Posted by
Chris Wood-5 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FW-Deconvolve-1-42-Components-Setup-now-OK-tp590745p590765.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalWe've been using LEDs in place of mercury lamps for around 4 years now,
and I can't see us ever going back to mercury lamps. They were absolutely
essential for what we were trying to do, which is measure Ca2+
fluctuations in the beating flagella of motile sperm - picking up a signal
from a sub-micron structure flailing around at 40-50 cycles per second was
no mean feat. Our only hope was stroboscopic epifluorescence, and LEDs
offered the perfect solution. Using them in pulsed mode we overdrove the
current 10x to maximise excitation, then set a illumination pulse duration
of 1 - 2 ms and streamed as fast as we could. The frame rate limitation
therefore boils down to how fast the camera empties its chip. We've
published a few articles with frame rates around 40 per s using a Quantix
57, but a while ago we upgraded to an iXon 887 and now we're routinely
collecting full frame epifluorescence images at around 200-400 fps.
Apart from the stroboscopic aspect allowing us to freeze flagellar
motility, another big advantage for us has been minimising photobleaching
and phototoxicity (to which sperm are extremely sensitive). Because the
illumination lasts 1-2 ms and the LED is switched off during the chip read-
out, the sperm are illuminated for only a small fraction of the duty
cycle. Even at extremely high frame rates, 250 fps say, we reduce overall
illumination times during an experiment by 75% compared to an HBO lamp.
At slower frame rates (40fps) the reduction was around 96%. Shuttering an
HBO lamp at these sort of frame rates is not a viable alternative believe
me. We published a technical note last year outlining these benefits
(Nishigaki et al, Biotechniques 41:191-7), I recommend a look at the
movies comparing the phototoxicty of the two techniques, it's an
impressive difference.
The system we built ourselves as there was no commercial option four years
ago, but it's essentially the same implementation as the OptoLED from
Cairn Research. Another group in our Institute has one of these on order
so I'll be nosing around once it arrives. Zeiss have implemented their own
LED illumination module, called the Colibri, but when I heard the price I
must confess my jaw hit the floor.
To sum up, we now use LED illumination on all our 'scopes, not just for
the sperm motility work but for all our routine epifluorescence. As I said
I can't see why we would ever go back to the arc lamps. LEDs will very
soon become the routine choice, and the technology is advancing fast.
Saludos
Chris
Dr Chris Wood
Instituto de Biotecnología
Universidad Nacional Autónoma de México
Av. Universidad 2001
Col. Chamilpa
Cuernavaca 62150
Morelos
México
On Tue, 6 Nov 2007 10:27:39 -0600, Barbara Foster <
[hidden email]> wrote:
>Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
>Dear Glen
>
>As a strategic consultant in microscopy, I get to see the latest
>technology and there is, indeed, a great deal of flurry about LED
>technology. In the summer of 2006, I had a chance to evaluate the
>AFTER/FluoLED from Fraen and was very impressed with the design, ease
>of use, and flexibility. I have been working on assignment with
>Fraen more recently and was surprised to see how much both LED
>technology and this product line had evolved. So here are
>observations on both LED technology in general, and the Fraen system
>in particular.
>
>Fraen's FluoLEDs are now available in UV (354nm), Royal blue (450nm),
>Blue (480nm), Cyan (505 nm), Green (535nm) Yellow (590nm) and red
>(630nm). While Fraen is a new name in the microscopy arena, most of
>you already know them: they are the world's largest manufacturer of
>the LEDs used for the pointers/indicators for the speedometers, gas
>gauges, etc., on the dashboard of your cars.
>
>Until recently Fraen's AFTER/FluoLEDs were only available in
>transmitted light version for upright microscopes, currently, over 17
>different models from all the major manufacturers and several of the
>smaller ones. For us "old timers", transmitted light has typically
>been seen as less efficient, but the superb images from FluoLED tell
>a very different story: Bright features against wonderfully velvet
>black background. In other words: great S/N. Fraen will be
>releasing the first systems for inverted stands next month and have
>begun work on an epi version as well.
>
>As with any technology, there is up side/down side to LEDs
>The good news is the consistency, lack of fuss, and economy of
>LEDs. When they are on, they are on. When they are off and you need
>them on, you can turn them on immediately - no cycle time.
>Also, they exhibit much less drop off over time than HBOs. That time
>factor is critical. Life expectancy of an HBO is on the order of
>200-300 hrs; for Fraen's LED's (I don't have figures on the others)
>30,000 hrs. No error in decimal points here: you can run them 8 hrs
>a day, 5 days a week, for 5 years without changing a lamp. If you
>plot drop-off versus time, a 100 fold increase in time is
>significant, especially for those of us doing long term experiments.
>When it comes time to switch out the lamp, there is no alignment, no
>disposal issue.
>The economy issue is also an interesting. Fraen's European office
>did the following calculations (Euros) for the LED cassette for a
>standard Blue excitation kit vs. an HBO arc lamp:
>Cost of LED cassette: Eu720 Cost of HBO lamp: 160
>Lifetime LED casette: 30,000hrs Lifetime HBO lamp: 300 hrs
>Eu/hr LED cassette: EU 0.024 Eu/hr HBO lamp: Eu 0.53
>Assumption: if you run both systems for 2000 hrs/year
>Cost of LED cassette/yr: Eu48 Cost of HBOs/year: Eu1060.
>Savings, using LEDs: Eu1012
>
>One more bit of good news: LEDs are also a much cooler source so
>there is dramatically less photobleaching.
>
>The down side really isn't very down, just something to be aware of.
>Because of the state of LED technology, green and yellow LEDs
>generate less power so the resulting images will be somewhat less
>bright than with HBO. This is not much of an issue when the
>fluorescence is viewed at magnifications up to about 60x but if you
>routinely use 100x objectives, you should run the test to see if it
>is a problem with your particular samples. The good news is (a) for
>green LEDs, research is powering ahead. Fraen expects to have new,
>brighter LEDs in Feb 08. (b) For Yellow (Texas red, etc.), research
>is slower. However, they also have a good news side: they exhibit
>better S/N ratio, even at the lower power, than HBO.
>
>The FluoLED family has a number of things to recommend it:
>a. They have engineered a clever "multi-cube" device so that you can
>have 1 LED, 2 LEDs, or 3 LEDs and can switch conveniently from one to
another
>b. For multi-user labs, the LED cassettes can be switched quickly and
>easily. This feature reminded me of the old Reichert Polyvars, one
>of my favorite microscopes, especially for teaching. The
>fluorescence (and reflected light DIC and Darkfield) cubes came on
>"lolly pop" sticks so that you could just slide in what you
>needed. FluoLED has mimicked that flexibility with their cassette
>approach. A lab can have a set of cassettes sitting in a drawer next
>to the microscope or each group can have what they need in their own
>area, so they can have whatever excitation/emission they need by just
>plugging in their cassette and tightening the locking
>screw. Immediate change out... no alignment!
>c. Fraen has engineered intelligent electronics into their
>controllers. Different wavelength LEDs require different amperages
>to drive them. With Fraen's system, when a cassette is plugged into
>position, the controller intelligently senses which LED is in the
>cassette and provides the appropriate amperage, even with the 3
>cassette system.
>d. The controller also allows the user to change intensity so that
>you can balance different channels for optimum imaging.
>e. Finally, and as a past high school teacher, I loved this one...
>Fraen has engineered less expensive "baby" systems in Blue and Royal
>blue, so that we can finally get fluorescence into teaching labs.
>
>That's the story. I hope it was helpful. I am at Neuroscience this
>week and LEDs are, indeed,grabbing a lot of interest.
>
>Best regards,
>Barbara Foster, President
>
>We've moved!
>Microscopy/Microscopy Education
>7101 Royal Glen Trail, Suite A
>McKinney TX 75070
>P: (972)924-5310
>Skype: fostermme
>W: www.MicroscopyEducation.com
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>December. Call us today for details.
>
>P. S.
>Need a good general reference or light microscopy text for next
>semester? Call us today to learn more about "Optimizing LIght
>Microscopy". Copies still available through MME... even for
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>At 07:21 AM 11/6/2007, Gerard Whoriskey wrote:
>>Search the CONFOCAL archive at
>>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>>
>>Hi Glen,
>>The argument for LED systems is very strong on reliability and
operational
>>costs and is continually improving with regard to performance, measured
in
>>choice of wavelengths and intensity.
>>I assume that in your confocal set-up you are only using the mercury
based
>>bulb system to check and align samples and that you only need excitation
>>regions that match the laser lines you are using. An LED system that you
>>can switch on and off as you please is ideal for such applications and a
>>very cost effective replacement to bulbs.
>>Commercial bit:
>>We have only recently included 445nm and 505nm options to our range. Now
>>users can choose from 7 options of 400nm, 445nm, 465nm, 505nm, 525nm,
>>595nm, and 635nm.
>>I will contact you directly with more commercial information.
>>
>>Best Regards,
>>
>>Gerry
>>
>>Gerard Whoriskey
>>Development Engineer
>>CoolLED Ltd
>>CIL House
>>Charlton Road
>>Andover
>>Hampshire
>>SP10 3JL
>>
>>Mob: 07789535762
>>Tel: +44 (0) 1264 321321
>>Dir: +44 (0)1264 320984
>>web site: www.coolled.com
>