How to measure objective transmission curves?
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Michael Weber-4 |
How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, one of the main differences of objectives is their transmission efficiency at certain wavelengths. One way to compare this is the trial-and-error method, however, this is not straight forward. My idea is to use a combination of spectrophotometer and a lamp with a more or less even spectra (i.e. Xenon) on some kind of optical bench. This would make the setup independent from the manufacturer. Beside the distance between the light source and the detector, there are obviously more things to consider: different diameter of the back focal plane, different focal lenghts... I would like to hear about your opinion about how to measure objective transmission. Have you ever done this in your lab? Did you find a setup that worked for you? cheers, Michael |
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Andrew Resnick |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We've done similar measurements, it's not too difficult. The main trick is handling the large NA lenses. Our setup was light source -> objective -> integrating sphere -> spectrometer. After normalizing to the source, we obtained really good data. It's pretty easy, actually. Andy At 08:45 AM 8/31/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear all, > >one of the main differences of objectives is their transmission >efficiency at certain wavelengths. One way to compare this is the >trial-and-error method, however, this is not straight forward. > >My idea is to use a combination of spectrophotometer and a lamp with >a more or less even spectra (i.e. Xenon) on some kind of optical >bench. This would make the setup independent from the manufacturer. >Beside the distance between the light source and the detector, there >are obviously more things to consider: different diameter of the >back focal plane, different focal lenghts... > >I would like to hear about your opinion about how to measure >objective transmission. Have you ever done this in your lab? Did you >find a setup that worked for you? > >cheers, >Michael Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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Rietdorf, Jens |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Michael & Andy, For Olympus lenses you can look it up on the web...the info for other manufacturers lenses would be interesting though; do you plan to publish the numbers? Andy, would you mind to disclose yours? regards, jens --- Dr. Jens Rietdorf Head Microscopy Novartis Research Foundation Friedrich-Miescher-Institute, wro1066.2.32 Maulbeerstr.66, CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737 Email:rietdorf(at)fmi.ch -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew Resnick Sent: Freitag, 31. August 2007 17:00 To: [hidden email] Subject: Re: How to measure objective transmission curves? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We've done similar measurements, it's not too difficult. The main trick is handling the large NA lenses. Our setup was light source -> objective -> integrating sphere -> spectrometer. After normalizing to the source, we obtained really good data. It's pretty easy, actually. Andy At 08:45 AM 8/31/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear all, > >one of the main differences of objectives is their transmission >efficiency at certain wavelengths. One way to compare this is the >trial-and-error method, however, this is not straight forward. > >My idea is to use a combination of spectrophotometer and a lamp with a >more or less even spectra (i.e. Xenon) on some kind of optical bench. >This would make the setup independent from the manufacturer. >Beside the distance between the light source and the detector, there >are obviously more things to consider: different diameter of the back >focal plane, different focal lenghts... > >I would like to hear about your opinion about how to measure objective >transmission. Have you ever done this in your lab? Did you find a setup >that worked for you? > >cheers, >Michael Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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Jennifer Waters |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Jens,
Can you tell us where to find the Olympus lenses transmission online? I looked on their website but couldn't find it.
Thanks, Jennifer
On 8/31/07, Rietdorf, Jens <[hidden email]> wrote:
Search the CONFOCAL archive at -- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Please check the link. http://www.olympusamerica.com/seg_section/uis2/seg_uis2_uplsapo_60xo.asp. It is Olympus UPLSAPO 60x oil lens. You could find "UPLSAPO", "PLAPON", "UPLFLN","LUCPLFLN" series lens spectrum data. Best regards Fan Chang -------------------------------------------------- Fan Chang National Center for Microscopy & Imaging Research University of California San Diego 9500 Gilman Drive Basic Science Building #1000 La Jolla, CA 92093-0608, USA Tel: 858 534-2583 Fax: 858 534-7497 -------------------------------------------------- Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Mark Cannell |
Re: How to measure objective transmission curves?
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In reply to this post by Andrew Resnick
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Andrew While I can see that method would work for lenses designed to work in air, wouldn't total internal reflection be a problem for lenses with an NA over 1.0? The only way I can think of doing it is to use two objectives of the same type looking at each other at a common focal point with the right immersion medium between them. Then the overall transmission would be half that of a single lens... Cheers Mark Andrew Resnick wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We've done similar measurements, it's not too difficult. > > The main trick is handling the large NA lenses. Our setup was light > source -> objective -> integrating sphere -> spectrometer. After > normalizing to the source, we obtained really good data. It's pretty > easy, actually. > > Andy > > > At 08:45 AM 8/31/2007, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear all, >> >> one of the main differences of objectives is their transmission >> efficiency at certain wavelengths. One way to compare this is the >> trial-and-error method, however, this is not straight forward. >> >> My idea is to use a combination of spectrophotometer and a lamp with >> a more or less even spectra (i.e. Xenon) on some kind of optical >> bench. This would make the setup independent from the manufacturer. >> Beside the distance between the light source and the detector, there >> are obviously more things to consider: different diameter of the back >> focal plane, different focal lenghts... >> >> I would like to hear about your opinion about how to measure >> objective transmission. Have you ever done this in your lab? Did you >> find a setup that worked for you? >> >> cheers, >> Michael > > Andrew Resnick, Ph. D. > Instructor > Department of Physiology and Biophysics > Case Western Reserve University > 216-368-6899 (V) > 216-368-4223 (F) |
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Sudipta Maiti |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you are interested in the transmission characterisitics of the material of the objective, the NA should not be a big issue. You can put an iris in the beam and make it thin before entering the objective. Do take the iris transmission as the baseline (this of course will tell you nothing about the transmission of the peripheral, high angle rays).. With an iris and with no integrating sphere available, you should still be able to get a reasonable estimate of the wavelength dependent transmission from a simple spectrophotometer. Sudipta On Mon, 3 Sep 2007, Mark Cannell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Andrew > > While I can see that method would work for lenses designed to work in air, > wouldn't total internal reflection be a problem for lenses with an NA over > 1.0? The only way I can think of doing it is to use two objectives of the > same type looking at each other at a common focal point with the right > immersion medium between them. Then the overall transmission would be half > that of a single lens... > > Cheers Mark > > Andrew Resnick wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> We've done similar measurements, it's not too difficult. >> >> The main trick is handling the large NA lenses. Our setup was light >> source -> objective -> integrating sphere -> spectrometer. After >> normalizing to the source, we obtained really good data. It's pretty >> easy, actually. >> >> Andy >> >> >> At 08:45 AM 8/31/2007, you wrote: >> > Search the CONFOCAL archive at >> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> > >> > Dear all, >> > >> > one of the main differences of objectives is their transmission >> > efficiency at certain wavelengths. One way to compare this is the >> > trial-and-error method, however, this is not straight forward. >> > >> > My idea is to use a combination of spectrophotometer and a lamp with a >> > more or less even spectra (i.e. Xenon) on some kind of optical bench. >> > This would make the setup independent from the manufacturer. Beside the >> > distance between the light source and the detector, there are obviously >> > more things to consider: different diameter of the back focal plane, >> > different focal lenghts... >> > >> > I would like to hear about your opinion about how to measure objective >> > transmission. Have you ever done this in your lab? Did you find a setup >> > that worked for you? >> > >> > cheers, >> > Michael >> >> Andrew Resnick, Ph. D. >> Instructor >> Department of Physiology and Biophysics >> Case Western Reserve University >> 216-368-6899 (V) >> 216-368-4223 (F) > > -- Sudipta Maiti Dept. of Chemical Sciences Tata Institute of Fundamental Research Homi Bhabha Road, Colaba, Mumbai 400005, India 91-22-2278-2716 www.tifr.res.in/~biophotonics |
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Andrew Resnick |
Re: How to measure objective transmission curves?
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In reply to this post by Mark Cannell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Mark, That's a good question. We attached a coverslip with a small drop of oil to the objective as a way to deal with immersion objectives. I don't know what it means that the "sample" is in air; if some light is then totally internally reflected back into the coverslip, which could be a spectral issue due to dispersion. Using two matched objectives is probably a better method, but I imagine considerably more difficult due to alignment constraints and obtaining matched objectives. The objectives we had obeyed the 'sine condition', which I believe means that there are fewer high-NA rays than low NA rays- this would help mitigate our experimental error as well. At 04:19 PM 9/2/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi Andrew > >While I can see that method would work for lenses designed to work >in air, wouldn't total internal reflection be a problem for lenses >with an NA over 1.0? The only way I can think of doing it is to use >two objectives of the same type looking at each other at a common >focal point with the right immersion medium between them. Then the >overall transmission would be half that of a single lens... > >Cheers Mark > >Andrew Resnick wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>We've done similar measurements, it's not too difficult. >> >>The main trick is handling the large NA lenses. Our setup was >>light source -> objective -> integrating sphere -> >>spectrometer. After normalizing to the source, we obtained really >>good data. It's pretty easy, actually. >> >>Andy >> >> >>At 08:45 AM 8/31/2007, you wrote: >>>Search the CONFOCAL archive at >>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>Dear all, >>> >>>one of the main differences of objectives is their transmission >>>efficiency at certain wavelengths. One way to compare this is the >>>trial-and-error method, however, this is not straight forward. >>> >>>My idea is to use a combination of spectrophotometer and a lamp >>>with a more or less even spectra (i.e. Xenon) on some kind of >>>optical bench. This would make the setup independent from the >>>manufacturer. Beside the distance between the light source and the >>>detector, there are obviously more things to consider: different >>>diameter of the back focal plane, different focal lenghts... >>> >>>I would like to hear about your opinion about how to measure >>>objective transmission. Have you ever done this in your lab? Did >>>you find a setup that worked for you? >>> >>>cheers, >>>Michael >> >>Andrew Resnick, Ph. D. >>Instructor >>Department of Physiology and Biophysics >>Case Western Reserve University >>216-368-6899 (V) >>216-368-4223 (F) > >Andrew Resnick, Ph. D. >Instructor >Department of Physiology and Biophysics >Case Western Reserve University >216-368-6899 (V) >216-368-4223 (F) |
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Andrew Resnick |
Re: How to measure objective transmission curves?
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In reply to this post by Rietdorf, Jens
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Jens, I could not find the data- I must have left it behind. I don;t recall anything odd; the transmission curves were uniformly high in the visible and dropped off to about 50%, IIRC, at 1064 nm (the laser tweezer wavelength). Andy At 10:04 AM 8/31/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear Michael & Andy, > >For Olympus lenses you can look it up on the web...the info for other >manufacturers lenses would be interesting though; do you plan to publish >the numbers? Andy, would you mind to disclose yours? > >regards, jens > >--- >Dr. Jens Rietdorf >Head Microscopy >Novartis Research Foundation >Friedrich-Miescher-Institute, wro1066.2.32 >Maulbeerstr.66, CH-4058 Basel, Switzerland >phone +41(61)69-75172 mobil +41 798284737 >Email:rietdorf(at)fmi.ch > >-----Original Message----- >From: Confocal Microscopy List [mailto:[hidden email]] On >Behalf Of Andrew Resnick >Sent: Freitag, 31. August 2007 17:00 >To: [hidden email] >Subject: Re: How to measure objective transmission curves? > >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >We've done similar measurements, it's not too difficult. > >The main trick is handling the large NA lenses. Our setup was light >source -> objective -> integrating sphere -> spectrometer. After >normalizing to the source, we obtained really good data. It's pretty >easy, actually. > >Andy > > >At 08:45 AM 8/31/2007, you wrote: > >Search the CONFOCAL archive at > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > >Dear all, > > > >one of the main differences of objectives is their transmission > >efficiency at certain wavelengths. One way to compare this is the > >trial-and-error method, however, this is not straight forward. > > > >My idea is to use a combination of spectrophotometer and a lamp with a > >more or less even spectra (i.e. Xenon) on some kind of optical bench. > >This would make the setup independent from the manufacturer. > >Beside the distance between the light source and the detector, there > >are obviously more things to consider: different diameter of the back > >focal plane, different focal lenghts... > > > >I would like to hear about your opinion about how to measure objective > >transmission. Have you ever done this in your lab? Did you find a setup > > >that worked for you? > > > >cheers, > >Michael > >Andrew Resnick, Ph. D. >Instructor >Department of Physiology and Biophysics >Case Western Reserve University >216-368-6899 (V) >216-368-4223 (F) Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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Mark Cannell |
Re: How to measure objective transmission curves?
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In reply to this post by Andrew Resnick
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Andrew All rays whose effective NA >1 get reflected back by the system you describe. There are more high NA rays than low NA rays (due to the area of an annulus increasing with it's radius : pi(2r dr). As I see it, one can't measure the true throughput with axial rays as the marginal rays encouner very different glass thicknesses and there may be internal field stops (or even phase rings!). The measurement of throughput is certainly not going to be easy. I don't see how this is related to the sine condition, which I believe simply defines a geometric constraint for an undistorted image viz: http://en.wikipedia.org/wiki/Abbe_sine_condition Cheers Mark Andrew Resnick wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Mark, > > That's a good question. We attached a coverslip with a small drop of > oil to the objective as a way to deal with immersion objectives. I > don't know what it means that the "sample" is in air; if some light is > then totally internally reflected back into the coverslip, which could > be a spectral issue due to dispersion. > > Using two matched objectives is probably a better method, but I > imagine considerably more difficult due to alignment constraints and > obtaining matched objectives. The objectives we had obeyed the 'sine > condition', which I believe means that there are fewer high-NA rays > than low NA rays- this would help mitigate our experimental error as > well. > > > At 04:19 PM 9/2/2007, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi Andrew >> >> While I can see that method would work for lenses designed to work in >> air, wouldn't total internal reflection be a problem for lenses with >> an NA over 1.0? The only way I can think of doing it is to use two >> objectives of the same type looking at each other at a common focal >> point with the right immersion medium between them. Then the overall >> transmission would be half that of a single lens... >> >> Cheers Mark >> >> Andrew Resnick wrote: >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> We've done similar measurements, it's not too difficult. >>> >>> The main trick is handling the large NA lenses. Our setup was light >>> source -> objective -> integrating sphere -> spectrometer. After >>> normalizing to the source, we obtained really good data. It's >>> pretty easy, actually. >>> >>> Andy >>> >>> >>> At 08:45 AM 8/31/2007, you wrote: >>>> Search the CONFOCAL archive at >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >>>> Dear all, >>>> >>>> one of the main differences of objectives is their transmission >>>> efficiency at certain wavelengths. One way to compare this is the >>>> trial-and-error method, however, this is not straight forward. >>>> >>>> My idea is to use a combination of spectrophotometer and a lamp >>>> with a more or less even spectra (i.e. Xenon) on some kind of >>>> optical bench. This would make the setup independent from the >>>> manufacturer. Beside the distance between the light source and the >>>> detector, there are obviously more things to consider: different >>>> diameter of the back focal plane, different focal lenghts... >>>> >>>> I would like to hear about your opinion about how to measure >>>> objective transmission. Have you ever done this in your lab? Did >>>> you find a setup that worked for you? >>>> >>>> cheers, >>>> Michael >>> >>> Andrew Resnick, Ph. D. >>> Instructor >>> Department of Physiology and Biophysics >>> Case Western Reserve University >>> 216-368-6899 (V) >>> 216-368-4223 (F) >> >> Andrew Resnick, Ph. D. >> Instructor >> Department of Physiology and Biophysics >> Case Western Reserve University >> 216-368-6899 (V) >> 216-368-4223 (F) |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
People do seem to be making
rather heavy weather of this,
which would seem fairly simple to me.
First off, there's
no need to use oil - the acceptance angle
of the lens is
not affected in any way by the presence or
absence of
oil (resolution and aberration correction
are a different
matter!). The acceptance angle
of an NA 1.4 lens is
67.5 deg. One could put an
integrating sphere over the
lens if one is really worried that
transmission for paraxial
and abaxial rays is different. One
could also test this point
by using a parallel beam and measuring
first on the optic
axis and then at an angle of (say) 60
deg.
In fact, this would be quite useful to know
since such an
effect would impact on effective NA in
confocal and MP
microscopy.
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) Electron Microscope Unit, 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 From: Confocal Microscopy List on behalf of Mark Cannell Sent: Wed 5/09/2007 9:15 AM To: [hidden email] Subject: Re: How to measure objective transmission curves? Search the CONFOCAL archive at Hi Andrew All rays whose effective NA >1 get reflected back by
the system you http://en.wikipedia.org/wiki/Abbe_sine_condition Cheers Mark Andrew Resnick wrote: |
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Mark Cannell |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Guy Cox wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > People do seem to be making rather heavy weather of this, > which would seem fairly simple to me. First off, there's > no need to use oil - the acceptance angle of the lens is > not affected in any way by the presence or absence of > oil (resolution and aberration correction are a different > matter!). That's true but the reflective losses from the lens will depend on the medium contacting the front lens. Of course this could be estimated from the Fresnel equations and for a high NA oil immersion lens this will add ( I guess) about a 10%. > The acceptance angle of an NA 1.4 lens is > 67.5 deg. One could put an integrating sphere over the > lens if one is really worried that transmission for paraxial > and abaxial rays is different. One could also test this point > by using a parallel beam and measuring first on the optic > axis and then at an angle of (say) 60 deg. Hi Guy, I don't think that will work because of the apertures present. If you shine a // beam into the front of a high NA lens the beam will be highly focussed within the lens body and the rear aperture may block some of the light coming out (it depends where the back focal plane of the objective is -for highly complex high NA lenses I'm not sure that that is at the rear aperture). In addition I think reflective losses within the lens will change as the input is not a cone of light with the correct NA (i.e. the path of most of the rays is now completely different). Also, real integrating spheres are _not_ Lambertian and comparing a highly divergent beam to a highly collimated beam will also likely give rise to an error (this is a well known problem). If you have any lens design software you can simulate the problem quite easily. I'd say the problem is so 'not simple' and manufacturers avoided publishing transmission curves beacuse they are very hard to measure correctly. I'll guess that that the curves now published by manufacturers are actually produced by the lens design programs they use. Modern optical design programs can incorportae the anti-replection coatings to be applied and so the need for measurement is reduced/simplified. However, if one just wants to compare two lenses with the same NA then your method could work -but it will be necessary to make sure the input beam is not clipped. Cheers Mark > > > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, 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 > </exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List on behalf of Mark Cannell > *Sent:* Wed 5/09/2007 9:15 AM > *To:* [hidden email] > *Subject:* Re: How to measure objective transmission curves? > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Andrew > > All rays whose effective NA >1 get reflected back by the system you > describe. There are more high NA rays than low NA rays (due to the area > of an annulus increasing with it's radius : pi(2r dr). As I see it, one > can't measure the true throughput with axial rays as the marginal rays > encouner very different glass thicknesses and there may be internal > field stops (or even phase rings!). The measurement of throughput is > certainly not going to be easy. I don't see how this is related to the > sine condition, which I believe simply defines a geometric constraint > for an undistorted image viz: > > http://en.wikipedia.org/wiki/Abbe_sine_condition > > Cheers Mark > > > Andrew Resnick wrote: > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Mark, > > > > That's a good question. We attached a coverslip with a small drop of > > oil to the objective as a way to deal with immersion objectives. I > > don't know what it means that the "sample" is in air; if some light is > > then totally internally reflected back into the coverslip, which could > > be a spectral issue due to dispersion. > > > > Using two matched objectives is probably a better method, but I > > imagine considerably more difficult due to alignment constraints and > > obtaining matched objectives. The objectives we had obeyed the 'sine > > condition', which I believe means that there are fewer high-NA rays > > than low NA rays- this would help mitigate our experimental error as > > well. > > > > > > At 04:19 PM 9/2/2007, you wrote: > >> Search the CONFOCAL archive at > >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >> > >> Hi Andrew > >> > >> While I can see that method would work for lenses designed to work in > >> air, wouldn't total internal reflection be a problem for lenses with > >> an NA over 1.0? The only way I can think of doing it is to use two > >> objectives of the same type looking at each other at a common focal > >> point with the right immersion medium between them. Then the overall > >> transmission would be half that of a single lens... > >> > >> Cheers Mark > >> > >> Andrew Resnick wrote: > >>> Search the CONFOCAL archive at > >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >>> > >>> We've done similar measurements, it's not too difficult. > >>> > >>> The main trick is handling the large NA lenses. Our setup was light > >>> source -> objective -> integrating sphere -> spectrometer. After > >>> normalizing to the source, we obtained really good data. It's > >>> pretty easy, actually. > >>> > >>> Andy > >>> > >>> > >>> At 08:45 AM 8/31/2007, you wrote: > >>>> Search the CONFOCAL archive at > >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >>>> > >>>> Dear all, > >>>> > >>>> one of the main differences of objectives is their transmission > >>>> efficiency at certain wavelengths. One way to compare this is the > >>>> trial-and-error method, however, this is not straight forward. > >>>> > >>>> My idea is to use a combination of spectrophotometer and a lamp > >>>> with a more or less even spectra (i.e. Xenon) on some kind of > >>>> optical bench. This would make the setup independent from the > >>>> manufacturer. Beside the distance between the light source and the > >>>> detector, there are obviously more things to consider: different > >>>> diameter of the back focal plane, different focal lenghts... > >>>> > >>>> I would like to hear about your opinion about how to measure > >>>> objective transmission. Have you ever done this in your lab? Did > >>>> you find a setup that worked for you? > >>>> > >>>> cheers, > >>>> Michael > >>> > > > |
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James Pawley |
Re: How to measure objective transmission curves?
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In reply to this post by Andrew Resnick
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, If it doesn't sound too commercial, I would like to note that a lot of objective transmission curves are published in the 3rd Edition. In fact I take some pride in the fact that the earlier editions of the Handbook contain some of the first published transmission data on commercial objectives. One trick to get "all the light" out of an high NA is to oil-couple it to the flat side of a very small hemispherical lens. Even a small glass bead, with half of its thickness sticking out of the oil will do. But you have to hold your light sensor really close to it, and if you aren't careful, because the light diverging from the focus in the bead will leave it at such a large angle to the horizontal that a lot of it may be reflected from the glass covering your sensor (or miss it all together). An optometrical integrating sphere is better. I just measure transmission of the system (mostly losses in the fiber and filters) with the 10x lens and hope that the immersion lenses "are what they are". However, this won't show up a smear on the tube lens that only obscures high-NA rays. I use the focusing ability of the Bertrand lens (phase lens) to look for this by focusing up and down through the optical column. Amazing how much dust etc. you can see this way!!. Cheers, Jim P. >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Jens, > >I could not find the data- I must have left it behind. I don;t >recall anything odd; the transmission curves were uniformly high in >the visible and dropped off to about 50%, IIRC, at 1064 nm (the >laser tweezer wavelength). > >Andy > >At 10:04 AM 8/31/2007, you wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>Dear Michael & Andy, >> >>For Olympus lenses you can look it up on the web...the info for other >>manufacturers lenses would be interesting though; do you plan to publish >>the numbers? Andy, would you mind to disclose yours? >> >>regards, jens >> >>--- >>Dr. Jens Rietdorf >>Head Microscopy >>Novartis Research Foundation >>Friedrich-Miescher-Institute, wro1066.2.32 >>Maulbeerstr.66, CH-4058 Basel, Switzerland >>phone +41(61)69-75172 mobil +41 798284737 >>Email:rietdorf(at)fmi.ch >> >>-----Original Message----- >>From: Confocal Microscopy List [mailto:[hidden email]] On >>Behalf Of Andrew Resnick >>Sent: Freitag, 31. August 2007 17:00 >>To: [hidden email] >>Subject: Re: How to measure objective transmission curves? >> >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>We've done similar measurements, it's not too difficult. >> >>The main trick is handling the large NA lenses. Our setup was light >>source -> objective -> integrating sphere -> spectrometer. After >>normalizing to the source, we obtained really good data. It's pretty >>easy, actually. >> >>Andy >> >> >>At 08:45 AM 8/31/2007, you wrote: >>>Search the CONFOCAL archive at >>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>Dear all, >>> >>>one of the main differences of objectives is their transmission >>>efficiency at certain wavelengths. One way to compare this is the >>>trial-and-error method, however, this is not straight forward. >>> >>>My idea is to use a combination of spectrophotometer and a lamp with a >>>more or less even spectra (i.e. Xenon) on some kind of optical bench. >>>This would make the setup independent from the manufacturer. >>>Beside the distance between the light source and the detector, there >>>are obviously more things to consider: different diameter of the back >>>focal plane, different focal lenghts... >>> >>>I would like to hear about your opinion about how to measure objective >>>transmission. Have you ever done this in your lab? Did you find a setup >> >>>that worked for you? >>> >>>cheers, >>>Michael >> >>Andrew Resnick, Ph. D. >>Instructor >>Department of Physiology and Biophysics >>Case Western Reserve University >>216-368-6899 (V) >>216-368-4223 (F) > >Andrew Resnick, Ph. D. >Instructor >Department of Physiology and Biophysics >Case Western Reserve University >216-368-6899 (V) >216-368-4223 (F) -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-262-9083 250 N. Mills St., Madison, WI, 53706 [hidden email] "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 |
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Mark Cannell |
Re: How to measure objective transmission curves?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes Jim! That's a good method and if the hemispherical lens is well AR coated then it should let the light out accurately. We used a drop of water on top of a water lens a while back but that won't work for a horizontal measuring system. Not sure about the detector angular dependence tho'. It might be interesting to compare the 'two lens method' I suggested (which will have no detector problems) with your integrating sphere method... Anyone here fancy getting a paper in microsc. techniques or optics letters? Cheers Mark though. the James Pawley wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi all, > > If it doesn't sound too commercial, I would like to note that a lot of > objective transmission curves are published in the 3rd Edition. In > fact I take some pride in the fact that the earlier editions of the > Handbook contain some of the first published transmission data on > commercial objectives. > > One trick to get "all the light" out of an high NA is to oil-couple it > to the flat side of a very small hemispherical lens. Even a small > glass bead, with half of its thickness sticking out of the oil will do. > > But you have to hold your light sensor really close to it, and if you > aren't careful, because the light diverging from the focus in the bead > will leave it at such a large angle to the horizontal that a lot of it > may be reflected from the glass covering your sensor (or miss it all > together). An optometrical integrating sphere is better. > > I just measure transmission of the system (mostly losses in the fiber > and filters) with the 10x lens and hope that the immersion lenses "are > what they are". However, this won't show up a smear on the tube lens > that only obscures high-NA rays. I use the focusing ability of the > Bertrand lens (phase lens) to look for this by focusing up and down > through the optical column. Amazing how much dust etc. you can see > this way!!. > > |
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John Oreopoulos |
Confusion about the equation for numerical perture
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In reply to this post by Mark Cannell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi, I'm a little confused about the traditional equation for a microscope's numerical aperture:
NA = n x sin(theta) where theta is the maximum half angle subtended by the lens's light collection cone. It's the "n", the refractive index I'm confused about. I've now read several sources and books that say n is the refractive index of the medium that the sample is embedded in (above the coverslip), and in other places, I read that n is the refractive index of the immersion fluid, say oil (below the coverslip). I understand that in the ideal cases, both of these refractive indexes should be matched for the best possible imaging, but in reality they never really are, right? So which medium does the "n" refer too? And what about in the case for TIRF microscopy, where it is required that you have a oil immersion objective to image a sample in water for example? Which n should I use to calculate my objective NA? John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 |
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Jennifer Waters |
Re: Confusion about the equation for numerical perture
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Search the CONFOCAL archive at
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For NA, n = the refractive index of the media between the lens and coverslip (ie, immersion oil). The refractive index of the specimen is used to calculate axial resolution.
Best, Jennifer
On 9/10/07, John Oreopoulos <[hidden email]> wrote:
Search the CONFOCAL archive at <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School |
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In reply to this post by John Oreopoulos
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John, The perfect imaging plane for a typical objective is right after the coverglass, and the coverglass is usually compensated through the objective collar. So the refractive index n is referring to the immersion fluid, which helps to "bend" the light for a higher NA. -- Peng Dantus Research Group Department of Chemistry Michigan State University East Lansing, MI 48824 Tel: (517) 355-9715 x319 Email: [hidden email] http://www.msu.edu/~xipeng/ John Oreopoulos wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I'm a > little confused about the traditional equation for a microscope's > numerical aperture: > > NA = n x sin(theta) > > where theta is the maximum half angle subtended by the lens's light > collection cone. It's the "n", the refractive index I'm confused > about. I've now read several sources and books that say n is the > refractive index of the medium that the sample is embedded in (above > the coverslip), and in other places, I read that n is the refractive > index of the immersion fluid, say oil (below the coverslip). I > understand that in the ideal cases, both of these refractive indexes > should be matched for the best possible imaging, but in reality they > never really are, right? So which medium does the "n" refer too? And > what about in the case for TIRF microscopy, where it is required that > you have a oil immersion objective to image a sample in water for > example? Which n should I use to calculate my objective NA? > > > John Oreopoulos, BSc, > > PhD Candidate > > University of Toronto > > Institute For Biomaterials and Biomedical Engineering > > Centre For Studies in Molecular Imaging > > > Tel: W:416-946-5022 > > > |
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John Oreopoulos |
How to measure the actual numerical aperture of a microscope objective?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello all again,
In my research, I am now faced with a situation where I need to obtain an actual measurement of the numerical aperture (NA) of an Olympus oil immersion objective. Can anyone tell me or point me to a reference that explains a simple method to determine the actual NA of a microscope objective? If the measurement is difficult and not simple (ie requiring sophisticated optical instruments and opto-mechanical apparatus), can someone tell me how reliable the number written on the barrel of the objective is or what it's actual uncertainty is? The NA written on my objective is 1.45. How accurate is this number? Thanks in advance for any help! John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 |
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lechristophe |
Re: How to measure the actual numerical aperture of a microscope objective?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
http://support.svi.nl/wiki/NaNumericalApertureExperimentalPsfBlurConeImpFaq One can have surprises in the resulting number (I got around 1.15 from a 1.40 100X objective). However, one has to try to match RI of the mounting medium. By the way, the SVI wiki has many great advices about deconvolution and microscopy. Christophe John Oreopoulos a écrit : Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all again, |
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In reply to this post by John Oreopoulos
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal In principle it's remarkably easy to measure the NA of an objective. We get students to do it with nothing more sophisticated than an old microscope, a board marked in degrees, and a piece of string. The idea is that if you look through an objective using a phase telescope, you are imaging the back focal plane which is where the lens will form an image of an object at infinity. So if the lens has an unobstructed view (which of course you have to arrange) you'll see an image of the room around you. The edge of what you can see (it will be quite a fish-eye view) is determined by the maximum acceptance angle of the objective, and with a suitable target you can measure this. With your NA 1.45 lens it should be 73 degrees from the optic axis (=straight ahead). Remarkably unsophisticated - and surprisingly accurate. 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) Electron Microscope Unit, 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 ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos Sent: Tuesday, 11 September 2007 6:29 AM To: [hidden email] Subject: How to measure the actual numerical aperture of a microscope objective? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all again, In my research, I am now faced with a situation where I need to obtain an actual measurement of the numerical aperture (NA) of an Olympus oil immersion objective. Can anyone tell me or point me to a reference that explains a simple method to determine the actual NA of a microscope objective? If the measurement is difficult and not simple (ie requiring sophisticated optical instruments and opto-mechanical apparatus), can someone tell me how reliable the number written on the barrel of the objective is or what it's actual uncertainty is? The NA written on my objective is 1.45. How accurate is this number? Thanks in advance for any help! John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.485 / Virus Database: 269.13.14/999 - Release Date: 10/09/2007 5:43 PM No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.485 / Virus Database: 269.13.14/999 - Release Date: 10/09/2007 5:43 PM |
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