confocal microscope efficiency measurement
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fhenriques |
confocal microscope efficiency measurement
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Dear all,
First of all, hello everyone. I am new to the mailling list and the microscopy world. I'm a portughese student and am currently doing my master thesis at the Instituto Gulbenkian de Ciência under the guidance of Nuno Moreno. We are trying to measure the light capturing efficiency of a confocal microscope but aren't sure of the methodology and were hoping you could shed some light on it. The efficiency we want to measure is the fraction of photons emitted from a light source that is detected in the final image, being: efficiency= np/ne, where np is the number of detected photons and ne is the photons emitted by a calibrated light source(LED). To calculate np we assumed there was no amplification noise, and used the mean of the image M and it's variance V. M=np*g and V=np*g^2. Being g the average pixel intensity resulting from the detection of one photon, which is proportional to the gain of the PMT and subsequent elecronics. And np the average detected photons per pixel. Our doubt is in the calculation of ne. Is each pixel a representation of all the light that reaches the lens during the pixel dwell time? If so, ne should be the number of photons that reach the lens during the dwell time. If not, if each pixel represents only a portion of the light that reaches the lens during the dwell time, than ne should be a fraction of the number of photons that reach the lens during the dwell time. I have searched, but I am unable to find any kind of tutorial to help me understand this concept. If somebody could point me in the direction of a good source to understand how this should be done, I would be very grateful. Many thanks Francisco Henriques |
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Jason Swedlow |
Re: confocal microscope efficiency measurement
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Hi Francisco-
Great project! Actually, knowing the number of photons emitted from a fluorescent source (knowing in the sense of having a standard where the exact production of photons is "known") is very difficult. Not impossible, but difficult. You can get the efficiencies of the individual elements in the light path, and multiply them together. This will give you an estimate of the efficiency. John Murray did this in his chapter in Goldman and Spector's "Live Cell Imaging". However, you can imagine coming up with a standard sample, and using that to compare different microscopes. This was done recently: search Pubmed for PMID: 18045334. This might give you an idea for a standard sample you might use. Good luck! Cheers, Jason On Fri, Sep 11, 2009 at 4:42 PM, Francisco Henriques <[hidden email]> wrote: Dear all, -- ************************** Wellcome Trust Centre for Gene Regulation & Expression College of Life Sciences MSI/WTB/JBC Complex University of Dundee Dow Street Dundee DD1 5EH United Kingdom phone (01382) 385819 Intl phone: 44 1382 385819 FAX (01382) 388072 email: [hidden email] Lab Page: http://www.dundee.ac.uk/lifesciences/swedlow/ Open Microscopy Environment: http://openmicroscopy.org ************************** |
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Louis Villeneuve |
Fixation with frozen Paraformaldehyde
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In reply to this post by fhenriques
Bonjour à tous, Can Paraformaldehyde 2-4% can be frozen for future use without lost of fixation's efficiency? Thanks for your help? Louis
Dear all, First of all, hello everyone. I am new to the mailling list and the microscopy world. I'm a portughese student and am currently doing my master thesis at the Instituto Gulbenkian de Ciência under the guidance of Nuno Moreno. We are trying to measure the light capturing efficiency of a confocal microscope but aren't sure of the methodology and were hoping you could shed some light on it. The efficiency we want to measure is the fraction of photons emitted from a light source that is detected in the final image, being: efficiency= np/ne, where np is the number of detected photons and ne is the photons emitted by a calibrated light source(LED). To calculate np we assumed there was no amplification noise, and used the mean of the image M and it's variance V. M=np*g and V=np*g^2. Being g the average pixel intensity resulting from the detection of one photon, which is proportional to the gain of the PMT and subsequent elecronics. And np the average detected photons per pixel. Our doubt is in the calculation of ne. Is each pixel a representation of all the light that reaches the lens during the pixel dwell time? If so, ne should be the number of photons that reach the lens during the dwell time. If not, if each pixel represents only a portion of the light that reaches the lens during the dwell time, than ne should be a fraction of the number of photons that reach the lens during the dwell time. I have searched, but I am unable to find any kind of tutorial to help me understand this concept. If somebody could point me in the direction of a good source to understand how this should be done, I would be very grateful. Many thanks Francisco Henriques |
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Ian Montgomery |
Re: Fixation with frozen Paraformaldehyde
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Louis, Yes, do it all the time. Ian. Dr. Ian Montgomery, Histotechnology, I.B.L.S. Support Unit, G12 8QQ. From:
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Cameron, Lisa |
Re: Fixation with frozen Paraformaldehyde
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In reply to this post by Louis Villeneuve
In my post-doc lab, we used to make the fixation solution
fresh, use it, and then freeze it at -20C once.
Anything left over after thawing it once was
discarded.
If you typically use a certain volume of fixation, you
could freeze aliquots of the appropriate size for maximal
use.
Cheers -
Lisa
--------------------------------------- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email] Sent: Monday, September 21, 2009 9:35 AM To: [hidden email] Subject: [CONFOCALMICROSCOPY] Fixation with frozen Paraformaldehyde Bonjour à tous, Can Paraformaldehyde 2-4% can be frozen for future use without lost of fixation's efficiency? Thanks for your help? Louis The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. |
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Sylvie Le Guyader-2 |
Re: Fixation with frozen Paraformaldehyde
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I routinely freeze 10mL aliquots of PFA
(freshly made from powder) 8% or 4%. I have used my stock without any problem
for 2 years. I thaw a working stock that I keep in the
fridge for up to one month. I only warm up (if needed) the volume I need (so
the aliquot stays in the fridge) and add it 2x to the cell culture with the
same volume of warm medium. This perfectly preserves filopodia which are
fragile structures. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 Huddinge Sweden +46 (0)8 608 9240 From:
In my post-doc lab, we used to make the
fixation solution fresh, use it, and then freeze it at -20C once. Anything left over after thawing it once
was discarded. If you typically use a certain volume of
fixation, you could freeze aliquots of the appropriate size for maximal use. Cheers - Lisa ---------------------------------------
From:
The information in this e-mail is intended only for the person to whom it isaddressed. If you believe this e-mail was sent to you in error and the e-mailcontains patient information, please contact the Partners Compliance HelpLine athttp://www.partners.org/complianceline . If the e-mail was sent to you in errorbut does not contain patient information, please contact the sender and properlydispose of the e-mail. |
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