Re: Help with EMCCD evaluation*Commercial Response*

[Gerard Whoriskey-3]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

On the phototoxicity point due to delays causing cells to be exposed to
light for too long.

One elegant solution would be to have no mechanical elements in the system
that need to be triggered. Instead just trigger the light source and have
no moving parts. This can now be done with some LED systems which means
that there should be no need for slow mechanical shutters or filter
wheels. Or faster expensive ones either.

The CoolLed precisExcite LED system can be controlled by TTL and will
switch on and off in microseconds. Filters can also be incorporated into
the unit so removing the need for filter wheels altogether. The narrow
band peaks from the LED source also means no need for extra UV blocking
filters when looking at sensitive live cells and percentage control means
no need for ND filters.  

However this still does not get around the problem of the software delay
activating the trigger. I assume the solution is to trigger the light in
parallel or even after the camera. The CoolLED system would illuminate the
full field at once. So you would not get one side of the image exposed
longer than the other, like if you tried this with a slow shutter.

Please feel free to contact off line for more details.

Regards,

Gerry

Gerard Whoriskey
Development Engineer
CoolLED Ltd
CIL House
Charlton Road
Andover
Hampshire
SP10 3JL
 
Mob: [phone]
Tel: [phone]
Dir: [phone]
web site: www.coolled.com



On Wed, 28 Nov 2007 13:59:55 +0000, Nuno Moreno <[hidden email]>
wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi!
>
>If it is for cell imaging you might consider a BT camera.
>
>Forcing the previous point, which I thing is very important:
>
>I'm having the same problem with an ORCA AG with ImagePro Plus.  What I
>have now is a small program called by macros that controls an external
>shutter via TTL, which makes a delay till it opens the shutter. I found
>out that the driver takes almost 500ms to get the camera taking a
>picture which is unacceptable! However this solution is far from being
>elegant and sometimes the pseudo synchronization fails! Is this a
>Hamamatsu problem? With a Hamamatsu BT1024 which has a QE>90% it
>happends the same thing with metamorph.
>
>Regarding to the previous commercial response, does anyone have a
>cheaper and more flexible solution?
>
>Regards,
>NM
>
>
>
>
>
>Nowell, Cameron wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi Neville,
>>               While an EMCCD camera will decrease your exposure time,

you may find that it will not really help solve your phototoxicity
problem. The time your cells are exposed to fluorecent light will not
neccesarily be dependant on the exposure time of your camera, but on how
fast your entire sytem (microscope, shutters, filters, etc.) shuffles
everything around.
>>
>> What software etc are you using to capture your images?
>>
>> We have a wide filed live cell rig here that uses an Orca ER camera on
an IX81 microscope and it is all driven by metamorph. Even when the camera
is capturing at 100ms exposures, the samples are exposed to flourecent
light for about 2 seconds. To get rid of these sort of delays you need to
move to using filter wheels, or for even shorter exposuers something like
olympus' CellR system

>>
>>
>> Cheers
>>
>>
>> Cam
>>
>>
>> Cameron Nowell B. Sc (Hons)
>>
>>
>>
>> Microscopy Imaging and Research Core Facility
>>
>> Peter MacCallum Cancer Centre
>>
>> 7 St Andrews Place
>>
>> East Melbourne, Victoria 3002
>>
>> AUSTRALIA
>>
>>
>>
>> Phone: [phone]
>>
>> Mobile: [phone]
>>
>> Fax: [phone]
>>
>>
>>
>> ________________________________
>>
>> From: Confocal Microscopy List on behalf of Neville Sanjana
>> Sent: Wed 28/11/2007 7:57 AM
>> To: [hidden email]
>> Subject: Help with EMCCD evaluation
>>
>>
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi,
>>
>> I am in the market for an EMCCD for widefield fluorescence
>> time-lapse imaging of genetically-labeled cultured cells. I
>> am acquiring images frequently (every 10 minutes) for
>> several days. The cells seem to be very sensitive to
>> phototoxicity and photobleaching (GFP is the fluorophore),
>> so I am hoping that replacing my Roper CoolSnapHQ CCD with
>> an EMCCD will allow me to use much shorter exposure times.
>>
>> I realize that this is a confocal list and my application is
>> widefield but since EMCCDs are also used with spinning disk
>> confocal, I thought I'd ask. Apologies if this question has
>> been hashed out before... I searched the archives without
>> finding definitive recommendations. (Maybe that's asking for
>> too much!) Any help on the following would be much appreciated:
>>
>> 1) What EMCCDs have people found to be the most *sensitive*
>> (nice picture in low-light) and the most *reliable* (driver
>> doesn't crash, etc.)? I'd especially be interested in any
>> experiences or opinions with Roper/Photometrics's CascadeII
>> or QuantEM and the Andor iXon cameras.
>>
>> 2) How have people been testing cameras? I've set up demos
>> of several cameras so I'd like to do some standard tests.
>> I've seen some references on the web for calculating dark
>> noise, read noise, and gain, such as:
>> http://www.qsimaging.com/ccd_noise_measure.html
>> http://www.mirametrics.com/tech_note_ccdgain.htm
>>

http://www.photomet.com/library/library_encyclopedia/library_enc_gain.php

>> but I'd love to hear what other tests people think are
>> relevant for these cameras. Obviously, I'll try imaging my
>> samples (using fiber-coupled LED as a constant illumination
>> source)... what else should I do? Fluorescent beads instead
>> of my cells perhaps?
>>
>> 3) For coupling the camera to my Olympus IX71, is there
>> something like the Zeiss Optovar (which, as I understand it,
>> has multiple selectable magnifications of the intermediate
>> lens) made by a third party? This would be helpful for
>> changing between achieving the full NA of the objective and
>> getting less resolution but a bigger field of view for
>> different experiments.
>>
>> Thanks for your help. Best,
>>
>> - Neville
>>
>> ---
>> Neville Sanjana
>> Dept. of Brain and Cognitive Science
>> Massachusetts Institute of Technology
>>
>>
>>
>>
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>
>--
>Nuno Moreno
>Cell Imaging Unit
>Instituto Gulbenkian de Ciência
>http://uic.igc.gulbekian.pt
>http://www.igc.gulbekian.pt
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