the at least one object relative to the receptacle, and (c) the controlled movement comprises a rotation of the at least one object by an influence of electrical field forces, said object being rotated around at least one of a predetermined axis and a predetermined rotation angle, and
evaluating the data sets to obtain quantitative three-dimensional information of the at least one object, wherein said quantitative three-dimensional information represents the three-dimensional shape of the at least one object.
20. The method according to
21. The method according to
The invention is related to methods and devices for high-resolution image recording of at least one object, in particular with a microscope.
A) Microscopy—in Biological Research.
The development of fluorescent bio-molecular probes—especially fluorescent proteins enabling the observation of sub-cellular process and structure inside living cells, has come to pass as a renaissance in light microscopy. Now a variety of evolving bio-chemical techniques incorporate and/or are based upon combining fluorescent molecular probes and light microscopy. They provide both qualitative and quantitative visualization of specific molecular dynamics underlying live cellular activities. However, inasmuch as it is now clear that these biological processes depend upon spatio-temporal compartmentalization, the major focus for development in light microscopy, is to overcome certain systematic problems that obstruct our ability to render simple, yet quantitatively accurate three-dimensional mapping of fluorescent signals inside living cells (viz. 3-D microscopy).
Three Major Systematic Problems in 3-D Microscopy
The ability to render three-dimensional maps of fluorescent signals from micro objects visualized by light microscopy is limited mainly by three systematic artifacts.
a) Axial Aberration—the Elongation Effect
A central issue in light microscopy arises from limitations due to the spatial resolution of images. This is dependent upon the objective lens used and the geometry of the light focused by the lens. In general, microscope objectives with a high magnification and a high numerical aperture are used for achieving the best resolution (e.g. 63×; 100×/N.A. 1.4 oil immersion). However, there exist physical limitations for light collection through the glass lens of any objective. In particular, the xy resolution is always (at least two times) greater than in the z-axis (a.k.a "optical" axis). Specifically, x,y plane resolution is around 100-150 nm, whereas the resolution along the z-axis is much lower (around 300 to 500 nm), and this fact results in a major systematic artifact of light microscopy—i.e. "axial aberration", whereby a spherical object at the focus of a microscope objective in fact appears to be elliptic in shape, with its largest extension along the z-axis, i.e. the light path. A schematic representation of this type of optical aberration (the "elongation" effect) is shown in
b) Chromatic Aberration—Axial Misalignment of Multiple Wavelengths.
In addition to the problems of optical aberration and diminished axial resolution that reduce the ability to visualize in 3-D on a light microscope, another problem is referred to as "chromatic aberration" or "axial chromatic aberration". Chromatic aberration occurs in applications where multi color images are acquired (Beyer). When performing z-scans at different wavelengths the light diffraction at the glass-to-medium-interface and within the whole microscope set-up depends upon the wavelength. As a consequence the focus for different wavelengths varies as a function of the z-axis displacement. Various types of correction are used to overcome chromatic aberration, for example, in confocal-microscopy (see below) a calibration of the "zero" position (unique to just one z-axis position) for all colors can be performed by an alignment of the light paths for the different wavelengths using multi colored (artificial) spatial-calibration samples (so-called "Focal Check Microparticles"). However, inasmuch as the calibration is unique to just one z-axis focal position, if a shift in focus occurs a systematic misalignment will again persist.
c) Out of Focus Light, and Diffraction Effects.
The third problem of three-dimensional fluorescence microscopy is that the micro-object itself comprises a complex three-dimensional form, and as such interferes with image visualization from the focal plane. This fact gives rise to a host of related problems for which there is no single general solution. Nonetheless, these problems are inextricably linked and must be considered critically in order to achieve true three-dimensional rendering. In general, these problems stem from light distortion caused by both the object itself, and the non-linear characteristics of light diffusion between different focal planes. These sorts of problem become critical in fluorescence microscopy where so-called "out-of-focus-light", (light from parts of an object laying outside the focal plane) contribute to what is observed at the focal plane. This light compromises the crispness of the image, inasmuch as it introduces an out-of-focus "haze" into the image focal plane.
There are two ways of eliminating or reducing out-of-focus haze: i) confocal microscopy (see below), and ii) deconvolution. The latter is a calculation-intensive, algorithm-based mathematical method for sharpening images (from any source) that contain out-of-focus light. In general, the method requires that an axial stack of images be collected from the sample, at small (e.g. 50-500 nm) steps. The axial stack may then be processed using special algorithms that take into account a variety of optical parameters including the objective lens, and excitation/emission wavelengths. The corrected stack of images is then converted into a three-dimensional model of the object by either removing or reassigning the identified out-of-focus light (Egner, Markham). Using adequate graphics computing power, this three-dimensional model may then be rendered into animations allowing the object to be observed from any arbitrary viewing point.
Disadvantages of this approach are that it is calculation intensive (which costs time and processing power), and that it suffers inaccuracies and artifacts due to its extensive dependence upon calculation-based assumptions and/or corrections that must be applied at multiple stages during the processing procedure. It must also be noted that the best types of deconvolution algorithm rely upon prior measurement of a so-called PSF (Point-Spread-Function) specific to any given optical configuration (ie the microscope set-up). Put simply, the PSF is a measure of light diffusion from a sub-resolution point within a given focal plane, and can therefore be used to "re-map" out-of-focus light back into its appropriate 3-D voxel. However, a major problem with applied algorithms using PSF, is that it is extremely difficult to measure a "good" PSF. In particular, a major problem arises from the fact that the PSF in any given sample is itself altered as a function of the axial distance through the sample (Sedat). This fact results in any single PSF being representative only of a single focal plane, and therefore distorts reconstructions based upon z-axis stacks where clearly the z-axis is deflected in order to scan throughout the volume of a given sample. Finally, in addition to out-of-focus light, the sample (as stated above) alters the diffusion of light through its own volume by diffraction. This gives rise to a further group of problems, whereby light emission from the focal plane is deteriorated due to shading or diffraction do light by optically dense regions within the object itself (e.g. cell) that lay in between the microscope lens and the fluorescent features being imaged.
Advanced Techniques for Improved 3-D Fluorescence Microscopy
Three-dimensional imaging of micro objects requires that the above problems be addressed, and this may in part be achieved using numerous types of novel approach. Herein, a brief description summarises some of the advanced microscope techniques at the cutting edge of what is currently available. However, it should be noted that all will be improved substantially by the utility of the invention described herein. On this point, as for conventional fluorescence microscopy, all these techniques (without exception) achieve 3-D rendering by mechanically scanning the focus through multiple z-axis acquisitions at small (nm) intervals, collected from the sample volume. As such the micro object must, therefore, be immobilized by adherence to an optically transparent surface substrate (normally a 150 micron thick glass cover-slip). The resulting image "z-stack" must then be treated by calculation-intensive processing to yield 3-D rendering.
Single-photon excitation confocal fluorescence microscopy uses focused laser light for fluorescence excitation and, in general, a pinhole in the path of fluorescence emission, which allows in focus light derived from the x,y image plane to pass, but effectively rejects out-of-focus light. Fluorescence light measured using pinhole systems is detected using photo-multipliers and a scanning device. By way of an alternative, some commercial confocal systems use a so-called "Spinning-Disc" (Nipkow disk) system that achieves much the same result by rejecting out-of-focus light. However, the detection system differs inasmuch as it comprises a CCD camera, affording greater speed of acquisition. Either way, the advantage of confocal microscopy is that out-of-focus haze is greatly reduced, and by performing a z-scan, stacks of confocal images can be generated from a sample volume, in order to build a three-dimensional rendering of the imaged volume. Note that this approach still suffers from chromatic and axial aberration problems.
Multi-Photon Excitation Confocal Fluorescence Microscopy
A method for improving resolution in fluorescence microscopy is based upon the use of multi photon laser excitation. Fluorescence excitation of a fluorophore occurs at a certain wavelength λ nominally determined by its specific excitation absorption maxima. Efficient absorption of a single photon at this wavelength results in excitation and emission of fluorescent light (conventional fluorescence microscopy). However, excitation may also be achieved by simultaneous absorption of two photons of lower energy, displaying wavelengths approximately half the excitation maxima. This mode of so-called "multiphoton" excitation is considered to be "biphotonic or two-photon" induced fluorescence, and is made possible by grace of high energy pulsed lasers. In general this mode of excitation can be considered a means to excite fluorescence from, for example, a blue-green absorbing fluorophore using multi-photon excitation from a near-infra-red laser emitting sub-microsecond pulses of light. Inasmuch as the two photons of near-IR light are aligned and collide only at the focal plane of the optical set-up, the energy density of this multi photon excitation is concentrated solely at a single femtoliter volume within the microscope's focal plane. As such, multi-photon excitation is intrinsically confocal by nature. In effect this approach gives a pure, and efficient image free from "out of focus" fluorescence. The disadvantage of multi photon fluorescence microscopy is the requirement for high energy pulsed lasers to be attached to the microscope, resulting in high cost and large, difficult to manage equipment assemblage, maintenance and application.
4Pi Confocal (Theta) Microscopy, Standing-Wave Microscope (SWM), Incoherent Illumination Interference Image Interference Microscopy (I5M)
The generation of higher resolved three-dimensional images of cells can be improved by a combination of the techniques mentioned above and modifications of the optomechanic set-up. The use of two separate objective lenses for excitation and collection of fluorescence emission light leads to a smaller detection volume element and an equilateral resolution some 4 times higher than for conventional fluorescence microscopy (Egner). This technique is used in combination with multi photon fluorescence microscopy. In a 4Pi confocal fluorescence microscope two opposing microscope objective lenses are used to illuminate a fluorescent object from both sides and to collect the fluorescent emissions on both sides. Constructive interference of either the illumination wave fronts in the common focus or the detection wave fronts in the common detector pinhole results in an axial resolution approximately four times higher than in a confocal fluorescence microscope (Hell). The excitation/observation volume can be considerably decreased when the detection axis is rotated by an angle Theta (e.g. 90°) relative to the illumination axis as in Theta Microscopy (Lindek). Both methods bring along substantial limitations for the sample carrier and the microscope objective, which can be used. In addition there is a huge effort involved when aligning the two focal volumes of the objective lenses, which has to be done with sub-micrometer precision.
B) Micro Electrode/Fluidics Chamber(s) for Three Dimensional Manipulation of Micro Objects
Holding and lifting micro objects by negative dielectrophoresis in a well defined electric field minimum has been described since 1992 (Fuhr, G. et al. "Biochim. Biophys. Acta" 1108, 1992, 215-223). First, planar two-dimensional arrangements of micro electrodes have been used. They contained for example four electrodes with a tip-to-tip distance of 100 to 200 micrometers. Holding and lifting objects in these so-called "field traps" was only possible using alternating fields. Rotational fields had only limited trapping efficiency and were very sensitive to hydrodynamic streaming (Schnelle, Th. et al. "J. Electrostatics" 46, 1993, 13-28, Schnelle, Th. et al. "J. Electrostatics" 50, 2000, 17-29, Schnelle, Th. et al. "Appl. Phys. B" 70, 2000, 267-274, Reichle, Ch. et al. "Biochim. Biophys. Acta" 1459, 2000, 218-229). The development of so-called CellProcessors—three-dimensional electrode arrangements led to "field cages" consisting of eight electrodes and building up closed electric field cages (Schnelle, Th. et al., 1993, see above, Müller, Th. et al. "Biosensors & Bioelectronics" 14, 1999, 247-256, Reichle, Ch. et al. "Electrophoresis" 22/2, 2001, 272-282). "Cell Processors" containing dielectric field cages (DFCs) have been used in combination with a variety of high resolution optical techniques applied to micro objects, such as fluorescence correlation spectroscopy (FCS, Schnelle, Th. et al. "Electrophoresis" 21, 2000, 66-73), force measurements using laser tweezers (Fuhr et al. "Appl. Phys. A." 67, 1998, 385-390), electro-rotation (Schnelle et al., see above), measurement of ligand-receptor binding forces (Reichle et al. 2001, see above) and confocal laser scanning microscopy (Müller, Th. et al. "European Biophysics Journal" 29/4-5, 2000, 12D-3 (Poster); Wissel, H. et al. "American Journal of Physiology Lung Cell Mol. Physiol." 281, 2001, L345-L360).
The object of the invention is to provide improved high-resolution measuring methods, in particular imaging methods avoiding the disadvantages of conventional methods. It is a particular object of the invention to provide a generally applicable solution that enhances high-resolution 3-D imaging methods by overcoming, diminishing or avoiding completely the systematic artifacts of light based microscopy described above. The invention shall provide an altogether new approach for the generation of three-dimensional image series describing both quantitatively and qualitatively a more accurate spatial map of any given fluorescent, bioluminescent, or auto-fluorescent micro-object—e.g. a live mammalian cell labelled with fluorescent molecules. Another object of the invention is to provide a device that may be implemented to any (and all) existing fluorescent microscope techniques (as described above).
Generally, the high-resolution image recording according to the invention comprises a positioning of at least one object in a receptacle in the optical axis of a microscope imaging system, generating at least two intermediate images of the object with different orientations in space and evaluating an object image from the intermediate images, wherein the different orientations of the object are provided by moving the object as such relative to the receptacle. The feature of moving only the object while the imaging system and the receptacle are kept at fixed positions (e.g. in a laboratory system) has the advantage of providing high-resolution images without troublesome changes of the opto-mechanical features of the imaging system. The term different orientation refers generally to different geometrical arrangements of the object relative to the optical axis. The arrangements can be obtained by translations and/or rotations.
The invention can avoid completely (1 and 2), or minimize (3) the following problems in 3-D light microscopy:
The invention also optimizes completely (4 and 5), and enhances greatly (6) the following procedures of 3-D light microscopy:
One basic idea of the invention described herein is to use in particular a DFC in order to freely translate and/or rotate the micro object itself in order to record different image planes and to keep the measurement volume itself motionless (or to move it in just one dimension e.g. scanning).
In the context of the current invention these movements of the micro object—e.g. a live suspended biological cell—are preferably realized by time dependent ac electric fields and negative dielectrophoretic forces. They are preferably generated in fluidic micro-chips containing DFC's that are customized to achieve specific movements of the micro object in three dimensional space. In this context we have implemented and substantiated that a DFC comprising eight micro electrodes forming a closed field cage, provides sufficient stability and control to achieve these movements. Ideally, the dimensions—diameter and distance between electrodes—are preferentially within the range of the object's size, i.e. between 1 and 1000 μm. The electrodes were fixed within a microfluidic chamber which was made of a transparent material suitable for high resolution microscopy, e.g. glass with 150 μm thickness. Negative dielectrophoresis was induced by applying electric ac fields in a frequency range of 100 kHz to 100 MHz. Field cages were generated, for example, by applying rotational fields to the two planes of four electrodes each with a phase shift of 90° between the electrodes within one plane and of 180° between the planes. During micro object rotation this mode yielded an efficient dielectric field cage and highly stable positioning in x-, y- and z-directions.
Thus, the invention is based on this surprising and unexpected result of the inventors according to which micro-objects suspended in a fluid can be manipulated with electric field forces with a precision sufficient for an evaluation of the object image from the intermediate images. The precision of rotation even was obtained despite asymmetry and/or gravitation effects. Thus, it has been found that the object can be positioned stably in x-, y- and z-directions. It can be rotated around a defined axis and oriented by addressing the electrodes with a suited signal characterized by time dependent amplitude, frequency and phase.
The physical reason for the rotation of a micro object is a polarization of its charges/dipoles within a rotational field. The torque is dependent upon the field strength, the field frequency and the passive electric properties of the object in relation to the suspending medium. The torque can be deliberately induced by using appropriate rotational fields, i.e. certain phase shifts between the electrodes and/or a certain geometry of the electrode arrangements. Computer/user controlled protocols will be adapted that allow the electrodes to be alternately triggered in order to achieve fixed protocols of micro object movement, and facile directional and speed control. The resulting device shall be capable of allowing the user to select and change the rotational axis, speed of rotation, and extent/angle of deflection.
With this device rotating the object can be realized in a continuous fashion or for defined time periods and angles. For example, a horizontal rotation can be changed to a vertical rotation or a "random" rotation while maintaining the same z-position. Small changes of the rotational axis can be achieved by varying the amplitude and/or the phase at least at a single electrode. Moreover, a well-defined variation of the z-position can be achieved by varying the amplitude and/or the phase within one electrode plane, and rotational axes can be changed without affecting the positioning stability.
For caging objects within a DFC without inducing a torque AC fields can be used. The dipole moment in z is then zero and forcing in z-direction—i.e. the positioning along the z-axis against the force of gravity—is achieved merely by higher moments, e.g. quadruple moment. This works best for a ratio of object diameter compared to tip-to-tip distance of the DFC electrodes of 1:4 to 1:10. Preferred field modulations are adapted for changing a horizontal rotation to a vertical rotation or a "random" rotation while maintaining the same z-position, applying small changes of the rotational axis—typically 1° to 10°—by varying the amplitude and/or the phase at least at one electrode, or varying the z-position—typically 50 nm to 10 μm—by varying the amplitude and/or the phase within one electrode plane. If the object to be investigated has an asymmetrical shape, the electrodes can be controlled such that the object is rotated relative to a fixed centre of rotation. A variation of angular velocity and/or centre of rotation can be compensated by a field amplitude modulation. In terms of a homogeneous compensation, the electric field forces preferably are generated by a plurality of electrodes (about 8 or more) surrounding the object.
According to an alternative embodiment of the invention the micro object may be moved by holding the object at a fixed position by means of electric field forces and by rotating the object by means of optical forces as generated e.g. with laser tweezers.
Image Series Generated from Micro Object Rotation Using the Invention Contain Characteristic 3-D Spatial Information that is Free from Artifacts.
Generally, the high-resolution image recording according to the invention comprises a positioning of at least one object in a receptacle in the optical axis of a microscope imaging system, generating at least two intermediate images of the object with different orientations in space and evaluating an object image from the intermediate images, wherein the different orientations of the object are provided by moving the object as such relative to the receptacle. Clearly, rotating a micro object while maintaining the imaging system, optics and sample receptacle in a fixed position has the advantage of generating an x,y image series that reveals implicitly 3-D details that are transiently axial to the focal plane, but without axial or chromatic aberrations. Moreover, this 3-D visualization is facile, inasmuch as it does not require troublesome axial changes in the focal plane that normally require complex opto-mechanical devices.
Thus, in effect the invention allows, on-line, "real-time" 3-D movies to be generated of micro-objects that should normally require "virtual" rendering by off-line, convoluted and expensive methodologies.
Utility and Further Advantages of the Invention:
The methods of the invention are implemented with an optical microscope imaging system as e.g. a modified conventional fluorescence microscope being equipped with a two-dimensional camera or with other detector elements being arranged point-wise, as a line or two-dimensionally. In the context of the present invention, "high-resolution imaging" is defined as spatially mapping micro-objects with maximum optical resolution (i.e. on the x,y plane of axes ˜100 nm) without recourse to z-axis deflection that should nominally introduce axial aberration and a loss of resolution. A micro-object to be imaged is a biological or synthetic object with a typical cross-section in the range of 1-100 μm. Imaging an object generally comprises mapping of topographical object features. The object features comprise the complete object or only parts thereof, they are located on the surface, or in the volume of the object. The 3-D object shape may be evaluated from the intermediate images, and by grace of the invention specific rotations of the suspended cell, around a fixed xyz coordinate gives optimal optical access to every location within the cell. Thus, the invention makes it possible to generate image planes from different angles and object planes without moving the microscope objective or the scanning table of the microscope. For the application of deconvolution algorithms using PSF based calibrations, this has the added advantage that only a single PSF (measured a fixed distance from the objective and coincident with the focal plane) needs to be applied.
Continuous imaging of controlled changes in position of the object by rotating it around at least one horizontal axis and one vertical axis is very useful for a reduction of the calculation effort required for a three dimensional imaging. A film series (see figures) of cell rotation imaged with a camera at relatively high speed (1-50 images/second) results in a "real" recording of three-dimensional structure, thereby replacing the virtual 3-D rendering generated from conventional optomechanical z-stacking acquisition (as described above). Depending on the phase shift of the electric fields at the eight electrodes of the field cage, arbitrary rotational axes can be realized. A rotation of a single cell around two different axes which run through its center—for example within the x,y plane of the microscope's field of view and around the z-axis—enables multiple sets of images to be repeatedly captured and the 3-D structure of the cell can be fully evaluated from all angles, limited only by the number of sampling repeats.
The invention can be combined easily with many different techniques of fluorescence microscopy including all confocal techniques as well as with methods for single molecule detection [e.g. fluorescence correlation spectroscopy, Fluorescence Intensity Distribution Analysis (FIDA), Moment Analysis of Fluorescence Intensity Distribution (MAFID)]. For example, it can be advantageous to move a light beam that passes the objective in one dimension like in a laser-scanning microscope (scanning). The object can be rotated slowly around a horizontal (xy) and/or vertical (z) axis, typically with a frequency of 0.5 rps (revolutions per second) to 1 rpm (revolutions per minute). Along the same lines, a rotation around the z-axis and simultaneous fixed-point scanning in a single direction (like applied in the "In-sight" reader of molecular fluorescence equipped with a linear beam scanner) yields a two-dimensional scan of a plane within the cell. Photon counting and signal processing techniques can then count fluorescent molecules within this specific plane. Along the same lines, the ability of the invention to move the suspended cell along the z-axis is of great utility. In particular, moving the cell eliminates the need to move the carrier in relation to the microscope objective. Thus, once the optical system is aligned for multi color observations, chromatic aberration due to different refractive behavior of different excitation wavelengths in multi color applications does not occur.
The Invention Enhances the Application of Existing 3-D Rendering PSF-Based Algorithms.
The ability to rotate a micro object is useful for sampling sub-compartmentalised domains. For example, by grace of the invention, a living cell may be suspended and imaged in its starting position, before being rotated around an axis within the x,y (equatorial) plane by a defined angle (e.g. 180°, 90° or less) and then imaged again in a second position. The same procedure is repeated for an axis perpendicular to the first in the z plane. This repeating procedure provides a series of images of the same cell from different angles which, in principal, can be used to map the whole 3D volume with a resolution and precision being dependent from the number of images and different angles included (see below). In combination with the ability to rotate the micro-object, the invention allows small (50 nm-50 μm) z-axis movements to be achieved. Thus, an object may be rotated and shifted on the z-axis in order to build a series of images from the same sub-volume, but from different views. Clearly, this type of combined rotational/axial manipulation allows objects to be introduced and orientated within the "optimal imaging space" i.e. at a focal plane whereby the application of PSF-based deconvolution and image reconstruction algorithms may be optimised according to corrections intended to minimise spatial and chromatic aberration. Such existing methods must always take into account the coordinates of the optical measurement volume, defined by the position and characteristics of the microscope objective relative to a fixed position sample. When the z-position changes (i.e. the focus is moved) all these parameters (i.e. those determining the PSF and axial volume) change and must be recalculated, re-measured or estimated. Inasmuch as the invention allows for the optical geometry to be fixed, it therefore greatly reduces the errors intrinsic to measurement and application of existing image algorithms used for deconvolution and volume reconstruction from z-stack image series.
The Invention Opens the Way for Development of New Three Dimensional Image Reconstruction Algorithms.
As mentioned in the preceding paragraph, in addition to enhancing the application of existing algorithms, the invention also opens the way for development of image processing algorithms previously applied to other problems. For example, in X-ray computer tomography objects are imaged by rotation of the detection system around the object. By taking a multiple image series of intermediate coordinates from around the object, it is possible to both render the object's three-dimensional volume, and increase resolution of the images. Thus, by extrapolation, it is in principal possible to apply an analogous algorithmic approach to rendering of micro-object rotational series generated using the invention described here (e.g. see Appendix I). As such, the invention underlies development of completely new "3D Image Reconstruction" methods. The primary advantage of this sort of approach is that the invention ensures that imaging uses solely the x,y plane of resolution to visualize a micro object from multiple view points. Given the absence of axial aberration and reduced z-axis sampling resolution, these new methods shall implicitly deliver 3D reconstruction representations with substantially higher resolution than for conventional reconstruction techniques. Indeed, such methods may also be combined with those already existing image algorithms techniques that use a combination of PSF and volume rendering.
Another subject of the invention is a device for high-resolution imaging of at least one object, which comprises an optical-microscopic imaging system with a receptacle for accommodating the object and a control circuit for controlling the generation of the intermediate images, wherein this imaging system is equipped with a driving device for moving the object relative to the receptacle. According to a preferred embodiment of the invention, the receptacle is provided by a fluidic microsystem with an arrangement of microelectrodes, wherein the driving device comprises the driving electrodes and a driving circuit within the control circuit.
According to a preferred embodiment of the invention, the control circuit contains a switching box being adapted for predetermined switching the rotation axis of the object. The switching box allows predetermined manipulations of the object as well as the implementation of a time trigger.
Further advantageous embodiments of the imaging system are characterized by a multi-electrode arrangement in the fluidic microsystem. The multi-electrodes arrangement comprises e.g. at least 3 electrodes within one plane surrounding the object. These electrodes can be controlled for an improved compensation of position or velocity variations of asymmetric objects.
Further advantages are derived from the fact that the object manipulation in the receptacle does not effect the object (e.g. the physiology of biological cell). High-resolution imaging according to the invention can be conducted even over a long period of time (e.g. 60 minutes).
The implementation of the invention is not restricted to the application with a microscope. Alternatively, the Method for high-resolution image recording of at least one object can be implemented with a measuring device comprising a predetermined measurement field. As an example, the measuring device can comprise an impedance measurement device and said measurement field being the receptacle itself.
Further details and advantages of the invention are described in the following with reference to the attached drawings, which show in:
Firstly, examples of a method according to the present invention are described with reference to
The position of the electrodes of the dielectric field cage is illustrated in
Reference numerals 1′, 2′, 3′, 4′ refer to electrode tips of the four electrodes within the lower electrode plane, i.e. on the lower glass substrate of the DFC channel which is facing towards the microscope objective. X, y and z are the axes of the optical set-up as described in the text.
Continuous Sampling
The method of the invention is based on the collection (sampling) of at least two intermediate images of the object. Sampling can be performed in a continuous fashion as described with reference to
The cell 3.1, having a dimension 3.2 of several micrometers, is continuously sampled during low velocity, high precision rotation of the cell within the z-plane 3.5, i.e. around the y-axis (y). Every feature (e.g. 3.3) of the cell will be within the focal plane 3.7, having a thickness of several hundred nanometers, at a certain time and will be imaged there with highest xy resolution. Discontinuous sampling (see below), i.e. a xy move of 90° (3.6) followed by a second rotation of the cell within a the z-plane (3.5) allows full sampling. This positions a feature with the start position 3.3 for the first scan to the start position 3.4.
Discontinuous Sampling
Examples of discontinuous sampling are illustrated in
Piezo-Electric Objective Sampler in Combination with Fixed Multiple Sampling Angles
The image-spin series of the invention can be combined to good effect with fixed and discontinuous sampling in an alternating-mode.
First, a z-scan is performed on a motionless cell at fixed position 5.1 using a piezo-electric objective sampler (schematically shown at 5.1.1). The z-position of the focus is varied in small steps, e.g. 0.1 to 1 μm (z-scan). Reference numeral 5.1.2 indicates an example of an actual focal plane. After taking a series of images, the cell is rotated around the y-axis 5.1.4 by a fixed angle of 90° (5.1.3) to position 5.2 followed by another z-scan (5.2.1). A second series of images is taken. A further rotation by 90° around the x-axis 5.2.2 to position 5.3 followed by another z-scan (5.3.1) completes the procedure.
According to the above principle, the feature 5.1.5 is sampled twice in two perpendicular axes. In position 5.1, x-y-images are taken in dependence on z:
5.1: (x,y→z)
After turn 5.1.3, the feature 5.1.5 has a new z-position in the x-y-images:
5.1.3, 5.2: (z→x, y)
Both scans 5.1.1 and 5.2.1 deliver a plurality of images, which allow a calculation of the x,y,z-volume of the feature 5.1.5. The feature 5.1.6 is not sampled until the second turn. The total object is reconstructed from all coordinate stacks after the second rotation around the x-axis only.
The receptacle (20) comprises a fluidic microsystem with a channel or chamber (21) for accommodating a liquid with at least one suspended object to be investigated. Microelectrodes (22) are arranged on the walls of the chamber (21) as it is known from conventional microsystem technology. The chamber (21) is closed with a cover glass (23) and carried by a support (24). The support (24) can be adjusted with a receptacle drive unit (25).
The control circuit (30) comprises a data set storage (31), a parameter storage (32), a switching box (33), a calculation circuit (34) and an object image storage (35). The control circuit (30) is connected with further input/output devices (40), e.g. with a display and a printer.
For conducting the method according to the invention, the object to be investigated is positioned in the optical axis of the microscope (11). The microelectrodes are controlled with a certain set of parameters, which are contained in the parameter storage (32). The parameters comprise e.g. the phase, amplitude and frequency of electrical fields, information about the control mode as well as object properties. According to the parameters, the object is moved within the receptacle (20) as described above. During the movement, a series of at least two data sets is recorded with the camera (13) and submitted to the data set storage (31). The data sets are evaluated in the calculation circuit (34) in dependence on the parameters (32) for obtaining the object image, which is submitted to the object image storage (35).
The switching box (33) is connected with the parameter storage (32) and the calculation circuit (34). The switching box (33) is arranged for submitting predetermined switching parameters to the parameter storage (32). According to the switching parameters, a movement mode of the object in the receptacle (20) is adjusted.
—A—Visualization of the Lamin Structure of HeLa Cells
As an example for studying a cellular protein by live three-dimensional microscopy fluorescent nuclear lamin proteins inside a live suspended cell were imaged using a prototype of the invention.
Lamins are the major components of the nuclear lamina, a two-dimensional filamentous network at the periphery of the nucleus in higher eukaryotes, directly underlying the inner nuclear membrane. In the course of cell-cycle-dependent dynamics of the nucleus in higher eukaryotes, lamins as well as lamin-binding proteins seem to possess important functions during various steps of post-mitotic nuclear reassembly and seem to play a role in various pathological processes in muscle (Emery-Dreifuss muscular dystrophy, EDMD; dilated cardiomyopathy and conduction system defect, DCM-CD; and limb-girdle muscular dystrophy, LGMD) and adipose tissue (Dunnigan-type familial partial lipodystrophy, FPLD). Lamins are more dynamic than originally thought. These findings altogether demonstrate the necessity to investigate the localization of lamins within the cell and their dynamics with great precision in order to learn about their functions and dysfunctions. HeLa cells were used for this purpose which overexpressed lamin protein fused to the fluorescent protein dsRed.
The cells were detached from the culture flask using trypsin and suspended in Cytocon™ Buffer II containing 30 mM salt/phosphate buffer balanced to normo-osmolar values using 0.3 M inositol solution. The cells were introduced into DFC4 chips and caged within the 30 μm field cage (distance between opposing electrode tips within one plane: 30 μm) using the Cytocon™ 300. The field cage was energized at 700 kHz and 1.9-2.6 Vrms. The cells were subjected to a rotational movement by using the "rot II" mode of the Cytocon™ 300. The DFC4 Chips was mounted on a Zeiss Axiovert 200 microscope equipped with the Photometrics Cool SNAP camera Hamamatsu and the T.I.L.L. Vision imaging system. The cell rotated at a constant speed of approximately 20 revolutions per minute and an image-spin series was generated at a speed of 10 frames per second with 50 ms exposure time. The axis of the rotation was selected by a controlled modulation of amplitudes of the upper and lower electrode plane of the field cage.
Image-spin series of the HeLa cell with overexpressed lamin protein fused to the fluorescent protein dsRed are illustrated in
—B—Rotation of Jurkat Cells Around Two Different Rotation Axes
Jurkat-cells were suspended in Cytocon™ Buffer II and introduced into a DFC Chip. A single cell was caged within the center 30 μm field cage of the chip using a Cytoman™ and a frequency of 700 kHz and an amplitude of 1.9-2.6 Vrms. The cell could be continuously rotated around a horizontal as well as a vertical axis without altering its center position. Spin frequency could be varied from 0.01 rps (revolutions per second) to 0.5 rps by altering the amplitude in a range of 1.5-3 Vrms and/or the frequency in a range of 0.3 MHz-8 MHz.
The Cytoman™ provides two different rotational field modes and one alternating field mode according to table 1. A switch box enabled to change between two different spin axes (around a vertical and a horizontal spin axis, see
A deconvolution, volume reconstruction operation bases itself on the following considerations: "A digital image (e.g. microscopic image of a distribution of fluorescent molecules) is a convoluted 2-dimensional representation of a 3-dimensional reality. The individual information given at each image pixel includes not only the true signal emerging from a single point in the focal plane, but also varying degrees of additional signal accumulated from fluorescent out-off-focus volumes, above and below the focal plane, but within the focal depth along z-axis" (from Shorte & Bolsover, 1999). Fluorescent specimens act as self-luminous objects in which point sources behave as independent sources. Neglecting the light scattering, the optical density g at each pixel can be written as:
The deconvolution is obtained in analogy to conventional reconstruction from radiographic projections (tomography). In standard tomography, the specimen does not move but the radiographic apparatus turns around it to get a set of images. The values c can be reconstructed as described by B. Chalmond et al. in "inverse problems", vol. 15, 1999, p. 399-411. According to the present invention, the situation is reversed. The object to be investigated is rotated whereas the microscope is fixed. The conventional tomography algorithms can be used after a numerical coordinate transformation of the microscope set up into the radiographic context.
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I'm a textbook INTJ. I can seem a bit socially retarded at times and I'm pretty sure that has something to do with it.
I'm in a fantastic, fulfilling (open, poly) relationship with smart, vivacious woman that I met right here on OkC. Being cool with that is obviously mandatory.


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I have being asked to blog supporting the march against Cuts for the Child Benefit. In case you haven't heard yet, the Government might cut Child Benefit from €140 to €100 per child.
Before I go on about other related stuff, my opinion is that it is wrong to cut Child Benefit to ALL families regardless of their income. My blood boils when I think about this, because I cannot understand for the life of me why the heck it is not MEANS TESTED
MEANS TESTED BENEFITS is the only fair way to do things! WHY are they NOT doing this????
Why are they going to cut the child benefit regardless of the family situation? Cuts for all! It doesn't matter if you are a single working mother earning 30k or less per year or a couple earning excess 70k per year. It doesn't make any sense!
(Sorry, I told you my blood boils)
Apart from the means tested detail, It is wrong to cut child benefit to all families, essentially, because the top cats are still living the life of kings, bondholders are still getting their money back and all the money we are saving in tax, cuts and increase VAT is going towards banks, bankers, politicians and nothing towards making this country a better place to live in. This is why is WRONG. They didn't invest sht in public services during the Celtic Tiger and you can be sure, they won't invest now for a long, long time. So we have to put up with the horrendous health service, crap roads, crap public transportation, overflowing bins full of more crap because they can't afford to pay someone to empty them, crap playgrounds for kids, shortage of midwives, shortage of beds in hospitals, shortage of special needs assistants and no adequate equipment to treat sick children, I mean that is top of the pops. You need to know no more.
Today, this post has been published by http://pacub.blogspot.ie/ (who is the one behind the march against Child Benefit cuts which takes place on Saturday the 3rd of November) talking about why means-testing is not a good idea. I SO don't agree with it. People seem to be scared of thinking about putting a threshold on family income to decide who gets the benefit and who doesn't. Let me do it for you. What about at least 100k per year per family? Im sure there are still plenty.
If the base of your argument is that all children are equal, so they all deserve the benefit, well, let me tell you, no child is equal because their life will be completely different depending on their parent's situation. A well off family will have no problem covering the costs of raising their children. It is their responsibility to do so. They shouldn't need money from the estate for this.
Many people seem to think the benefit is actually not enough. I made some calculations (Please be free to tell me if I made a mistake!)
During 2008 and 2009, child benefit was between €160/€166 per month. That is nearly 2000€ per year per child.
The Early Childcare allowance (payable until 6 years old was a total of 1000€ per month PER CHILD, paid 3 times a year)
So that equals a total of nearly €3000 Per year, PER CHILD. A fecking fortune. I couldn't believe it.
The Early Childcare Allowance stopped at the end of 2009. Child Benefit has been cut during those years and now it stands at €140.
"In 2012 Child Benefit is €140 per month for each of the first two children. From 1 Jaunary 2012 the rate for the third child is €148 and for the 4th and each subsequent child is €160.
Additional monthly payment for twins and triplets will continue but the grant of €635 paid at birth, at 4 years of age and at 12 years of age for these multiple births has ceased from 1 January 2012. " Source
€140 per month is €1680 per year.
2 kids = €3360 per year
3 Kids = €5136
4 kids = €7056
Holy Moly. That is a crazy amount of money.
Please don't get me wrong. I'm not in favour of cutting the benefit, and I know many, many families are struggling (Heck, I'm struggling. I work full time but my partner is unemployed. We don't have a mortgage and we don't have debts. We only have one child. I would like to have another child, but you know what, right now I can't because of my economic situation, and we have to suck it up. I think people have to start taking some responsibility of what they do with their lives….), but I am completely AGAINST universal benefits. It is just wrong.
I thought it would be interesting to look at the child benefit from the angle of a Spaniard living here (As I am Spanish living in Ireland).
I decided to ask some Spanish friends who have been living or are still living in Ireland, and had children here and in Spain and see what they think about this subject, and what country they think is cheaper to raise a child.
In general they are all delighted to live in Ireland and receive the child benefit. Basically, nowadays, in Spain you get nothing. Spanish women think basic needs such as clothes, infant formula, baby bottles and nappies are cheaper over here. (Penneys, Dunnes yaiii!)
Primary School Education is more expensive in Spain because parents pay about €300 for books! And they must pay minimum €120 for the canteen (if the child eats at the school. Remember, children in Spain stay in school until about 5pm every day with 2 hours break for lunch time)
Childcare in Spain is much cheaper. But Spanish women only get 4 months maternity leave. (Full paid by employer). They have a right to work part time for a while. Salaries are lower. Public Healthcare IS Public Healthcare which means GP is free. Which brings me to:
Last year I spent close to €500 on GP's and medicine for my son, who was getting sick every couple of weeks. AND I have private insurance (fecking useless to be honest). If Child Benefit is cut, again, for ALL families regardless of their income, I can assure there are going to be kids getting sick that won't be seen by GP's because their parents can't afford to dish out €50 per GP visit, just to write the name of the antibiotics your child needs or worse, just to tell you: "just keep doing what you are doing, Calpol and Nurofen, €50 please :D"
So much more to moan about (Actually, it is not moaning, people, it is fighting for your rights!) but Ill stop now.
10 Irish parenting bloggers have joined forces in a "BlogMarch" to raise awareness of the crippling impact that cuts to child benefit will have on Irish families if introduced in December's budget. The bloggers are publishing a blog post each over ten days to highlight the negative impact that child benefit cuts will have.
If you'd like to lend your support, you can sign the online petition here.
You can also share your thoughts with us on Twitter at #BlogMarch or Like our Facebook page.


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Akc yorkie males for 600.00 and females for 700.00. They are utd on vaccination and worming. We guarantee their health. Check out face book under Puppy Dog City.


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Groups of students hover around desks in an Oklahoma City Community College classroom, conferring with each other about the math problems on the worksheets in front of them.
Today, the lesson is on factoring equations. Instructors walk from one group to another, talking to each about multiples, products and greatest common factors.
Strictly speaking, this isn't a college-level math class. Credit won't count toward students' degrees, and their GPAs won't reflect how they did.
But experts say courses like this one — and how they're conducted — are a critical factor in whether a student graduates with a degree or leaves college empty-handed.


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