Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Guidance-wanted-on-illumination-stability-tp5784748p5792413.html
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>Prior to Dr. Hell's excellent 2007 paper cited
>below using laser light pulses in the ns range,
>Dr. Nishigaki et al, in 2006 used the LED
>approach successfully, though with more modest
>gains. They custom built a pulsed diode with
>broader duty cycle (0.5 to 2ms) that reduced
>bleaching of organic dyes and associated cell
>toxicity. Apparently driving diodes much
>faster than this raises heating concerns.
>
>Or is this no longer true?
>
>Stroboscopic illumination using light-emitting
>diodes reduces phototoxicity in fluorescence
>cell imaging. Nishigaki T, Wood CD, Shiba K,
>Baba SA, Darszon A. Biotechniques. 2006
>Aug;41(2):191-7.
>
>Mike Ignatius
>Molecular Probes, Life Technologies
Hi Mike,
Actually, I have some reservations about your
interpretation of this paper. I am not sure that
was a fair fight. First of all, according to the
authors, the Hg arc used for continuous
illumination was originally designed for UV
uncaging. This means that, except for the
excitation filter and the dichroic BS, the optics
were capable of conveying UV to the specimen". I
would have liked to see a UV absorbing filter in
there somewhere just to be sure. Hg produces a
lot of UV and it can reflect around (or even
through) filter systems that aren't properly
designed.
More to the point, as I understand it, the LED
was only on during 1ms of the 2ms that the CCD
camera was recording the image at a rate of 10
images/second (i.e., for only 1% of each second
of recording time). The Hg source seems to have
been on for the full 7 minutes even though the
camera was still on for only the same 10, 2ms
exposures per second. Quote:
The difference between the two samples is
therefore most likely due to the 100- fold
increase in overall exposure time to illumination
light from the mercury lamp (images/s = 10;
duration of individual LED pulse = 1 ms;
therefore total exposure to LED illumination/s =
10 ms, compared with full 1000 ms exposure to
mercury lamp).
In the Discussion, the authors mention that one
might expect improvements if one used the
mechanical shutter on the Hg source to turn off
the excitation except during the 20ms/second when
the the camera was recording but that they hadn't
done so to reduce vibration of the system and
wear-and-tear on the shutter.
I agree. If you use 100x more light, you will do
more damage. I don't think that pulse-length was
important. What was important was duty cycle. In
the LED case, they "used" all (ok, not all, but
all they could collect) of the light that they
excited. In the Hg case they didn't.
In other quibbles, they didn't attempt to measure
the number of mW.s or excitation light at the
specimen from the two sources but assumed that
they must be similar if they produced a similar
level of exposure in their camera, given a fixed
2ms exposure. While this may be convenient and
probably true, we can all think of a number of
factors that may have made this assumption unsafe.
But not unsafe on the same order as a 100x change in total light dose.
So I think that this paper just restates what we
have known for some time (certainly as far back
as the time that time-lapse was recorded on 16mm
film), namely that it is better to turn off the
light when we aren't recording the image. And in
addition, that if your object is moving so fast
that you must record 10 frames/s, the easiest way
to do this is with an LED source rather than a Hg
arc plus an Ultrablitz mechanical shutter (On the
other hand, a suitably synchronized rotary
shutter might do fine.).
Cheers,
Jim P.
*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research
Building, FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info:
http://www.3dcourse.ubc.ca/
Applications still being accepted
"If it ain't diffraction, it must be statistics." Anon.
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:
[hidden email]] On
>Behalf Of Jeremy Adler
>Sent: Tuesday, November 30, 2010 7:27 AM
>To:
[hidden email]
>Subject: Re: Guidance wanted on illumination stability
>
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>It might also be useful to have pulsatile LEDs operating in the
>nanosecond range - there was a Stefan Hell paper that showed very
>significant reductions in photobleaching when the gap between pulses
>was longer than the fluorescent lifetime. Is this possible with LEDs ?
>
>
>Quoting Gordon Scott <
[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>>
>> Hi Guy,
>>
>> No the copy strategy didn't work. Of course one only finds that out
>> after the event. Ho Hum. :-(
>>
>> My original thoughts on video were as yours, however I _think_ most
>> cameras take a full image and then the lines are downloaded
>> sequentially, but I'm also very aware that that may not be the whole, or
>> even the correct story. If the line rate _is_ important, and I guess if
>> the data is downloaded from the camera `live' line-by-line, rather than
>> as a complete image, then 100kHz will definitely show artefacts and the
>> case is closed .. I need to keep the linear control.
>>
>> The main aim is actually to avoid unnecessary waste heat, though saving
>> money is always nice. I'm not sure it would make a particularly
>> significant effect on price, but it may. Any waste heat I have I then
>> have to get rid of. LEDs must not get as hot as bulbs and indeed we
>> actively cool them to get the best out of them, so getting the excess
>> heat out of the boxes needs heat sinks and fans or similar. At present
>> that waste heat puts a frustrating limit on what's feasible with the
>> units, and I'd like to remove that frustration.
>>
>> Thanks for your comments.
>>
>> Kind regards,
>> Gordon.
> > --
>> Gordon Scott Design Engineering
>> Custom Interconnect Ltd.
http://www.cil-uk.co.uk>> CoolLED
http://www.coolled.com>> CoolLED is a division of Custom Interconnect Ltd.
>> Phone +44-1264-321321
>> CIL House, Charlton Road, Andover SP10 3JL, UK
>>
>>
>>> -----Original Message-----
>>> From: Guy Cox [mailto:
[hidden email]]
>>> Sent: 29 November 2010 23:15
>>> To: Confocal Microscopy List
>>> Cc: Gordon Scott
>>> Subject: RE: Guidance wanted on illumination stability
>>>
>>> Gordon,
>>>
>>> Your strategy of copying to the list didn't seem to work.
>>> Anyway, I think that lots of list members, having seen the
>>> question, like to see the answers.
>>>
>>> If someone is taking conventional images with a 1
>>> second exposure 100kHz ripple will not be noticeable. But if
>>> you are taking video at 25 fps 525 line (international video)
>>> your line rate is about
>>> 13 kHz (US video about 14kHz) so I'd imagine there would be
>>> rather unwelcome diagonal stripes on the image and you'd be
>>> getting angry phone calls from your customers.
>>>
>>> You haven't told us the other side of the
>>> trade-off. Do you want to eliminate the linear stage to save
>>> money - if so, how much cheaper would it make a CooLED
>>> illuminator? Or is it to save power?
>>> How much would that save? Given that an LED source already
>>> uses hugely less power that an HBO 100 mercury lamp, would
>>> anyone care?
>>>
>>> Guy
>>>
>>> Optical Imaging Techniques in Cell Biology
>>> by Guy Cox CRC Press / Taylor & Francis
>>>
http://www.guycox.com/optical.htm>>> ______________________________________________
>>> Associate Professor Guy Cox, MA, DPhil(Oxon) Australian
>>> Centre for Microscopy & Microanalysis, Madsen Building F09,
> >> University of Sydney, NSW 2006
>>>
>>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>>> Mobile 0413 281 861
>>> ______________________________________________
>>>
http://www.guycox.net>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:
[hidden email]]
>>> On Behalf Of Gordon Scott
>>> Sent: Tuesday, 30 November 2010 2:34 AM
>>> To:
[hidden email]
>>> Subject: Guidance wanted on illumination stability
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> Hi Guys,
>>>
>>> I'm looking at ways to further improve the performance and
>>> efficiency of our light sources.
>>>
>>> There are always tradeoffs when doing this and I'd like to
>>> better understand what tradeoffs are acceptable for real
>>> microscopy users.
>>>
>>> Our present illumination sources all use a switched-mode
>>> pre-regulation and a linear final regulation for the LED
>>> power, so ripple is very low, but at a cost for us of some
>>> power wasted in the linear stages.
>>>
>>> I can improve that efficiency and reduce the waste by
>>> foregoing the linear stage and regulating directly with the
>>> switching mode, but the tradeoffs are a longer On/Off
>>> switching time than is feasible with linear, and a
>>> high-frequency ripple.
>>>
>>> My simulations suggest switch-on and switch-off times of
>>> around 50us and a ripple of around 25% at 100kHz, which would
>>> be reasonable from an electrical/energy point of view.
>>>
>>>
>>>
>>> The question is, of course, would any of the people likely to
>>> use it find that performance difficult or unacceptable?
>>>
>>>
>>>
>>> I've copied to the list rather than posting direct, so
>>> hopefully the replies will come to me rather than cluttering the list.
>>>
>>> Thanks for considering the question, even if you need not, or
>>> choose not, to answer.
>>>
>>> Kind regards,
>>> Gordon.
>>> --
>>> Gordon Scott Design Engineering
>>> Custom Interconnect Ltd.
http://www.cil-uk.co.uk>>> CoolLED
http://www.coolled.com>>> CoolLED is a division of Custom Interconnect Ltd.
>>> Phone +44-1264-321321
>>> CIL House, Charlton Road, Andover SP10 3JL, UK
>>>
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>>
>
>
>
>Jeremy Adler
>Genetics & Pathology
>Rudbeckslaboratoriet
>Daghammersköljdsväg 20
>751 85 Uppsala
>Sweden
>
>0046 (0)18 471 4607
--
*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research
Building, FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info:
http://www.3dcourse.ubc.ca/
Applications still being accepted
"If it ain't diffraction, it must be statistics." Anon.