Setting up epifluorescence
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I am building my own epifluorescence setup with a monochromator (400-700 nm) as light source. The light guide from the monochromator has an N.A. of 0.22. The objective is an Olympus LMPlan IR 50X, N.A. 0.55. What would I need, in terms of correcting optics between my light guide and the objective rear aperture? And how should the light be focused on the objective rear aperture.
Thomas ------------------------------------------------------------- Thomas Aabo Phone: +45 35333636 M.Sc., Ph.D student, [hidden email] University of Copenhagen Department of Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark www.ifv.life.ku.dk ------------------------------------------------------------- |
 
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Kevin Braeckmans |
Re: Setting up epifluorescence
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Hi,
For widefield imaging, basically you just have to project an (enlarged) image of the light guide output at the objective back focal plane (or a conjugate plane, such as where the aperture diaphragm is located). So all you need is an achromat lens with a suitable focal length that images your light guide output on the aperture diaphragm. Best regards, Kevin > -----Oorspronkelijk bericht----- > Van: Confocal Microscopy List [mailto:[hidden email]] > Namens Thomas Aabo > Verzonden: donderdag 23 oktober 2008 14:27 > Aan: [hidden email] > Onderwerp: Setting up epifluorescence > > I am building my own epifluorescence setup with a monochromator (400- > 700 nm) as light source. The light guide from the monochromator has an > N.A. of 0.22. The objective is an Olympus LMPlan IR 50X, N.A. 0.55. > What would I need, in terms of correcting optics between my light guide > and the objective rear aperture? And how should the light be focused on > the objective rear aperture. > > Thomas > > ------------------------------------------------------------- > Thomas Aabo > Phone: +45 35333636 > M.Sc., Ph.D student, [hidden email] > University of Copenhagen > Department of Food Science > Rolighedsvej 30 > 1958 Frederiksberg C > Denmark > www.ifv.life.ku.dk > ------------------------------------------------------------- |
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Thomas Aabo |
Svar: Re: Setting up epifluorescence
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Thank you, for the quick reply. How tightly focussed (N.A.) should the light be on the rear aperture of the objective? Does it matter what the N.A. is of the objective is?
thomas >>> Kevin Braeckmans <[hidden email]> 10/23/08 2:41 >>> Hi, For widefield imaging, basically you just have to project an (enlarged) image of the light guide output at the objective back focal plane (or a conjugate plane, such as where the aperture diaphragm is located). So all you need is an achromat lens with a suitable focal length that images your light guide output on the aperture diaphragm. Best regards, Kevin > -----Oorspronkelijk bericht----- > Van: Confocal Microscopy List [mailto:[hidden email]] > Namens Thomas Aabo > Verzonden: donderdag 23 oktober 2008 14:27 > Aan: [hidden email] > Onderwerp: Setting up epifluorescence > > I am building my own epifluorescence setup with a monochromator (400- > 700 nm) as light source. The light guide from the monochromator has an > N.A. of 0.22. The objective is an Olympus LMPlan IR 50X, N.A. 0.55. > What would I need, in terms of correcting optics between my light guide > and the objective rear aperture? And how should the light be focused on > the objective rear aperture. > > Thomas > > ------------------------------------------------------------- > Thomas Aabo > Phone: +45 35333636 > M.Sc., Ph.D student, [hidden email] > University of Copenhagen > Department of Food Science > Rolighedsvej 30 > 1958 Frederiksberg C > Denmark > www.ifv.life.ku.dk > ------------------------------------------------------------- |
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Chris Tully |
Re: Svar: Re: Setting up epifluorescence
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Thomas,
The image of your light source (arc lamp, lamp filament or light guide) should be diffuse (no sharp edges of the lamp filament for example) and completely fill the back aperture of the objective. I concur with Kevin that an achromat lens is sufficient for this purpose. The critical point is that any areas of the back focal plane that are not illuminated (or are dimly illuminated) will produce dark areas in the image sent to the eyes or camera. Chris Tully -- Chris Tully Microscopy and Image Analysis Expert [hidden email] 240-888-1021 http://www.linkedin.com/in/christully On Thu, Oct 23, 2008 at 8:59 AM, Thomas Aabo <[hidden email]> wrote: Thank you, for the quick reply. How tightly focussed (N.A.) should the light be on the rear aperture of the objective? Does it matter what the N.A. is of the objective is? |
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Kevin Braeckmans |
Re: Svar: Re: Setting up epifluorescence
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In reply to this post by Thomas Aabo
If you focus the light to a small spot at the back focal plane, than
basically the NA of the light beam at that point will just determine the size of the field of illumination (and thus the light density, i.e. flux). The NA of the light beam at that point should not exceed the NA of the objective lens - else you will just lose part of the excitation light. In your case, since you have a light source with very limited NA, it is not useful to fill the back aperture of the objective lens since points at the border of the image at the back focal plane will send light at too high an angle (make a ray tracing diagram). You could, however, use a (holographic) diffuser with a high NA achromat lens to increase the available NA of the illumination source. Best regards, Kevin > -----Oorspronkelijk bericht----- > Van: Confocal Microscopy List [mailto:[hidden email]] > Namens Thomas Aabo > Verzonden: donderdag 23 oktober 2008 15:00 > Aan: [hidden email] > Onderwerp: Svar: Re: Setting up epifluorescence > > Thank you, for the quick reply. How tightly focussed (N.A.) should the > light be on the rear aperture of the objective? Does it matter what the > N.A. is of the objective is? > thomas > > >>> Kevin Braeckmans <[hidden email]> 10/23/08 2:41 >>> > Hi, > > For widefield imaging, basically you just have to project an (enlarged) > image of the light guide output at the objective back focal plane (or a > conjugate plane, such as where the aperture diaphragm is located). So > all > you need is an achromat lens with a suitable focal length that images > your > light guide output on the aperture diaphragm. > > Best regards, > > Kevin > > > > > -----Oorspronkelijk bericht----- > > Van: Confocal Microscopy List > [mailto:[hidden email]] > > Namens Thomas Aabo > > Verzonden: donderdag 23 oktober 2008 14:27 > > Aan: [hidden email] > > Onderwerp: Setting up epifluorescence > > > > I am building my own epifluorescence setup with a monochromator (400- > > 700 nm) as light source. The light guide from the monochromator has > an > > N.A. of 0.22. The objective is an Olympus LMPlan IR 50X, N.A. 0.55. > > What would I need, in terms of correcting optics between my light > guide > > and the objective rear aperture? And how should the light be focused > on > > the objective rear aperture. > > > > Thomas > > > > ------------------------------------------------------------- > > Thomas Aabo > > Phone: +45 35333636 > > M.Sc., Ph.D student, [hidden email] > > University of Copenhagen > > Department of Food Science > > Rolighedsvej 30 > > 1958 Frederiksberg C > > Denmark > > www.ifv.life.ku.dk > > ------------------------------------------------------------- |
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In reply to this post by Thomas Aabo
Choose your lens based on the NA of the fiber (this will tell you the angle of the cone of light coming from the fiber tip) and the diameter of the back aperture of the objective. This assumes an infinity corrected objective which expects a collimated beam. Basically you want to choose a lens such that when it is focused on the fiber tip it produces a collimated beam the same diameter as the back aperture of the objective. If you have power to spare, you might want to slightly overfill the aperture so you have more uniform illumination.
Craig On Thu, Oct 23, 2008 at 6:41 AM, Kevin Braeckmans <[hidden email]> wrote: Hi, |
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