Posted by
Andrea Latini on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585191.html
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Hi Rafael,
Confocal System limitation:
your confocal system max acquisition speed is determined by disk rotation speed and total
number of even detector FOV pinholes overlap for single disk revolution;
in your case: 10.000 RPM, 24 even overlaps per revolution, means 6ms/24= 250us for an
even sample pinholes overlap.
hence, exposure time set at minimum of 250us gives you a maximum frame rate of 4000fps
and exposure time set at 250us * N (N integer), gives you a striping free acquisition.
the disk motor has an accuracy of 10.000 RPM +/- 50RPM (0.5%) and the 250us must be
fine tuned on your system's precise rotation speed.
with that said, in more than 7 years I've seen only one experimental setup and sample whit
a fluorescence signal bright enough to be detected at 1ms exposure time on an EMCCD
detector with Region of Interest and Readout Time fine tuning.
in other words, the maximum frame rate you will achieve will depend on:
- sample fluorescence photon flux
- optical power of your excitation source
- Magnification and NA of your objective
- throughput of your Confocal System (for both excitation and signal collection)
it's a balance and optimization of each of the above variables, mostly, that will give you a
good experimental result and an increased knowledge of your Confocal System capabilities
and limitations.
Andrea Latini
President
CrestOptics Srl
On Fri, 20 May 2016 15:40:59 -0400, Rafael Jaimes III <
[hidden email]> wrote:
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>Post images on
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>
>Hi Andrea,
>
>Thanks for your reply! It means a lot to hear from the president of the
>company.
>
>I was also wondering -- is there an easy way to determine the maximum
>acquisition speed for my disk? I have the 10,000 RPM variant. One of the
>other users mentioned that 10ms exposure / 100 fps may be too fast, at
>least regarding the light levels.​ Do you typically use EMCCD for these
>disks to accommodate the loss of light? I am worried that my sCMOS is not
>sensitive enough.
>
>As some others requested, I will certainly post some pictures to imgur when
>I get a chance.. please stand by.
>
>Rafael
>
>On Fri, May 20, 2016 at 2:45 PM, Andrea Latini <
[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> Post images on
http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear Rafael,
>> thank you for raising the question to the Confocal List and sorry for my
>> late response. Luckily
>> John Oreopoulos answers/explanations are a very good starting point on how
>> to optimize your
>> confocal system and better evaluate your experimental results and of
>> course I fully agree with
>> him.
>> I'll be pleased to help you on pinholes focusing and overall system setup
>> and to directly
>> evaluate your acquisitions.
>>
>> regards.
>>
>> Andrea Latini
>> President
>> CrestOptics srl
>>