Phase Contrast Microscopy
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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. ***** I posted this message on the Microscopy listserv and got such confusing answers that I thought I would try this list to see if anyone can clarify. I thought I understood how phase contrast microscopy works, but then I was reading MicroscopyU (https://www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-contrast-microscopy<https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.microscopyu.com%2Ftechniques%2Fphase-contrast%2Fintroduction-to-phase-contrast-microscopy&data=02%7C01%7Cdavid.knecht%40uconn.edu%7C80f17dd0f2dd4d83409c08d7e6c53275%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C0%7C637231606945304315&sdata=LkYN7e343%2B7073PGh%2FePzDsc3tWc4gO3E5G0s1Q%2Fo8Q%3D&reserved=0>) and other sites and now I am confused. My understanding is that it works because of refractive index differences in different parts of the light path (cells vs. surround), leading to retardation of light refracted by cells (slowed about ¼ wavelength) and eventual phase differences relative to un-refracted (surround) light that passes through the thinned area of the phase plate relative to the thick area (another ¼ wavelength) in the objective. However, in the web site indicated and others, they use refracted light and diffracted light almost interchangeably in explaining phase contrast. To my “biologist” level understanding, diffraction and refraction are very different phenomenon and I did not think that diffraction changed the optical path length like refractive index differences so would not lead to interference in the same way. Refraction makes total sense to me in the context of phase contrast, but I don’t see how diffraction is relevant. Can someone explain what I am missing? Thanks, Dave Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 |
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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. ***** Hi Dave, Perhaps, the confusing part here is that we are used that diffraction occurs at sharp boundaries between transparent and opaque. But diffraction can also be caused by boundaries between parts with different refractive indices. So, phase contrast utilizes diffraction which is caused by refraction... Such is at least my understanding Best Mike ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Knecht, David <[hidden email]> Sent: Thursday, April 30, 2020 12:32 PM To: [hidden email] <[hidden email]> Subject: Phase Contrast Microscopy ***** To join, leave or search the confocal microscopy listserv, go to: https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cmmodel%40KENT.EDU%7Cb65991bae6c242c021a108d7ed242d5d%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637238612050242101&sdata=E0YDD3ZRZfeJqlHznwBy5W%2BFUXsB59MOq0yg5bQPeI0%3D&reserved=0 Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=02%7C01%7Cmmodel%40KENT.EDU%7Cb65991bae6c242c021a108d7ed242d5d%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637238612050242101&sdata=yXP0WzX3cKrBkbp5RKF5iMJ%2BTXmhm6jPMQG%2FsGioP%2Bo%3D&reserved=0 and include the link in your posting. ***** I posted this message on the Microscopy listserv and got such confusing answers that I thought I would try this list to see if anyone can clarify. I thought I understood how phase contrast microscopy works, but then I was reading MicroscopyU (https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.microscopyu.com%2Ftechniques%2Fphase-contrast%2Fintroduction-to-phase-contrast-microscopy&data=02%7C01%7Cmmodel%40KENT.EDU%7Cb65991bae6c242c021a108d7ed242d5d%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637238612050242101&sdata=%2Fobnoagky1PeWQiZvAdXCOzLuLMCwRRO6j8gay5sDlI%3D&reserved=0<https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.microscopyu.com%2Ftechniques%2Fphase-contrast%2Fintroduction-to-phase-contrast-microscopy&data=02%7C01%7Cmmodel%40KENT.EDU%7Cb65991bae6c242c021a108d7ed242d5d%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637238612050242101&sdata=%2Fobnoagky1PeWQiZvAdXCOzLuLMCwRRO6j8gay5sDlI%3D&reserved=0>) and other sites and now I am confused. My understanding is that it works because of refractive index differences in different parts of the light path (cells vs. surround), leading to retardation of light refracted by cells (slowed about ¼ wavelength) and eventual phase differences relative to un-refracted (surround) light that passes through the thinned area of the phase plate relative to the thick area (another ¼ wavelength) in the objective. However, in the web site indicated and others, they use refracted light and diffracted light almost interchangeably in explaining phase contrast. To my “biologist” level understanding, diffraction and refraction are very different phenomenon and I did not think that diffraction changed the optical path length like refractive index differences so would not lead to interference in the same way. Refraction makes total sense to me in the context of phase contrast, but I don’t see how diffraction is relevant. Can someone explain what I am missing? Thanks, Dave Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 |
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In reply to this post by Knecht, David
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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. ***** Hi David, I think that you understanding of phase contrast is correct and that you have explained it yourself properly and in a concise way. Diffraction and refraction are different indeed. It is in fact the diffraction of light that plays an important role in phase contrast microscopy. Have a look at the Leica tutorial: https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast and figure 4 in the link you shared: 1. The illumination light passes through the annual ring in the condenser, resulting in a hollow cone of illumination. 2.1 A higher refractive index in the sample causes retardation, on average generating a phase shift of -1/4 λ in the light that interacted with the specimen compared to freely passing light. 2.2 Light interacting with the specimen (cell, granule, nucleus...) is diffracted to the outside of the illuminating light cone.The smaller the object, the larger the angle of diffraction [1]. 2.3 Light that does not interact with the specimen is not diffracted and hence stays on the inside of the illumination cone. 3. In the phase plate, only the light on the inside of the light-cone is then advanced +1/4 λ . 4. This result in a phase difference between illuminating light and sample light of 1/2 λ, generating destructive interference and hence maximum contrast in the imaging plane wherever there was a structure in the sample Here is also a very nice iBology talk on the subject: https://www.ibiology.org/talks/phase-contrast-microscopy Hope this helps! [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc |
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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. ***** I've found the best way to understand phase contrast is on a phase contrast microscope. On a phase contrast microscope, take out one of the oculars so you can see the focal planes. In the path without the ocular, you should see a ring of light perfectly aligned to an annular neutral density filter (switch between brightfield and phase contrast to see how the illumination perfectly aligns with the ND filter). If they are not aligned, then the annulus is not setup properly and you are not getting phase contrast (check your manual to fix this). Next. make a slide with a Kim-Wipe or lens paper on it, or grab an unstained test slide (although tissue paper makes this effect very clear) . As you move the sample into the light path, you will see all the diffracted light miss the annular ring of light and fill the rest of the focal plane. Move the sample out of the light path, and you will see this diffracted light disappear. Next, get a poor phase object like adherent cells or anything else that is hard to see with bright field, and put it under the phase contrast microscope. Once you are focused on the sample, look at the focal plane (side without the occular) and slide the annulus out of the way just a bit (it should look like the sun just peaking around the moon after a solar eclipse). Now look at your sample, and it should look like just brightfield (i.e. poorly contrasting). Then put the annulus back into place, while looking at the sample, and you see a sudden jump in contrast. This is one of the striking features of phase contrast where it really is all or none, and if the annulus is at all out of alignment you just get brightfield. As others have said, phase contrast is fixing a fundamental issue with brightfield imaging of poorly diffractive objects (such as adherent cells). The issue is that bright field microscopy works by having the diffracted light undergo a phase shift relative to the undiffracted light (as it follows a different path length in the microscope). Then, these rays are allowed to interfere with each other at the image plane, producing an image. You can see this effect in bright field in a similar manner to the phase contrast trick described above, but instead close the aperture stop and you will again be able to see the diffracted light separated from the non-diffracted light in the focal plane. The problem with poorly diffracting objects is two fold, 1) very little light gets diffracted leaving not much light for interference, and 2) the phase shift of the diffracted light is very small. The end result being that samples like adherent cells only cause a very small decreases in intensity, and since we perceive light on a logarithmic scale, this is very hard for us to see. Therefore, phase contrast fixes both of these issues. One way (which you can see by looking at the focal planes) is that it uses a cone of light as the illumination pattern (due to the annulus at the front focal plane), and this cone perfectly lines up with an annular neutral density filter (the phase plate) at the back focal plane. This ring illumination is a clever way to spatially separate the diffracted light from the undiffracted light at the focal plane, allowing us to attenuate the undiffracted light without impacting the diffracted light, balancing the amount of diffracted and undiffracted light. Why use a ring to do this instead of say an aperture stop? The ring illumination simply preserves more of the illumination NA and therefore preserves more of your resolution. However, the phase plate doesn't stop there, as its name suggests, it also enhances the phase difference between the diffracted and undiffracted light (ideally such that the highest diffractive orders are phase shifted to 180°) such that the sharpest edges get perfect destructive interference. Now at this point, you may be asking yourself, how do they know how much to attenuate the undiffracted light (i.e. how dark to make the annular ND filter) and how much of a phase shift to impose, as some samples may be more diffractive than others. And this is why there are actually many different phase contrast objectives with different phase plates. In life sciences, most of the time we're only using phase contrast to check cell culture, so we wind up only ever dealing with just one phase plate optimized for that task. Hope this helps, and I really do recommend checking it out on your own microscope. I've found with students, looking at the correlation between the focal plane and image plane with and without sample can really inspire that "aha" moment. Cheers, Ben Smith On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[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. > ***** > > Hi David, > > I think that you understanding of phase contrast is correct and that you > have explained it yourself properly and in a concise way. > Diffraction and refraction are different indeed. It is in fact the > diffraction of light that plays an important role in phase contrast > microscopy. > > Have a look at the Leica tutorial: > https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast > and figure 4 in the link you shared: > > 1. The illumination light passes through the annual ring in the condenser, > resulting in a hollow cone of illumination. > 2.1 A higher refractive index in the sample causes retardation, on average > generating a phase shift of -1/4 λ in the light that interacted with the > specimen compared to freely passing light. > 2.2 Light interacting with the specimen (cell, granule, nucleus...) is > diffracted to the outside of the illuminating light cone.The smaller the > object, the larger the angle of diffraction [1]. > 2.3 Light that does not interact with the specimen is not diffracted and > hence stays on the inside of the illumination cone. > 3. In the phase plate, only the light on the inside of the light-cone is > then advanced +1/4 λ . > 4. This result in a phase difference between illuminating light and sample > light of 1/2 λ, generating destructive interference and hence maximum > contrast in the imaging plane wherever there was a structure in the sample > > Here is also a very nice iBology talk on the subject: > https://www.ibiology.org/talks/phase-contrast-microscopy > Hope this helps! > > [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc > -- Benjamin E. Smith, Ph. D. Imaging Specialist, Vision Science University of California, Berkeley 195 Life Sciences Addition Berkeley, CA 94720-3200 Tel (510) 642-9712 Fax (510) 643-6791 e-mail: [hidden email] https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/ |
 
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George McNamara |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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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. ***** Ben wrote: "As others have said, phase contrast is fixing a fundamental issue with brightfield imaging of poorly diffractive objects (such as adherent cells)." In defense of brightfield microscopy: A correctly working brightfield microscope with a video camera or digital camera, or the transmitted light path of a confocal microscope, is perfectly capable of imaging very thin objects (such as very flat adherent cells or very thin unlabeled tissue sections). The camera on a smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe Photoshop (has had math capability for a long time), etc, to be able to subtract background, and add back offset. They key is contrast control. Optional: unsharp masking. Some of the (GPU) deconvolution software vendors even offer deconvolution of brightfield image data. Bonus: no halo. Reference: Shinya Inoue and Ken Spring 1997 Video Microscopy https://www.springer.com/gp/book/9780306455315 I don't see the 1997 book online, the 1st edition, 1986 is https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 See also Shinya's publications ... online or Collected Works, https://www.worldscientific.com/worldscibooks/10.1142/6315 Bob and Nina Allen's papers. Also nanovid papers (single molecule imaging before STORM, PALM, etc). To quote (and add to) Yogi Berra: "You can see a lot by just looking" ... especially if you switch the light path from your (hopefully not covid-19 contaminated) eyepiece to your camera port ... and then use your instrumentation well. For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 disclosure: I wrote most of the article (took over from Ted Inoue, whose self-portrait may - or not - be inside the monitor figure ... "Acquisition rules for Quantitative Fluorescence" section has stood up pretty well since circa 1993). On 5/1/2020 12:35 PM, Benjamin Smith 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. > ***** > > I've found the best way to understand phase contrast is on a phase contrast > microscope. On a phase contrast microscope, take out one of the oculars so > you can see the focal planes. In the path without the ocular, you should > see a ring of light perfectly aligned to an annular neutral density filter > (switch between brightfield and phase contrast to see how the illumination > perfectly aligns with the ND filter). If they are not aligned, then the > annulus is not setup properly and you are not getting phase contrast (check > your manual to fix this). > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > unstained test slide (although tissue paper makes this effect very clear) . > As you move the sample into the light path, you will see all the diffracted > light miss the annular ring of light and fill the rest of the focal plane. > Move the sample out of the light path, and you will see this diffracted > light disappear. > > Next, get a poor phase object like adherent cells or anything else that is > hard to see with bright field, and put it under the phase contrast > microscope. Once you are focused on the sample, look at the focal plane > (side without the occular) and slide the annulus out of the way just a bit > (it should look like the sun just peaking around the moon after a solar > eclipse). Now look at your sample, and it should look like just > brightfield (i.e. poorly contrasting). Then put the annulus back into > place, while looking at the sample, and you see a sudden jump in contrast. > This is one of the striking features of phase contrast where it really is > all or none, and if the annulus is at all out of alignment you just get > brightfield. > > As others have said, phase contrast is fixing a fundamental issue with > brightfield imaging of poorly diffractive objects (such as adherent > cells). The issue is that bright field microscopy works by having the > diffracted light undergo a phase shift relative to the undiffracted light > (as it follows a different path length in the microscope). Then, these > rays are allowed to interfere with each other at the image plane, producing > an image. You can see this effect in bright field in a similar manner to > the phase contrast trick described above, but instead close the aperture > stop and you will again be able to see the diffracted light separated from > the non-diffracted light in the focal plane. The problem with poorly > diffracting objects is two fold, 1) very little light gets diffracted > leaving not much light for interference, and 2) the phase shift of the > diffracted light is very small. The end result being that samples like > adherent cells only cause a very small decreases in intensity, and since we > perceive light on a logarithmic scale, this is very hard for us to see. > > Therefore, phase contrast fixes both of these issues. One way (which you > can see by looking at the focal planes) is that it uses a cone of light as > the illumination pattern (due to the annulus at the front focal plane), and > this cone perfectly lines up with an annular neutral density filter (the > phase plate) at the back focal plane. This ring illumination is a clever > way to spatially separate the diffracted light from the undiffracted light > at the focal plane, allowing us to attenuate the undiffracted light without > impacting the diffracted light, balancing the amount of diffracted and > undiffracted light. Why use a ring to do this instead of say an aperture > stop? The ring illumination simply preserves more of the illumination NA > and therefore preserves more of your resolution. > > However, the phase plate doesn't stop there, as its name suggests, it also > enhances the phase difference between the diffracted and undiffracted light > (ideally such that the highest diffractive orders are phase shifted to > 180°) such that the sharpest edges get perfect destructive interference. > > Now at this point, you may be asking yourself, how do they know how much to > attenuate the undiffracted light (i.e. how dark to make the annular ND > filter) and how much of a phase shift to impose, as some samples may be > more diffractive than others. And this is why there are actually many > different phase contrast objectives with different phase plates. In life > sciences, most of the time we're only using phase contrast to check cell > culture, so we wind up only ever dealing with just one phase plate > optimized for that task. > > Hope this helps, and I really do recommend checking it out on your own > microscope. I've found with students, looking at the correlation between > the focal plane and image plane with and without sample can really inspire > that "aha" moment. > > Cheers, > Ben Smith > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[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. >> ***** >> >> Hi David, >> >> I think that you understanding of phase contrast is correct and that you >> have explained it yourself properly and in a concise way. >> Diffraction and refraction are different indeed. It is in fact the >> diffraction of light that plays an important role in phase contrast >> microscopy. >> >> Have a look at the Leica tutorial: >> https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast >> and figure 4 in the link you shared: >> >> 1. The illumination light passes through the annual ring in the condenser, >> resulting in a hollow cone of illumination. >> 2.1 A higher refractive index in the sample causes retardation, on average >> generating a phase shift of -1/4 λ in the light that interacted with the >> specimen compared to freely passing light. >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is >> diffracted to the outside of the illuminating light cone.The smaller the >> object, the larger the angle of diffraction [1]. >> 2.3 Light that does not interact with the specimen is not diffracted and >> hence stays on the inside of the illumination cone. >> 3. In the phase plate, only the light on the inside of the light-cone is >> then advanced +1/4 λ . >> 4. This result in a phase difference between illuminating light and sample >> light of 1/2 λ, generating destructive interference and hence maximum >> contrast in the imaging plane wherever there was a structure in the sample >> >> Here is also a very nice iBology talk on the subject: >> https://www.ibiology.org/talks/phase-contrast-microscopy >> Hope this helps! >> >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >> > |
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Benjamin Smith |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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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. ***** It is true that bright field is still the resolution champion (especially with a DAPI filter cube in the light path). Although, why settle for poor visibility when you can use Rheinberg illumination and get the best of both worlds with both a high resolution brightfield image and a high contrast edge image (aka dark field). On Fri, May 1, 2020 at 3:15 PM George McNamara <[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. > ***** > > Ben wrote: "As others have said, phase contrast is fixing a fundamental > issue with brightfield imaging of poorly diffractive objects (such as > adherent cells)." > > In defense of brightfield microscopy: > > A correctly working brightfield microscope with a video camera or > digital camera, or the transmitted light path of a confocal microscope, > is perfectly capable of imaging very thin objects (such as very flat > adherent cells or very thin unlabeled tissue sections). The camera on a > smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe > Photoshop (has had math capability for a long time), etc, to be able to > subtract background, and add back offset. They key is contrast control. > Optional: unsharp masking. Some of the (GPU) deconvolution software > vendors even offer deconvolution of brightfield image data. Bonus: no halo. > > Reference: > > Shinya Inoue and Ken Spring 1997 Video Microscopy > > https://www.springer.com/gp/book/9780306455315 > > I don't see the 1997 book online, the 1st edition, 1986 is > > https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 > > See also Shinya's publications ... online or Collected Works, > https://www.worldscientific.com/worldscibooks/10.1142/6315 > > Bob and Nina Allen's papers. > > Also nanovid papers (single molecule imaging before STORM, PALM, etc). > > To quote (and add to) Yogi Berra: "You can see a lot by just looking" > ... especially if you switch the light path from your (hopefully not > covid-19 contaminated) eyepiece to your camera port ... and then use > your instrumentation well. > > For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 > disclosure: I wrote most of the article (took over from Ted Inoue, whose > self-portrait may - or not - be inside the monitor figure ... > "Acquisition rules for Quantitative Fluorescence" section has stood up > pretty well since circa 1993). > > > On 5/1/2020 12:35 PM, Benjamin Smith 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. > > ***** > > > > I've found the best way to understand phase contrast is on a phase > contrast > > microscope. On a phase contrast microscope, take out one of the oculars > so > > you can see the focal planes. In the path without the ocular, you should > > see a ring of light perfectly aligned to an annular neutral density > filter > > (switch between brightfield and phase contrast to see how the > illumination > > perfectly aligns with the ND filter). If they are not aligned, then the > > annulus is not setup properly and you are not getting phase contrast > (check > > your manual to fix this). > > > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > > unstained test slide (although tissue paper makes this effect very > clear) . > > As you move the sample into the light path, you will see all the > diffracted > > light miss the annular ring of light and fill the rest of the focal > plane. > > Move the sample out of the light path, and you will see this diffracted > > light disappear. > > > > Next, get a poor phase object like adherent cells or anything else that > is > > hard to see with bright field, and put it under the phase contrast > > microscope. Once you are focused on the sample, look at the focal plane > > (side without the occular) and slide the annulus out of the way just a > bit > > (it should look like the sun just peaking around the moon after a solar > > eclipse). Now look at your sample, and it should look like just > > brightfield (i.e. poorly contrasting). Then put the annulus back into > > place, while looking at the sample, and you see a sudden jump in > contrast. > > This is one of the striking features of phase contrast where it really is > > all or none, and if the annulus is at all out of alignment you just get > > brightfield. > > > > As others have said, phase contrast is fixing a fundamental issue with > > brightfield imaging of poorly diffractive objects (such as adherent > > cells). The issue is that bright field microscopy works by having the > > diffracted light undergo a phase shift relative to the undiffracted light > > (as it follows a different path length in the microscope). Then, these > > rays are allowed to interfere with each other at the image plane, > producing > > an image. You can see this effect in bright field in a similar manner to > > the phase contrast trick described above, but instead close the aperture > > stop and you will again be able to see the diffracted light separated > from > > the non-diffracted light in the focal plane. The problem with poorly > > diffracting objects is two fold, 1) very little light gets diffracted > > leaving not much light for interference, and 2) the phase shift of the > > diffracted light is very small. The end result being that samples like > > adherent cells only cause a very small decreases in intensity, and since > we > > perceive light on a logarithmic scale, this is very hard for us to see. > > > > Therefore, phase contrast fixes both of these issues. One way (which you > > can see by looking at the focal planes) is that it uses a cone of light > as > > the illumination pattern (due to the annulus at the front focal plane), > and > > this cone perfectly lines up with an annular neutral density filter (the > > phase plate) at the back focal plane. This ring illumination is a clever > > way to spatially separate the diffracted light from the undiffracted > light > > at the focal plane, allowing us to attenuate the undiffracted light > without > > impacting the diffracted light, balancing the amount of diffracted and > > undiffracted light. Why use a ring to do this instead of say an aperture > > stop? The ring illumination simply preserves more of the illumination NA > > and therefore preserves more of your resolution. > > > > However, the phase plate doesn't stop there, as its name suggests, it > also > > enhances the phase difference between the diffracted and undiffracted > light > > (ideally such that the highest diffractive orders are phase shifted to > > 180°) such that the sharpest edges get perfect destructive interference. > > > > Now at this point, you may be asking yourself, how do they know how much > to > > attenuate the undiffracted light (i.e. how dark to make the annular ND > > filter) and how much of a phase shift to impose, as some samples may be > > more diffractive than others. And this is why there are actually many > > different phase contrast objectives with different phase plates. In life > > sciences, most of the time we're only using phase contrast to check cell > > culture, so we wind up only ever dealing with just one phase plate > > optimized for that task. > > > > Hope this helps, and I really do recommend checking it out on your own > > microscope. I've found with students, looking at the correlation between > > the focal plane and image plane with and without sample can really > inspire > > that "aha" moment. > > > > Cheers, > > Ben Smith > > > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[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. > >> ***** > >> > >> Hi David, > >> > >> I think that you understanding of phase contrast is correct and that you > >> have explained it yourself properly and in a concise way. > >> Diffraction and refraction are different indeed. It is in fact the > >> diffraction of light that plays an important role in phase contrast > >> microscopy. > >> > >> Have a look at the Leica tutorial: > >> > https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast > >> and figure 4 in the link you shared: > >> > >> 1. The illumination light passes through the annual ring in the > condenser, > >> resulting in a hollow cone of illumination. > >> 2.1 A higher refractive index in the sample causes retardation, on > average > >> generating a phase shift of -1/4 λ in the light that interacted with the > >> specimen compared to freely passing light. > >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is > >> diffracted to the outside of the illuminating light cone.The smaller the > >> object, the larger the angle of diffraction [1]. > >> 2.3 Light that does not interact with the specimen is not diffracted and > >> hence stays on the inside of the illumination cone. > >> 3. In the phase plate, only the light on the inside of the light-cone is > >> then advanced +1/4 λ . > >> 4. This result in a phase difference between illuminating light and > sample > >> light of 1/2 λ, generating destructive interference and hence maximum > >> contrast in the imaging plane wherever there was a structure in the > sample > >> > >> Here is also a very nice iBology talk on the subject: > >> https://www.ibiology.org/talks/phase-contrast-microscopy > >> Hope this helps! > >> > >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc > >> > > > -- Benjamin E. Smith, Ph. D. Imaging Specialist, Vision Science University of California, Berkeley 195 Life Sciences Addition Berkeley, CA 94720-3200 Tel (510) 642-9712 Fax (510) 643-6791 e-mail: [hidden email] https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/ |
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Zdenek Svindrych-2 |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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*****
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. ***** According to this paper: https://www.researchgate.net/publication/280600830_High_resolution_optical_microscope provocatively titled " Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser " (spoiler alert: misleading title), a brightfield microscope with annular condenser has astonishing resolution. Well, in this case the most likely cause for these astonishing results is just misunderstanding of the calibration patterns, according to the official Richardson test slide pattern description ( https://www.grayfieldoptical.com/files/innerpattern.pdf ) the "100 nm" pattern has a period of 200 nm, the same factor of two applies to the "99 nm" horizontal and vertical bars. Arguably, a title stating "lambda/2.5 resolution" would not have the same distressing effect onto a regular microscopist... So, while transmitted light images look impressive with a well tuned instrument, it is well known that the resolution can match the resolution of fluorescence imaging only if the condenser NA is greater than or equal to the objective lens NA. That's perfectly doable, but not widely practiced these days... Best, zdenek On Fri, May 1, 2020 at 8:48 PM Benjamin Smith <[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. > ***** > > It is true that bright field is still the resolution champion (especially > with a DAPI filter cube in the light path). Although, why settle for poor > visibility when you can use Rheinberg illumination and get the best of both > worlds with both a high resolution brightfield image and a high contrast > edge image (aka dark field). > > On Fri, May 1, 2020 at 3:15 PM George McNamara <[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. > > ***** > > > > Ben wrote: "As others have said, phase contrast is fixing a fundamental > > issue with brightfield imaging of poorly diffractive objects (such as > > adherent cells)." > > > > In defense of brightfield microscopy: > > > > A correctly working brightfield microscope with a video camera or > > digital camera, or the transmitted light path of a confocal microscope, > > is perfectly capable of imaging very thin objects (such as very flat > > adherent cells or very thin unlabeled tissue sections). The camera on a > > smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe > > Photoshop (has had math capability for a long time), etc, to be able to > > subtract background, and add back offset. They key is contrast control. > > Optional: unsharp masking. Some of the (GPU) deconvolution software > > vendors even offer deconvolution of brightfield image data. Bonus: no > halo. > > > > Reference: > > > > Shinya Inoue and Ken Spring 1997 Video Microscopy > > > > https://www.springer.com/gp/book/9780306455315 > > > > I don't see the 1997 book online, the 1st edition, 1986 is > > > > https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 > > > > See also Shinya's publications ... online or Collected Works, > > https://www.worldscientific.com/worldscibooks/10.1142/6315 > > > > Bob and Nina Allen's papers. > > > > Also nanovid papers (single molecule imaging before STORM, PALM, etc). > > > > To quote (and add to) Yogi Berra: "You can see a lot by just looking" > > ... especially if you switch the light path from your (hopefully not > > covid-19 contaminated) eyepiece to your camera port ... and then use > > your instrumentation well. > > > > For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 > > disclosure: I wrote most of the article (took over from Ted Inoue, whose > > self-portrait may - or not - be inside the monitor figure ... > > "Acquisition rules for Quantitative Fluorescence" section has stood up > > pretty well since circa 1993). > > > > > > On 5/1/2020 12:35 PM, Benjamin Smith 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. > > > ***** > > > > > > I've found the best way to understand phase contrast is on a phase > > contrast > > > microscope. On a phase contrast microscope, take out one of the > oculars > > so > > > you can see the focal planes. In the path without the ocular, you > should > > > see a ring of light perfectly aligned to an annular neutral density > > filter > > > (switch between brightfield and phase contrast to see how the > > illumination > > > perfectly aligns with the ND filter). If they are not aligned, then > the > > > annulus is not setup properly and you are not getting phase contrast > > (check > > > your manual to fix this). > > > > > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > > > unstained test slide (although tissue paper makes this effect very > > clear) . > > > As you move the sample into the light path, you will see all the > > diffracted > > > light miss the annular ring of light and fill the rest of the focal > > plane. > > > Move the sample out of the light path, and you will see this diffracted > > > light disappear. > > > > > > Next, get a poor phase object like adherent cells or anything else that > > is > > > hard to see with bright field, and put it under the phase contrast > > > microscope. Once you are focused on the sample, look at the focal > plane > > > (side without the occular) and slide the annulus out of the way just a > > bit > > > (it should look like the sun just peaking around the moon after a solar > > > eclipse). Now look at your sample, and it should look like just > > > brightfield (i.e. poorly contrasting). Then put the annulus back into > > > place, while looking at the sample, and you see a sudden jump in > > contrast. > > > This is one of the striking features of phase contrast where it really > is > > > all or none, and if the annulus is at all out of alignment you just get > > > brightfield. > > > > > > As others have said, phase contrast is fixing a fundamental issue with > > > brightfield imaging of poorly diffractive objects (such as adherent > > > cells). The issue is that bright field microscopy works by having the > > > diffracted light undergo a phase shift relative to the undiffracted > light > > > (as it follows a different path length in the microscope). Then, these > > > rays are allowed to interfere with each other at the image plane, > > producing > > > an image. You can see this effect in bright field in a similar manner > to > > > the phase contrast trick described above, but instead close the > aperture > > > stop and you will again be able to see the diffracted light separated > > from > > > the non-diffracted light in the focal plane. The problem with poorly > > > diffracting objects is two fold, 1) very little light gets diffracted > > > leaving not much light for interference, and 2) the phase shift of the > > > diffracted light is very small. The end result being that samples like > > > adherent cells only cause a very small decreases in intensity, and > since > > we > > > perceive light on a logarithmic scale, this is very hard for us to see. > > > > > > Therefore, phase contrast fixes both of these issues. One way (which > you > > > can see by looking at the focal planes) is that it uses a cone of light > > as > > > the illumination pattern (due to the annulus at the front focal plane), > > and > > > this cone perfectly lines up with an annular neutral density filter > (the > > > phase plate) at the back focal plane. This ring illumination is a > clever > > > way to spatially separate the diffracted light from the undiffracted > > light > > > at the focal plane, allowing us to attenuate the undiffracted light > > without > > > impacting the diffracted light, balancing the amount of diffracted and > > > undiffracted light. Why use a ring to do this instead of say an > aperture > > > stop? The ring illumination simply preserves more of the illumination > NA > > > and therefore preserves more of your resolution. > > > > > > However, the phase plate doesn't stop there, as its name suggests, it > > also > > > enhances the phase difference between the diffracted and undiffracted > > light > > > (ideally such that the highest diffractive orders are phase shifted to > > > 180°) such that the sharpest edges get perfect destructive > interference. > > > > > > Now at this point, you may be asking yourself, how do they know how > much > > to > > > attenuate the undiffracted light (i.e. how dark to make the annular ND > > > filter) and how much of a phase shift to impose, as some samples may be > > > more diffractive than others. And this is why there are actually many > > > different phase contrast objectives with different phase plates. In > life > > > sciences, most of the time we're only using phase contrast to check > cell > > > culture, so we wind up only ever dealing with just one phase plate > > > optimized for that task. > > > > > > Hope this helps, and I really do recommend checking it out on your own > > > microscope. I've found with students, looking at the correlation > between > > > the focal plane and image plane with and without sample can really > > inspire > > > that "aha" moment. > > > > > > Cheers, > > > Ben Smith > > > > > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher < > [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. > > >> ***** > > >> > > >> Hi David, > > >> > > >> I think that you understanding of phase contrast is correct and that > you > > >> have explained it yourself properly and in a concise way. > > >> Diffraction and refraction are different indeed. It is in fact the > > >> diffraction of light that plays an important role in phase contrast > > >> microscopy. > > >> > > >> Have a look at the Leica tutorial: > > >> > > > https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast > > >> and figure 4 in the link you shared: > > >> > > >> 1. The illumination light passes through the annual ring in the > > condenser, > > >> resulting in a hollow cone of illumination. > > >> 2.1 A higher refractive index in the sample causes retardation, on > > average > > >> generating a phase shift of -1/4 λ in the light that interacted with > the > > >> specimen compared to freely passing light. > > >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is > > >> diffracted to the outside of the illuminating light cone.The smaller > the > > >> object, the larger the angle of diffraction [1]. > > >> 2.3 Light that does not interact with the specimen is not diffracted > and > > >> hence stays on the inside of the illumination cone. > > >> 3. In the phase plate, only the light on the inside of the light-cone > is > > >> then advanced +1/4 λ . > > >> 4. This result in a phase difference between illuminating light and > > sample > > >> light of 1/2 λ, generating destructive interference and hence maximum > > >> contrast in the imaging plane wherever there was a structure in the > > sample > > >> > > >> Here is also a very nice iBology talk on the subject: > > >> https://www.ibiology.org/talks/phase-contrast-microscopy > > >> Hope this helps! > > >> > > >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc > > >> > > > > > > > > -- > Benjamin E. Smith, Ph. D. > Imaging Specialist, Vision Science > University of California, Berkeley > 195 Life Sciences Addition > Berkeley, CA 94720-3200 > Tel (510) 642-9712 > Fax (510) 643-6791 > e-mail: [hidden email] > > https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/ > -- -- Zdenek Svindrych, Ph.D. Research Scientist - Microscopy Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth |
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George McNamara |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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*****
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. ***** and has had their marketing dept confused ever since https://cytoviva.com/products/microscopy-2/microscopy/ p.s. photo of the instrument looks a bit fuzzy to me. On 5/2/2020 1:46 AM, Zdenek Svindrych 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. > ***** > > According to this paper: > https://www.researchgate.net/publication/280600830_High_resolution_optical_microscope > > provocatively titled " Resolution of 90 nm (lambda/5) in an optical > transmission microscope with an annular condenser " (spoiler alert: > misleading title), a brightfield microscope with annular condenser has > astonishing resolution. > Well, in this case the most likely cause for these astonishing results is > just misunderstanding of the calibration patterns, according to the > official Richardson test slide pattern description ( > https://www.grayfieldoptical.com/files/innerpattern.pdf ) the "100 nm" > pattern has a period of 200 nm, the same factor of two applies to the "99 > nm" horizontal and vertical bars. > Arguably, a title stating "lambda/2.5 resolution" would not have the same > distressing effect onto a regular microscopist... > So, while transmitted light images look impressive with a well tuned > instrument, it is well known that the resolution can match the resolution > of fluorescence imaging only if the condenser NA is greater than or equal > to the objective lens NA. That's perfectly doable, but not widely practiced > these days... > Best, zdenek > > On Fri, May 1, 2020 at 8:48 PM Benjamin Smith <[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. >> ***** >> >> It is true that bright field is still the resolution champion (especially >> with a DAPI filter cube in the light path). Although, why settle for poor >> visibility when you can use Rheinberg illumination and get the best of both >> worlds with both a high resolution brightfield image and a high contrast >> edge image (aka dark field). >> >> On Fri, May 1, 2020 at 3:15 PM George McNamara <[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. >>> ***** >>> >>> Ben wrote: "As others have said, phase contrast is fixing a fundamental >>> issue with brightfield imaging of poorly diffractive objects (such as >>> adherent cells)." >>> >>> In defense of brightfield microscopy: >>> >>> A correctly working brightfield microscope with a video camera or >>> digital camera, or the transmitted light path of a confocal microscope, >>> is perfectly capable of imaging very thin objects (such as very flat >>> adherent cells or very thin unlabeled tissue sections). The camera on a >>> smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe >>> Photoshop (has had math capability for a long time), etc, to be able to >>> subtract background, and add back offset. They key is contrast control. >>> Optional: unsharp masking. Some of the (GPU) deconvolution software >>> vendors even offer deconvolution of brightfield image data. Bonus: no >> halo. >>> Reference: >>> >>> Shinya Inoue and Ken Spring 1997 Video Microscopy >>> >>> https://www.springer.com/gp/book/9780306455315 >>> >>> I don't see the 1997 book online, the 1st edition, 1986 is >>> >>> https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 >>> >>> See also Shinya's publications ... online or Collected Works, >>> https://www.worldscientific.com/worldscibooks/10.1142/6315 >>> >>> Bob and Nina Allen's papers. >>> >>> Also nanovid papers (single molecule imaging before STORM, PALM, etc). >>> >>> To quote (and add to) Yogi Berra: "You can see a lot by just looking" >>> ... especially if you switch the light path from your (hopefully not >>> covid-19 contaminated) eyepiece to your camera port ... and then use >>> your instrumentation well. >>> >>> For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 >>> disclosure: I wrote most of the article (took over from Ted Inoue, whose >>> self-portrait may - or not - be inside the monitor figure ... >>> "Acquisition rules for Quantitative Fluorescence" section has stood up >>> pretty well since circa 1993). >>> >>> >>> On 5/1/2020 12:35 PM, Benjamin Smith 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. >>>> ***** >>>> >>>> I've found the best way to understand phase contrast is on a phase >>> contrast >>>> microscope. On a phase contrast microscope, take out one of the >> oculars >>> so >>>> you can see the focal planes. In the path without the ocular, you >> should >>>> see a ring of light perfectly aligned to an annular neutral density >>> filter >>>> (switch between brightfield and phase contrast to see how the >>> illumination >>>> perfectly aligns with the ND filter). If they are not aligned, then >> the >>>> annulus is not setup properly and you are not getting phase contrast >>> (check >>>> your manual to fix this). >>>> >>>> Next. make a slide with a Kim-Wipe or lens paper on it, or grab an >>>> unstained test slide (although tissue paper makes this effect very >>> clear) . >>>> As you move the sample into the light path, you will see all the >>> diffracted >>>> light miss the annular ring of light and fill the rest of the focal >>> plane. >>>> Move the sample out of the light path, and you will see this diffracted >>>> light disappear. >>>> >>>> Next, get a poor phase object like adherent cells or anything else that >>> is >>>> hard to see with bright field, and put it under the phase contrast >>>> microscope. Once you are focused on the sample, look at the focal >> plane >>>> (side without the occular) and slide the annulus out of the way just a >>> bit >>>> (it should look like the sun just peaking around the moon after a solar >>>> eclipse). Now look at your sample, and it should look like just >>>> brightfield (i.e. poorly contrasting). Then put the annulus back into >>>> place, while looking at the sample, and you see a sudden jump in >>> contrast. >>>> This is one of the striking features of phase contrast where it really >> is >>>> all or none, and if the annulus is at all out of alignment you just get >>>> brightfield. >>>> >>>> As others have said, phase contrast is fixing a fundamental issue with >>>> brightfield imaging of poorly diffractive objects (such as adherent >>>> cells). The issue is that bright field microscopy works by having the >>>> diffracted light undergo a phase shift relative to the undiffracted >> light >>>> (as it follows a different path length in the microscope). Then, these >>>> rays are allowed to interfere with each other at the image plane, >>> producing >>>> an image. You can see this effect in bright field in a similar manner >> to >>>> the phase contrast trick described above, but instead close the >> aperture >>>> stop and you will again be able to see the diffracted light separated >>> from >>>> the non-diffracted light in the focal plane. The problem with poorly >>>> diffracting objects is two fold, 1) very little light gets diffracted >>>> leaving not much light for interference, and 2) the phase shift of the >>>> diffracted light is very small. The end result being that samples like >>>> adherent cells only cause a very small decreases in intensity, and >> since >>> we >>>> perceive light on a logarithmic scale, this is very hard for us to see. >>>> >>>> Therefore, phase contrast fixes both of these issues. One way (which >> you >>>> can see by looking at the focal planes) is that it uses a cone of light >>> as >>>> the illumination pattern (due to the annulus at the front focal plane), >>> and >>>> this cone perfectly lines up with an annular neutral density filter >> (the >>>> phase plate) at the back focal plane. This ring illumination is a >> clever >>>> way to spatially separate the diffracted light from the undiffracted >>> light >>>> at the focal plane, allowing us to attenuate the undiffracted light >>> without >>>> impacting the diffracted light, balancing the amount of diffracted and >>>> undiffracted light. Why use a ring to do this instead of say an >> aperture >>>> stop? The ring illumination simply preserves more of the illumination >> NA >>>> and therefore preserves more of your resolution. >>>> >>>> However, the phase plate doesn't stop there, as its name suggests, it >>> also >>>> enhances the phase difference between the diffracted and undiffracted >>> light >>>> (ideally such that the highest diffractive orders are phase shifted to >>>> 180°) such that the sharpest edges get perfect destructive >> interference. >>>> Now at this point, you may be asking yourself, how do they know how >> much >>> to >>>> attenuate the undiffracted light (i.e. how dark to make the annular ND >>>> filter) and how much of a phase shift to impose, as some samples may be >>>> more diffractive than others. And this is why there are actually many >>>> different phase contrast objectives with different phase plates. In >> life >>>> sciences, most of the time we're only using phase contrast to check >> cell >>>> culture, so we wind up only ever dealing with just one phase plate >>>> optimized for that task. >>>> >>>> Hope this helps, and I really do recommend checking it out on your own >>>> microscope. I've found with students, looking at the correlation >> between >>>> the focal plane and image plane with and without sample can really >>> inspire >>>> that "aha" moment. >>>> >>>> Cheers, >>>> Ben Smith >>>> >>>> On Fri, May 1, 2020 at 12:35 AM Kai Schleicher < >> [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. >>>>> ***** >>>>> >>>>> Hi David, >>>>> >>>>> I think that you understanding of phase contrast is correct and that >> you >>>>> have explained it yourself properly and in a concise way. >>>>> Diffraction and refraction are different indeed. It is in fact the >>>>> diffraction of light that plays an important role in phase contrast >>>>> microscopy. >>>>> >>>>> Have a look at the Leica tutorial: >>>>> >> https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast >>>>> and figure 4 in the link you shared: >>>>> >>>>> 1. The illumination light passes through the annual ring in the >>> condenser, >>>>> resulting in a hollow cone of illumination. >>>>> 2.1 A higher refractive index in the sample causes retardation, on >>> average >>>>> generating a phase shift of -1/4 λ in the light that interacted with >> the >>>>> specimen compared to freely passing light. >>>>> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is >>>>> diffracted to the outside of the illuminating light cone.The smaller >> the >>>>> object, the larger the angle of diffraction [1]. >>>>> 2.3 Light that does not interact with the specimen is not diffracted >> and >>>>> hence stays on the inside of the illumination cone. >>>>> 3. In the phase plate, only the light on the inside of the light-cone >> is >>>>> then advanced +1/4 λ . >>>>> 4. This result in a phase difference between illuminating light and >>> sample >>>>> light of 1/2 λ, generating destructive interference and hence maximum >>>>> contrast in the imaging plane wherever there was a structure in the >>> sample >>>>> Here is also a very nice iBology talk on the subject: >>>>> https://www.ibiology.org/talks/phase-contrast-microscopy >>>>> Hope this helps! >>>>> >>>>> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >>>>> >> >> -- >> Benjamin E. Smith, Ph. D. >> Imaging Specialist, Vision Science >> University of California, Berkeley >> 195 Life Sciences Addition >> Berkeley, CA 94720-3200 >> Tel (510) 642-9712 >> Fax (510) 643-6791 >> e-mail: [hidden email] >> >> https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/ >> > |
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0000001ed7f52e4a-dmarc-request |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by George McNamara
*****
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. ***** This is an interesting discussion, Our microbiologists love the 100x 1.4 NA phase contrast objectives for combined phase and widefield fluorescence imaging. I always wondered how the phase ring on the objective is actually made and what influence it has on the fluorescence signal? When it just shifts the phase, it should be relatively transparent and let the fluorescence pass without losses, but when I look at the ring from the back, it is quite reflective, indicating that some of the fluorescence signal will be lost. HAs anyone compared objectives with and without phase ring? What will be the influence of the phase shift on the PSF? best wishes Andreas -----Original Message----- From: George McNamara <[hidden email]> To: [hidden email] Sent: Fri, 1 May 2020 18:24 Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy ***** 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. ***** Ben wrote: "As others have said, phase contrast is fixing a fundamental issue with brightfield imaging of poorly diffractive objects (such as adherent cells)." In defense of brightfield microscopy: A correctly working brightfield microscope with a video camera or digital camera, or the transmitted light path of a confocal microscope, is perfectly capable of imaging very thin objects (such as very flat adherent cells or very thin unlabeled tissue sections). The camera on a smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe Photoshop (has had math capability for a long time), etc, to be able to subtract background, and add back offset. They key is contrast control. Optional: unsharp masking. Some of the (GPU) deconvolution software vendors even offer deconvolution of brightfield image data. Bonus: no halo. Reference: Shinya Inoue and Ken Spring 1997 Video Microscopy https://www.springer.com/gp/book/9780306455315 I don't see the 1997 book online, the 1st edition, 1986 is https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 See also Shinya's publications ... online or Collected Works, https://www.worldscientific.com/worldscibooks/10.1142/6315 Bob and Nina Allen's papers. Also nanovid papers (single molecule imaging before STORM, PALM, etc). To quote (and add to) Yogi Berra: "You can see a lot by just looking" ... especially if you switch the light path from your (hopefully not covid-19 contaminated) eyepiece to your camera port ... and then use your instrumentation well. For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 disclosure: I wrote most of the article (took over from Ted Inoue, whose self-portrait may - or not - be inside the monitor figure ... "Acquisition rules for Quantitative Fluorescence" section has stood up pretty well since circa 1993). On 5/1/2020 12:35 PM, Benjamin Smith 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. > ***** > > I've found the best way to understand phase contrast is on a phase contrast > microscope. On a phase contrast microscope, take out one of the oculars so > you can see the focal planes. In the path without the ocular, you should > see a ring of light perfectly aligned to an annular neutral density filter > (switch between brightfield and phase contrast to see how the illumination > perfectly aligns with the ND filter). If they are not aligned, then the > annulus is not setup properly and you are not getting phase contrast (check > your manual to fix this). > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > unstained test slide (although tissue paper makes this effect very clear) . > As you move the sample into the light path, you will see all the diffracted > light miss the annular ring of light and fill the rest of the focal plane. > Move the sample out of the light path, and you will see this diffracted > light disappear. > > Next, get a poor phase object like adherent cells or anything else that is > hard to see with bright field, and put it under the phase contrast > microscope. Once you are focused on the sample, look at the focal plane > (side without the occular) and slide the annulus out of the way just a bit > (it should look like the sun just peaking around the moon after a solar > eclipse). Now look at your sample, and it should look like just > brightfield (i.e. poorly contrasting). Then put the annulus back into > place, while looking at the sample, and you see a sudden jump in contrast. > This is one of the striking features of phase contrast where it really is > all or none, and if the annulus is at all out of alignment you just get > brightfield. > > As others have said, phase contrast is fixing a fundamental issue with > brightfield imaging of poorly diffractive objects (such as adherent > cells). The issue is that bright field microscopy works by having the > diffracted light undergo a phase shift relative to the undiffracted light > (as it follows a different path length in the microscope). Then, these > rays are allowed to interfere with each other at the image plane, producing > an image. You can see this effect in bright field in a similar manner to > the phase contrast trick described above, but instead close the aperture > stop and you will again be able to see the diffracted light separated from > the non-diffracted light in the focal plane. The problem with poorly > diffracting objects is two fold, 1) very little light gets diffracted > leaving not much light for interference, and 2) the phase shift of the > diffracted light is very small. The end result being that samples like > adherent cells only cause a very small decreases in intensity, and since we > perceive light on a logarithmic scale, this is very hard for us to see. > > Therefore, phase contrast fixes both of these issues. One way (which you > can see by looking at the focal planes) is that it uses a cone of light as > the illumination pattern (due to the annulus at the front focal plane), and > this cone perfectly lines up with an annular neutral density filter (the > phase plate) at the back focal plane. This ring illumination is a clever > way to spatially separate the diffracted light from the undiffracted light > at the focal plane, allowing us to attenuate the undiffracted light without > impacting the diffracted light, balancing the amount of diffracted and > undiffracted light. Why use a ring to do this instead of say an aperture > stop? The ring illumination simply preserves more of the illumination NA > and therefore preserves more of your resolution. > > However, the phase plate doesn't stop there, as its name suggests, it also > enhances the phase difference between the diffracted and undiffracted light > (ideally such that the highest diffractive orders are phase shifted to > 180°) such that the sharpest edges get perfect destructive interference. > > Now at this point, you may be asking yourself, how do they know how much to > attenuate the undiffracted light (i.e. how dark to make the annular ND > filter) and how much of a phase shift to impose, as some samples may be > more diffractive than others. And this is why there are actually many > different phase contrast objectives with different phase plates. In life > sciences, most of the time we're only using phase contrast to check cell > culture, so we wind up only ever dealing with just one phase plate > optimized for that task. > > Hope this helps, and I really do recommend checking it out on your own > microscope. I've found with students, looking at the correlation between > the focal plane and image plane with and without sample can really inspire > that "aha" moment. > > Cheers, > Ben Smith > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[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. >> ***** >> >> Hi David, >> >> I think that you understanding of phase contrast is correct and that you >> have explained it yourself properly and in a concise way. >> Diffraction and refraction are different indeed. It is in fact the >> diffraction of light that plays an important role in phase contrast >> microscopy. >> >> Have a look at the Leica tutorial: >> https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast >> and figure 4 in the link you shared: >> >> 1. The illumination light passes through the annual ring in the condenser, >> resulting in a hollow cone of illumination. >> 2.1 A higher refractive index in the sample causes retardation, on average >> generating a phase shift of -1/4 λ in the light that interacted with the >> specimen compared to freely passing light. >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is >> diffracted to the outside of the illuminating light cone.The smaller the >> object, the larger the angle of diffraction [1]. >> 2.3 Light that does not interact with the specimen is not diffracted and >> hence stays on the inside of the illumination cone. >> 3. In the phase plate, only the light on the inside of the light-cone is >> then advanced +1/4 λ . >> 4. This result in a phase difference between illuminating light and sample >> light of 1/2 λ, generating destructive interference and hence maximum >> contrast in the imaging plane wherever there was a structure in the sample >> >> Here is also a very nice iBology talk on the subject: >> https://www.ibiology.org/talks/phase-contrast-microscopy >> Hope this helps! >> >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >> > |
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Arne Seitz |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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*****
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. ***** The phase ring in an objective has two functions. It should (as the name suggests) shift the phase of the light. At the same time is also decreases the intensity (as far as I know it is just a grey filter). The rational behind that is that for maximal contrast after interference the intensity of the individual waves must have same or similar intensities. Therefore it is necessary to decrease the intensity of the so called surrounding light which is typically more intense than the so called scattered light. Therefore these objectives are less suited for fluorescence imaging. Part of the emitted photons will be absorbed by the neutral density filter inside the objective. But I have to admit that I have never measured the loss itself. Best Arne -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer Sent: Sonntag, 3. Mai 2020 14:01 To: [hidden email] Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy ***** 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. ***** This is an interesting discussion, Our microbiologists love the 100x 1.4 NA phase contrast objectives for combined phase and widefield fluorescence imaging. I always wondered how the phase ring on the objective is actually made and what influence it has on the fluorescence signal? When it just shifts the phase, it should be relatively transparent and let the fluorescence pass without losses, but when I look at the ring from the back, it is quite reflective, indicating that some of the fluorescence signal will be lost. HAs anyone compared objectives with and without phase ring? What will be the influence of the phase shift on the PSF? best wishes Andreas -----Original Message----- From: George McNamara <[hidden email]> To: [hidden email] Sent: Fri, 1 May 2020 18:24 Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy ***** 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. ***** Ben wrote: "As others have said, phase contrast is fixing a fundamental issue with brightfield imaging of poorly diffractive objects (such as adherent cells)." In defense of brightfield microscopy: A correctly working brightfield microscope with a video camera or digital camera, or the transmitted light path of a confocal microscope, is perfectly capable of imaging very thin objects (such as very flat adherent cells or very thin unlabeled tissue sections). The camera on a smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe Photoshop (has had math capability for a long time), etc, to be able to subtract background, and add back offset. They key is contrast control. Optional: unsharp masking. Some of the (GPU) deconvolution software vendors even offer deconvolution of brightfield image data. Bonus: no halo. Reference: Shinya Inoue and Ken Spring 1997 Video Microscopy https://www.springer.com/gp/book/9780306455315 I don't see the 1997 book online, the 1st edition, 1986 is https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 See also Shinya's publications ... online or Collected Works, https://www.worldscientific.com/worldscibooks/10.1142/6315 Bob and Nina Allen's papers. Also nanovid papers (single molecule imaging before STORM, PALM, etc). To quote (and add to) Yogi Berra: "You can see a lot by just looking" ... especially if you switch the light path from your (hopefully not covid-19 contaminated) eyepiece to your camera port ... and then use your instrumentation well. For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 disclosure: I wrote most of the article (took over from Ted Inoue, whose self-portrait may - or not - be inside the monitor figure ... "Acquisition rules for Quantitative Fluorescence" section has stood up pretty well since circa 1993). On 5/1/2020 12:35 PM, Benjamin Smith 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. > ***** > > I've found the best way to understand phase contrast is on a phase > contrast microscope. On a phase contrast microscope, take out one of > the oculars so you can see the focal planes. In the path without the > ocular, you should see a ring of light perfectly aligned to an annular > neutral density filter (switch between brightfield and phase contrast > to see how the illumination perfectly aligns with the ND filter). If > they are not aligned, then the annulus is not setup properly and you > are not getting phase contrast (check your manual to fix this). > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > unstained test slide (although tissue paper makes this effect very clear) . > As you move the sample into the light path, you will see all the > diffracted light miss the annular ring of light and fill the rest of the focal plane. > Move the sample out of the light path, and you will see this > diffracted light disappear. > > Next, get a poor phase object like adherent cells or anything else > that is hard to see with bright field, and put it under the phase > contrast microscope. Once you are focused on the sample, look at the > focal plane (side without the occular) and slide the annulus out of > the way just a bit (it should look like the sun just peaking around > the moon after a solar eclipse). Now look at your sample, and it > should look like just brightfield (i.e. poorly contrasting). Then put > the annulus back into place, while looking at the sample, and you see a sudden jump in contrast. > This is one of the striking features of phase contrast where it really > is all or none, and if the annulus is at all out of alignment you just > get brightfield. > > As others have said, phase contrast is fixing a fundamental issue with > brightfield imaging of poorly diffractive objects (such as adherent > cells). The issue is that bright field microscopy works by having the > diffracted light undergo a phase shift relative to the undiffracted > light (as it follows a different path length in the microscope). > Then, these rays are allowed to interfere with each other at the image > plane, producing an image. You can see this effect in bright field in > a similar manner to the phase contrast trick described above, but > instead close the aperture stop and you will again be able to see the > diffracted light separated from the non-diffracted light in the focal > plane. The problem with poorly diffracting objects is two fold, 1) > very little light gets diffracted leaving not much light for > interference, and 2) the phase shift of the diffracted light is very > small. The end result being that samples like adherent cells only > cause a very small decreases in intensity, and since we perceive light on a logarithmic scale, this is very hard for us to see. > > Therefore, phase contrast fixes both of these issues. One way (which > you can see by looking at the focal planes) is that it uses a cone of > light as the illumination pattern (due to the annulus at the front > focal plane), and this cone perfectly lines up with an annular neutral > density filter (the phase plate) at the back focal plane. This ring > illumination is a clever way to spatially separate the diffracted > light from the undiffracted light at the focal plane, allowing us to > attenuate the undiffracted light without impacting the diffracted > light, balancing the amount of diffracted and undiffracted light. Why > use a ring to do this instead of say an aperture stop? The ring > illumination simply preserves more of the illumination NA and therefore preserves more of your resolution. > > However, the phase plate doesn't stop there, as its name suggests, it > also enhances the phase difference between the diffracted and > undiffracted light (ideally such that the highest diffractive orders > are phase shifted to > 180°) such that the sharpest edges get perfect destructive interference. > > Now at this point, you may be asking yourself, how do they know how > much to attenuate the undiffracted light (i.e. how dark to make the > annular ND > filter) and how much of a phase shift to impose, as some samples may > be more diffractive than others. And this is why there are actually > many different phase contrast objectives with different phase plates. > In life sciences, most of the time we're only using phase contrast to > check cell culture, so we wind up only ever dealing with just one > phase plate optimized for that task. > > Hope this helps, and I really do recommend checking it out on your own > microscope. I've found with students, looking at the correlation > between the focal plane and image plane with and without sample can > really inspire that "aha" moment. > > Cheers, > Ben Smith > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher > <[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. >> ***** >> >> Hi David, >> >> I think that you understanding of phase contrast is correct and that >> you have explained it yourself properly and in a concise way. >> Diffraction and refraction are different indeed. It is in fact the >> diffraction of light that plays an important role in phase contrast >> microscopy. >> >> Have a look at the Leica tutorial: >> https://www.leica-microsystems.com/science-lab/the-principles-of-phas >> e-contrast >> and figure 4 in the link you shared: >> >> 1. The illumination light passes through the annual ring in the >> condenser, resulting in a hollow cone of illumination. >> 2.1 A higher refractive index in the sample causes retardation, on >> average generating a phase shift of -1/4 λ in the light that >> interacted with the specimen compared to freely passing light. >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) >> is diffracted to the outside of the illuminating light cone.The >> smaller the object, the larger the angle of diffraction [1]. >> 2.3 Light that does not interact with the specimen is not diffracted >> and hence stays on the inside of the illumination cone. >> 3. In the phase plate, only the light on the inside of the light-cone >> is then advanced +1/4 λ . >> 4. This result in a phase difference between illuminating light and >> sample light of 1/2 λ, generating destructive interference and hence >> maximum contrast in the imaging plane wherever there was a structure >> in the sample >> >> Here is also a very nice iBology talk on the subject: >> https://www.ibiology.org/talks/phase-contrast-microscopy >> Hope this helps! >> >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >> > |
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George McNamara |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by 0000001ed7f52e4a-dmarc-request
*****
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. ***** The canonical phase contrast hardware is illustrated and explained at https://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phase.html For fluorescence, the resolution is dictated by the highest NA photons - hence the phase ring in the objective lens does not go to the edge. Loss of intensity for fluorescence should be pretty much the area of the phase ring and its optical density ('partially absorbing material'). This would also be true for the transmitted light path (rotate out the condenser annular ring), as long as the optical path is well adjusted to avoid glare ... but no good comparator other than an identical objective lens without phase stuff in lens. On some (old) microscopes the phase contrast components are external -- I'm thinking certain tissue culture microscopes. So, could check your tissue culture room(s). Smartphone on the eyepiece port (or on a cradle on the eyepiece port) and/or take the phase ring and scan it on a flatbed scanner with a transparency illuminator (or lay on a slide, and tile scan). On 5/3/2020 8:00 AM, Andreas Bruckbauer 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. > ***** > > This is an interesting discussion, > > Our microbiologists love the 100x 1.4 NA phase contrast objectives for combined phase and widefield fluorescence imaging. I always wondered how the phase ring on the objective is actually made and what influence it has on the fluorescence signal? When it just shifts the phase, it should be relatively transparent and let the fluorescence pass without losses, but when I look at the ring from the back, it is quite reflective, indicating that some of the fluorescence signal will be lost. HAs anyone compared objectives with and without phase ring? What will be the influence of the phase shift on the PSF? > > best wishes > > Andreas > > -----Original Message----- > From: George McNamara <[hidden email]> > To: [hidden email] > Sent: Fri, 1 May 2020 18:24 > Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy > > ***** > 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. > ***** > > Ben wrote: "As others have said, phase contrast is fixing a fundamental > issue with brightfield imaging of poorly diffractive objects (such as > adherent cells)." > > In defense of brightfield microscopy: > > A correctly working brightfield microscope with a video camera or > digital camera, or the transmitted light path of a confocal microscope, > is perfectly capable of imaging very thin objects (such as very flat > adherent cells or very thin unlabeled tissue sections). The camera on a > smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe > Photoshop (has had math capability for a long time), etc, to be able to > subtract background, and add back offset. They key is contrast control. > Optional: unsharp masking. Some of the (GPU) deconvolution software > vendors even offer deconvolution of brightfield image data. Bonus: no halo. > > Reference: > > Shinya Inoue and Ken Spring 1997 Video Microscopy > > https://www.springer.com/gp/book/9780306455315 > > I don't see the 1997 book online, the 1st edition, 1986 is > > https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 > > See also Shinya's publications ... online or Collected Works, > https://www.worldscientific.com/worldscibooks/10.1142/6315 > > Bob and Nina Allen's papers. > > Also nanovid papers (single molecule imaging before STORM, PALM, etc). > > To quote (and add to) Yogi Berra: "You can see a lot by just looking" > ... especially if you switch the light path from your (hopefully not > covid-19 contaminated) eyepiece to your camera port ... and then use > your instrumentation well. > > For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 > disclosure: I wrote most of the article (took over from Ted Inoue, whose > self-portrait may - or not - be inside the monitor figure ... > "Acquisition rules for Quantitative Fluorescence" section has stood up > pretty well since circa 1993). > > > On 5/1/2020 12:35 PM, Benjamin Smith 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. >> ***** >> >> I've found the best way to understand phase contrast is on a phase contrast >> microscope. On a phase contrast microscope, take out one of the oculars so >> you can see the focal planes. In the path without the ocular, you should >> see a ring of light perfectly aligned to an annular neutral density filter >> (switch between brightfield and phase contrast to see how the illumination >> perfectly aligns with the ND filter). If they are not aligned, then the >> annulus is not setup properly and you are not getting phase contrast (check >> your manual to fix this). >> >> Next. make a slide with a Kim-Wipe or lens paper on it, or grab an >> unstained test slide (although tissue paper makes this effect very clear) . >> As you move the sample into the light path, you will see all the diffracted >> light miss the annular ring of light and fill the rest of the focal plane. >> Move the sample out of the light path, and you will see this diffracted >> light disappear. >> >> Next, get a poor phase object like adherent cells or anything else that is >> hard to see with bright field, and put it under the phase contrast >> microscope. Once you are focused on the sample, look at the focal plane >> (side without the occular) and slide the annulus out of the way just a bit >> (it should look like the sun just peaking around the moon after a solar >> eclipse). Now look at your sample, and it should look like just >> brightfield (i.e. poorly contrasting). Then put the annulus back into >> place, while looking at the sample, and you see a sudden jump in contrast. >> This is one of the striking features of phase contrast where it really is >> all or none, and if the annulus is at all out of alignment you just get >> brightfield. >> >> As others have said, phase contrast is fixing a fundamental issue with >> brightfield imaging of poorly diffractive objects (such as adherent >> cells). The issue is that bright field microscopy works by having the >> diffracted light undergo a phase shift relative to the undiffracted light >> (as it follows a different path length in the microscope). Then, these >> rays are allowed to interfere with each other at the image plane, producing >> an image. You can see this effect in bright field in a similar manner to >> the phase contrast trick described above, but instead close the aperture >> stop and you will again be able to see the diffracted light separated from >> the non-diffracted light in the focal plane. The problem with poorly >> diffracting objects is two fold, 1) very little light gets diffracted >> leaving not much light for interference, and 2) the phase shift of the >> diffracted light is very small. The end result being that samples like >> adherent cells only cause a very small decreases in intensity, and since we >> perceive light on a logarithmic scale, this is very hard for us to see. >> >> Therefore, phase contrast fixes both of these issues. One way (which you >> can see by looking at the focal planes) is that it uses a cone of light as >> the illumination pattern (due to the annulus at the front focal plane), and >> this cone perfectly lines up with an annular neutral density filter (the >> phase plate) at the back focal plane. This ring illumination is a clever >> way to spatially separate the diffracted light from the undiffracted light >> at the focal plane, allowing us to attenuate the undiffracted light without >> impacting the diffracted light, balancing the amount of diffracted and >> undiffracted light. Why use a ring to do this instead of say an aperture >> stop? The ring illumination simply preserves more of the illumination NA >> and therefore preserves more of your resolution. >> >> However, the phase plate doesn't stop there, as its name suggests, it also >> enhances the phase difference between the diffracted and undiffracted light >> (ideally such that the highest diffractive orders are phase shifted to >> 180°) such that the sharpest edges get perfect destructive interference. >> >> Now at this point, you may be asking yourself, how do they know how much to >> attenuate the undiffracted light (i.e. how dark to make the annular ND >> filter) and how much of a phase shift to impose, as some samples may be >> more diffractive than others. And this is why there are actually many >> different phase contrast objectives with different phase plates. In life >> sciences, most of the time we're only using phase contrast to check cell >> culture, so we wind up only ever dealing with just one phase plate >> optimized for that task. >> >> Hope this helps, and I really do recommend checking it out on your own >> microscope. I've found with students, looking at the correlation between >> the focal plane and image plane with and without sample can really inspire >> that "aha" moment. >> >> Cheers, >> Ben Smith >> >> On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[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. >>> ***** >>> >>> Hi David, >>> >>> I think that you understanding of phase contrast is correct and that you >>> have explained it yourself properly and in a concise way. >>> Diffraction and refraction are different indeed. It is in fact the >>> diffraction of light that plays an important role in phase contrast >>> microscopy. >>> >>> Have a look at the Leica tutorial: >>> https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast >>> and figure 4 in the link you shared: >>> >>> 1. The illumination light passes through the annual ring in the condenser, >>> resulting in a hollow cone of illumination. >>> 2.1 A higher refractive index in the sample causes retardation, on average >>> generating a phase shift of -1/4 λ in the light that interacted with the >>> specimen compared to freely passing light. >>> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is >>> diffracted to the outside of the illuminating light cone.The smaller the >>> object, the larger the angle of diffraction [1]. >>> 2.3 Light that does not interact with the specimen is not diffracted and >>> hence stays on the inside of the illumination cone. >>> 3. In the phase plate, only the light on the inside of the light-cone is >>> then advanced +1/4 λ . >>> 4. This result in a phase difference between illuminating light and sample >>> light of 1/2 λ, generating destructive interference and hence maximum >>> contrast in the imaging plane wherever there was a structure in the sample >>> >>> Here is also a very nice iBology talk on the subject: >>> https://www.ibiology.org/talks/phase-contrast-microscopy >>> Hope this helps! >>> >>> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >>> |
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Lutz Schaefer |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by 0000001ed7f52e4a-dmarc-request
*****
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. ***** Andreas, the phase ring has a profound impact on the PSF. For deconvolution with a modelled PSF you need to considerer this extra contribution in the complex valued intensities of the back focal plane! We had done some work on that a long time ago (~18 years...) on Zeiss objectives. They have 3 different kinds of phase rings. If you are interested in the physical dimensions and attenuations I can provide them to you off the list. Best Regards Lutz Schaefer __________________________________ L u t z S c h a e f e r Research Scientist Mathematical modeling / Computational microscopy Advanced Imaging Methodology Consultation 16-715 Doon Village Rd. Kitchener, ON, N2P 2A2, Canada Phone/Fax: +1 519 894 8870 Mobile: +1 519 722 8870 Email: [hidden email] Website: http://home.golden.net/~lschafer/ ___________________________________ -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer Sent: Sunday, May 03, 2020 8:01 AM To: [hidden email] Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy ***** 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. ***** This is an interesting discussion, Our microbiologists love the 100x 1.4 NA phase contrast objectives for combined phase and widefield fluorescence imaging. I always wondered how the phase ring on the objective is actually made and what influence it has on the fluorescence signal? When it just shifts the phase, it should be relatively transparent and let the fluorescence pass without losses, but when I look at the ring from the back, it is quite reflective, indicating that some of the fluorescence signal will be lost. HAs anyone compared objectives with and without phase ring? What will be the influence of the phase shift on the PSF? best wishes Andreas -----Original Message----- From: George McNamara <[hidden email]> To: [hidden email] Sent: Fri, 1 May 2020 18:24 Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy ***** 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. ***** Ben wrote: "As others have said, phase contrast is fixing a fundamental issue with brightfield imaging of poorly diffractive objects (such as adherent cells)." In defense of brightfield microscopy: A correctly working brightfield microscope with a video camera or digital camera, or the transmitted light path of a confocal microscope, is perfectly capable of imaging very thin objects (such as very flat adherent cells or very thin unlabeled tissue sections). The camera on a smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe Photoshop (has had math capability for a long time), etc, to be able to subtract background, and add back offset. They key is contrast control. Optional: unsharp masking. Some of the (GPU) deconvolution software vendors even offer deconvolution of brightfield image data. Bonus: no halo. Reference: Shinya Inoue and Ken Spring 1997 Video Microscopy https://www.springer.com/gp/book/9780306455315 I don't see the 1997 book online, the 1st edition, 1986 is https://link.springer.com/book/10.1007%2F978-1-4757-6925-8 See also Shinya's publications ... online or Collected Works, https://www.worldscientific.com/worldscibooks/10.1142/6315 Bob and Nina Allen's papers. Also nanovid papers (single molecule imaging before STORM, PALM, etc). To quote (and add to) Yogi Berra: "You can see a lot by just looking" ... especially if you switch the light path from your (hopefully not covid-19 contaminated) eyepiece to your camera port ... and then use your instrumentation well. For MetaMorph, see http://mdc.custhelp.com/app/answers/detail/a_id/18800 disclosure: I wrote most of the article (took over from Ted Inoue, whose self-portrait may - or not - be inside the monitor figure ... "Acquisition rules for Quantitative Fluorescence" section has stood up pretty well since circa 1993). On 5/1/2020 12:35 PM, Benjamin Smith 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. > ***** > > I've found the best way to understand phase contrast is on a phase > contrast microscope. On a phase contrast microscope, take out one of > the oculars so you can see the focal planes. In the path without the > ocular, you should see a ring of light perfectly aligned to an annular > neutral density filter (switch between brightfield and phase contrast > to see how the illumination perfectly aligns with the ND filter). If > they are not aligned, then the annulus is not setup properly and you > are not getting phase contrast (check your manual to fix this). > > Next. make a slide with a Kim-Wipe or lens paper on it, or grab an > unstained test slide (although tissue paper makes this effect very clear) . > As you move the sample into the light path, you will see all the > diffracted light miss the annular ring of light and fill the rest of the focal plane. > Move the sample out of the light path, and you will see this > diffracted light disappear. > > Next, get a poor phase object like adherent cells or anything else > that is hard to see with bright field, and put it under the phase > contrast microscope. Once you are focused on the sample, look at the > focal plane (side without the occular) and slide the annulus out of > the way just a bit (it should look like the sun just peaking around > the moon after a solar eclipse). Now look at your sample, and it > should look like just brightfield (i.e. poorly contrasting). Then put > the annulus back into place, while looking at the sample, and you see a sudden jump in contrast. > This is one of the striking features of phase contrast where it really > is all or none, and if the annulus is at all out of alignment you just > get brightfield. > > As others have said, phase contrast is fixing a fundamental issue with > brightfield imaging of poorly diffractive objects (such as adherent > cells). The issue is that bright field microscopy works by having the > diffracted light undergo a phase shift relative to the undiffracted > light (as it follows a different path length in the microscope). > Then, these rays are allowed to interfere with each other at the image > plane, producing an image. You can see this effect in bright field in > a similar manner to the phase contrast trick described above, but > instead close the aperture stop and you will again be able to see the > diffracted light separated from the non-diffracted light in the focal > plane. The problem with poorly diffracting objects is two fold, 1) > very little light gets diffracted leaving not much light for > interference, and 2) the phase shift of the diffracted light is very > small. The end result being that samples like adherent cells only > cause a very small decreases in intensity, and since we perceive light on a logarithmic scale, this is very hard for us to see. > > Therefore, phase contrast fixes both of these issues. One way (which > you can see by looking at the focal planes) is that it uses a cone of > light as the illumination pattern (due to the annulus at the front > focal plane), and this cone perfectly lines up with an annular neutral > density filter (the phase plate) at the back focal plane. This ring > illumination is a clever way to spatially separate the diffracted > light from the undiffracted light at the focal plane, allowing us to > attenuate the undiffracted light without impacting the diffracted > light, balancing the amount of diffracted and undiffracted light. Why > use a ring to do this instead of say an aperture stop? The ring > illumination simply preserves more of the illumination NA and therefore preserves more of your resolution. > > However, the phase plate doesn't stop there, as its name suggests, it > also enhances the phase difference between the diffracted and > undiffracted light (ideally such that the highest diffractive orders > are phase shifted to > 180°) such that the sharpest edges get perfect destructive interference. > > Now at this point, you may be asking yourself, how do they know how > much to attenuate the undiffracted light (i.e. how dark to make the > annular ND > filter) and how much of a phase shift to impose, as some samples may > be more diffractive than others. And this is why there are actually > many different phase contrast objectives with different phase plates. > In life sciences, most of the time we're only using phase contrast to > check cell culture, so we wind up only ever dealing with just one > phase plate optimized for that task. > > Hope this helps, and I really do recommend checking it out on your own > microscope. I've found with students, looking at the correlation > between the focal plane and image plane with and without sample can > really inspire that "aha" moment. > > Cheers, > Ben Smith > > On Fri, May 1, 2020 at 12:35 AM Kai Schleicher > <[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. >> ***** >> >> Hi David, >> >> I think that you understanding of phase contrast is correct and that >> you have explained it yourself properly and in a concise way. >> Diffraction and refraction are different indeed. It is in fact the >> diffraction of light that plays an important role in phase contrast >> microscopy. >> >> Have a look at the Leica tutorial: >> https://www.leica-microsystems.com/science-lab/the-principles-of-phas >> e-contrast >> and figure 4 in the link you shared: >> >> 1. The illumination light passes through the annual ring in the >> condenser, resulting in a hollow cone of illumination. >> 2.1 A higher refractive index in the sample causes retardation, on >> average generating a phase shift of -1/4 λ in the light that >> interacted with the specimen compared to freely passing light. >> 2.2 Light interacting with the specimen (cell, granule, nucleus...) >> is diffracted to the outside of the illuminating light cone.The >> smaller the object, the larger the angle of diffraction [1]. >> 2.3 Light that does not interact with the specimen is not diffracted >> and hence stays on the inside of the illumination cone. >> 3. In the phase plate, only the light on the inside of the light-cone >> is then advanced +1/4 λ . >> 4. This result in a phase difference between illuminating light and >> sample light of 1/2 λ, generating destructive interference and hence >> maximum contrast in the imaging plane wherever there was a structure >> in the sample >> >> Here is also a very nice iBology talk on the subject: >> https://www.ibiology.org/talks/phase-contrast-microscopy >> Hope this helps! >> >> [1] Abbe Diffraction: https://www.youtube.com/watch?v=d8Tqoo0S6gc >> > |
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Catalin Chiritescu |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by George McNamara
*****
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. ***** You lose about 20-30% of light when a Phase Contrast objective is used in fluorescence. AFAIK Nikon Ti (don't know about TI2) has an external phase ring setup that you can use for Phase Contrast imaging with BF objectives - you can take it out of fluorescence light path to avoid the loss and PSF disruption. We considered that option for our Quantitative Phase Imaging SLIM systems. |
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In reply to this post by George McNamara
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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. ***** I've previously had several measuremenst on Olympus IX scopes and found loss of less than 10% (much lower than I expected). Dotan |
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Knecht, David |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by Catalin Chiritescu
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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. ***** I suspect the higher number (20%) is if you consider loss in both excitation and emission through the phase objective, but in the excitation path, you usually have plenty of extra power to compensate for a bit of loss in excitation efficiency. So I think the lower number is more realistic. Dr. David Knecht Professor, Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. U-3125 Storrs, CT 06269-3125 On May 4, 2020, at 2:20 AM, Catalin Chiritescu <[hidden email]<mailto:[hidden email]>> wrote: *Message sent from a system outside of UConn.* ***** To join, leave or search the confocal microscopy listserv, go to: https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cdavid.knecht%40UCONN.EDU%7C56d2844bdecd440320e908d7eff39826%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C1%7C637241701845002840&sdata=JWBT8MSlHh9M3kW80xTazSbdgLRvB47wUsCWDXRbDiw%3D&reserved=0 Post images on https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=02%7C01%7Cdavid.knecht%40UCONN.EDU%7C56d2844bdecd440320e908d7eff39826%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C1%7C637241701845012833&sdata=csyg33JFpeMv%2FG3zUCs4DbzgwETROE%2FqfXdfLW38m2c%3D&reserved=0 and include the link in your posting. ***** You lose about 20-30% of light when a Phase Contrast objective is used in fluorescence. AFAIK Nikon Ti (don't know about TI2) has an external phase ring setup that you can use for Phase Contrast imaging with BF objectives - you can take it out of fluorescence light path to avoid the loss and PSF disruption. We considered that option for our Quantitative Phase Imaging SLIM systems. |
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Stanislav Vitha-2 |
Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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In reply to this post by George McNamara
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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. ***** When I did a test some years ago on our old Zeiss Axio system, using the phase contrast Plan Neofluar objective versus the non-phase objective of the same NA (Plan Neofluar or a Plan Apo, I do not remember) resulted in about 25 or 30% lower signal. This was true for a 40x/0.75 dry and a 100x/1.3 oil objectives. I think one should expect that different manufacturers or even different types of phase contrast objectives (negative phase, positive phase, anoptral phase, ...) will have different combination of attenuation (and optionally a phase shift) on the phase ring, as well as phase shift in the area outside and inside the ring where the diffracted orders would pass through in Zernike phase contrast. Stan Vitha Microscopy and Imaging Center Texas A&M University On Mon, 4 May 2020 13:03:19 +0000, Knecht, David <[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. >***** > >I suspect the higher number (20%) is if you consider loss in both excitation and emission through the phase objective, but in the excitation path, you usually have plenty of extra power to compensate for a bit of loss in excitation efficiency. So I think the lower number is more realistic. > >Dr. David Knecht >Professor, Department of Molecular and Cell Biology >University of Connecticut >91 N. Eagleville Rd. >U-3125 >Storrs, CT 06269-3125 > > > >On May 4, 2020, at 2:20 AM, Catalin Chiritescu <[hidden email]<mailto:[hidden email]>> wrote: > >*Message sent from a system outside of UConn.* > > >***** >To join, leave or search the confocal microscopy listserv, go to: >https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cdavid.knecht%40UCONN.EDU%7C56d2844bdecd440320e908d7eff39826%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C1%7C637241701845002840&sdata=JWBT8MSlHh9M3kW80xTazSbdgLRvB47wUsCWDXRbDiw%3D&reserved=0 >Post images on https://nam10.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=02%7C01%7Cdavid.knecht%40UCONN.EDU%7C56d2844bdecd440320e908d7eff39826%7C17f1a87e2a254eaab9df9d439034b080%7C0%7C1%7C637241701845012833&sdata=csyg33JFpeMv%2FG3zUCs4DbzgwETROE%2FqfXdfLW38m2c%3D&reserved=0 and include the link in your posting. >***** > >You lose about 20-30% of light when a Phase Contrast objective is used in fluorescence. AFAIK Nikon Ti (don't know about TI2) has an external phase ring setup that you can use for Phase Contrast imaging with BF objectives - you can take it out of fluorescence light path to avoid the loss and PSF disruption. We considered that option for our Quantitative Phase Imaging SLIM systems. |
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