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Home » Courses » Microscopy Series » Contrast Generation for Transmitted Light

Darkfield and Phase Contrast Microscopy

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00:00:12.02 I’m here to talk today
00:00:13.23 about the principles of darkfield and phase contrast microscopy.
00:00:20.17 I want to begin by talking about
00:00:23.10 some experiments that Frits Zernike described,
00:00:27.22 that he did during the process of discovering
00:00:32.02 phase contrast microscopy,
00:00:33.27 for which he received a Nobel Prize.
00:00:39.04 These are described in a Science article
00:00:41.05 and also in a chapter in a book
00:00:44.15 in the reference list.
00:00:46.25 And the setup of the microscope
00:00:49.22 that he used in these experiments
00:00:51.13 is shown in the diagram here.
00:00:53.27 It’s set up for Kohler illumination,
00:00:57.08 with the following adjustments.
00:01:01.02 The iris diaphragm on the condenser
00:01:04.04 is closed down to a very small spot.
00:01:07.11 And as a result of that,
00:01:09.21 that illuminates through the condenser
00:01:12.08 and produces a beam of plane wave illumination
00:01:15.04 of the specimen.
00:01:17.03 And the specimen he initially used…
00:01:19.17 that he used in these experiments
00:01:22.18 was very fine carbon particles
00:01:25.13 that were sprinkled on the surface of a coverslip
00:01:27.23 mounted onto a glass slide.
00:01:29.29 And when the plane wave hits these fine carbon particles,
00:01:33.19 a lot of the illuminating light
00:01:36.13 just passes by
00:01:38.21 and is collected by the objective lens.
00:01:40.27 And then that illuminating light
00:01:43.06 becomes focused at the objective back focal plane.
00:01:46.18 It’s also called the back aperture
00:01:49.13 of the objective, here,
00:01:52.16 as a point.
00:01:54.23 And then that light then spreads out from that point
00:01:56.29 and becomes spread…
00:01:59.22 and even illumination up here,
00:02:02.00 across the image plane, right up in there.
00:02:06.26 On the other hand, the light that the specimen scatters,
00:02:11.07 or diffracts,
00:02:13.27 which… right here…
00:02:17.04 is collected by the objective
00:02:19.23 and is focused as a real image
00:02:22.05 up here on the image plane,
00:02:24.26 as a… as a…
00:02:29.26 by the microscope objective.
00:02:32.08 And what one sees by eye, if you look in the microscope,
00:02:35.06 is these very fine specks of black carbon particles.
00:02:39.09 Then he did the following experiment.
00:02:41.19 He had used an objective
00:02:43.24 that had a special slot in it
00:02:46.02 so that in the back aperture,
00:02:48.26 where the illuminating beam is in focus,
00:02:52.10 he could insert a stop that was either
00:03:00.13 a piece of shimstock with a tiny hole in it
00:03:03.20 that just let the illuminating beam through
00:03:06.06 or a very fine sliver of shimstock
00:03:08.17 that would block the illuminating beam
00:03:11.08 but then let the diffracted or scattered light through.
00:03:14.27 And then he looked at the image that was formed under these conditions,
00:03:18.07 and this is shown here on the bottom.
00:03:20.14 So, here is the image that’s formed
00:03:23.05 without any stops in the back aperture,
00:03:25.06 and you can see the fine black carbon particles.
00:03:29.16 This is the image that’s…
00:03:31.13 he got if he just let the illuminating beam come through
00:03:34.21 and none of the scattered light —
00:03:36.15 he saw no particles at all.
00:03:39.10 And finally, if he blocked the illuminating beam,
00:03:43.01 then he did see the particles
00:03:45.25 but now in a kind of a darkfield illumination situation,
00:03:55.04 in which the particles were now bright white
00:03:58.14 against the black background.
00:04:01.00 Okay.
00:04:02.21 So, what Zernike concluded from this is that…
00:04:06.28 these experiments is that the image,
00:04:09.27 even for absorbing particles like the carbon particles,
00:04:13.01 is formed by interference of the undiffracted light
00:04:15.12 with the diffracted light;
00:04:18.16 that blocking the diffracted light results
00:04:21.25 in the loss of the image of the carbon particles,
00:04:24.11 and one just sees uniform illumination of the fields,
00:04:26.23 as predicted by Abbe a number of years ago;
00:04:30.10 and that the image formation is a result of the interference
00:04:33.09 of the diffracted light from the specimen
00:04:35.08 with the undiffracted light at the image plane;
00:04:38.23 and blocking the undiffracted light
00:04:40.28 results in a darkfield image
00:04:43.20 generated by interference of the diffraction orders,
00:04:46.27 because we’ve now lost the background light,
00:04:49.01 the illuminating light that wasn’t diffracted;
00:04:51.04 and finally, the interesting part of this
00:04:55.05 that led to phase contrast,
00:04:57.05 that absorbing objects appeared to behave like transparent objects
00:05:01.07 that have a wavelength/2…
00:05:04.19 that is, a retardation relative to the undiffracted, direct light.
00:05:09.15 That means that their… their light is 180 degrees
00:05:12.14 out of phase and destructively interferes
00:05:15.08 at the image plane
00:05:17.13 to produce the black contrast of the carbon particles.
00:05:20.20 Now, darkfield as a microscopy technique
00:05:25.11 is not done the way Zernike did in his experiment.
00:05:29.15 We want to have some resolution, normally,
00:05:31.14 in darkfield microscopy.
00:05:33.21 The principle is the same,
00:05:35.29 but one uses condensers that have an annulus of illumination
00:05:39.12 whose numerical aperture…
00:05:44.06 angle of illumination…
00:05:48.10 the numerical aperture is the refractive index…
00:05:51.26 in the specimen times the sign of the angle of illumination.
00:05:55.18 And so, you can see that special condensers
00:06:00.12 are used here to generate this high angular illumination,
00:06:03.29 and you want the angle of illumination to produce
00:06:06.15 a hollow cone of light
00:06:08.19 that’s not capable of being accepted
00:06:10.22 by the objective numerical aperture,
00:06:12.23 or objective aperture.
00:06:15.08 And as a consequence, one has a darkfield.
00:06:18.00 And then the scattered light
00:06:20.02 that’s generated by this illumination
00:06:22.29 being focused on the specimen
00:06:24.27 is collected by the objective
00:06:27.09 and then is focused as spots in the image plane.
00:06:30.09 Now, the… as I mentioned,
00:06:34.04 this requires a special condenser
00:06:36.15 if you want to do the highest resolution
00:06:39.10 light microscopy.
00:06:41.08 And these have special hemispherical mirrors
00:06:47.11 that reflect the light off to other mirrored surfaces,
00:06:50.09 over here on the…
00:06:52.10 on the outside of the objective,
00:06:55.15 that finally give you this cone of light
00:06:57.16 that’s coming out.
00:06:59.16 And you can make a numerical aperture
00:07:01.15 of this cone of light almost as…
00:07:03.15 to about 1.3.
00:07:06.06 And the highest numerical apertures of our objectives
00:07:10.21 used to be about 1.4 —
00:07:13.03 it’s getting higher now, 1.45 to 1.6.
00:07:15.24 They won’t work because
00:07:19.06 they’ll collect this cone of light.
00:07:22.00 And so, objectives actually used for darkfield
00:07:24.07 at the highest resolution
00:07:27.18 have a diaphragm in their back focal plane
00:07:30.26 so that they can pull it down
00:07:34.07 and be sure to block the illumination…
00:07:37.05 illuminating beam.
00:07:39.24 Now, the advantage of the darkfield is that it’s very…
00:07:42.01 it can be… provide high sensitivity,
00:07:44.25 and it’s possible to see scattered light from very small objects,
00:07:47.10 you know, like 25 nanometer diameter objects,
00:07:50.22 and it’s excellent for low magnification outlines
00:07:54.07 of individual cells such as sperm and Chlamydomonas
00:07:56.28 and other protozoa that scatter light
00:07:59.24 very, very strongly.
00:08:02.05 And here’s an example of a stroboscopic darkfield image
00:08:05.15 from a time lapse series
00:08:07.15 for a swimming sea urchin sperm,
00:08:09.24 where they’ve indicated one mark on the flagella
00:08:13.04 during its beat pattern in order to analyze
00:08:17.05 just what the beat pattern looks like.
00:08:19.03 And down below, here, is another time lapse series
00:08:21.06 from a stroboscopic series and darkfield
00:08:26.11 on the beating pattern of the two flagella of Chlamydomonas.
00:08:29.12 And again, this is… in order to learn about the actual waveforms
00:08:33.17 and beating of these organelles,
00:08:36.27 darkfield microscopy has been very important.
00:08:40.17 Now, it has a lot of disadvantages, though,
00:08:43.08 which means that the NA…
00:08:45.19 and this is because the NA of the condenser is less than the NA of the objective…
00:08:48.08 you get a limit in the actual resolution that you can get in the image.
00:08:52.10 And there’s also a lot of scattered light in darkfield imaging
00:08:56.19 for any thickness in specimens,
00:08:59.00 which obscures fine structural detail.
00:09:02.10 And it has poor depth of field
00:09:05.01 because the scattered light
00:09:07.20 is carried up the optical axis of the microscope,
00:09:11.05 and images of internal cellular structures
00:09:14.08 are often inaccurate and confusing
00:09:16.20 because you’re missing the fidelity
00:09:20.06 that you get by having interference with the undiffracted light.
00:09:22.27 And you often need very special, very bright light sources
00:09:26.13 to get enough light to make a good image
00:09:29.17 in darkfield.
00:09:32.23 So, darkfield has had limited applications,
00:09:35.15 and the discovery of the phase contrast technique
00:09:38.11 had a big impact in biology
00:09:42.04 because it offered a method to view living cells
00:09:45.12 with a rather simple optical method
00:09:48.22 that didn’t require such bright light,
00:09:51.19 but in addition would allow you to use
00:09:53.12 the highest resolution numerical aperture objectives
00:09:56.10 that were available.
00:09:59.11 And I show you an example, here,
00:10:02.19 of one of my squamous cheek cells.
00:10:05.20 And on the left-hand side
00:10:08.08 is the view of this cell as seen
00:10:11.09 by fully illuminating the objective aperture
00:10:14.27 with just brightfield illumination.
00:10:17.19 And this is the same cell
00:10:20.12 when the microscope is set up for phase contrast.
00:10:24.09 And so, I don’t even think…
00:10:27.20 you can just barely pick out the nucleus, right here,
00:10:30.17 in brightfield,
00:10:34.07 and here you can see lots of fine structural detail as well as where the nucleus is
00:10:38.03 and some of these dead mitochondria and other things
00:10:39.12 in my cheek cell.
00:10:42.05 So, how is this done normally?
00:10:45.13 So, if we go back to the basic experimental scheme
00:10:48.12 that Zernike used,
00:10:51.21 we want to start here with a plane wave…
00:10:54.26 a parallel beam of light that produces a plane wave
00:10:58.13 that hits the specimen,
00:10:59.14 but now our specimen is going to be a transparent specimen,
00:11:02.07 not a carbon par… an absorbing carbon particle
00:11:04.23 but a transparent specimen
00:11:07.08 that has a refractive index just slightly higher
00:11:10.07 than the background media.
00:11:15.02 And the refractive index, as you will remember,
00:11:18.14 is a measure of the speed of light.
00:11:21.06 The higher the refractive index,
00:11:23.29 the slower is the speed of light.
00:11:25.15 And this is going to be a very thin specimen,
00:11:28.26 so although it has a higher refractive index
00:11:31.18 than the background,
00:11:34.08 light will move… it won’t be very thick in this experiment.
00:11:38.17 And so, the beam…
00:11:41.06 our illuminating beam hits this specimen,
00:11:44.09 and as before, the illumination light
00:11:47.18 becomes focused at the back focal plane of the…
00:11:53.21 of the microscope, right here,
00:11:56.12 and then becomes spread out at the image plane.
00:11:59.23 And the scattered light, which isn’t very much,
00:12:03.02 from this transparent specimen
00:12:06.03 — there’s still scattered light —
00:12:09.01 is collected by the objective
00:12:12.19 and then a real image becomes in focus up here
00:12:15.26 at the image plane.
00:12:18.24 So… so here’s what happens to our illuminating wavefront.
00:12:22.20 If we look right at the wavefront,
00:12:27.13 just as it’s coming into the specimen,
00:12:30.06 we have a plane wavefront in this setup.
00:12:33.05 And here’s our little specimen, here,
00:12:36.13 of refractive index that’s larger than the background.
00:12:39.29 It has a thickness, t.
00:12:43.04 And if we look at the wavefront just after it passes through the specimen,
00:12:46.01 you can see that the wavefront has been retarded in space
00:12:49.14 relative to the surrounding media
00:12:53.19 because of the higher refractive index
00:12:56.24 producing a slower velocity of light moving through the specimen.
00:12:57.12 And for an example, you can take an organelle
00:13:02.20 that has a refractive index of maybe 1.4,
00:13:06.01 and the cytosol is 1.36, here, right?…
00:13:09.19 and if the thickness is 1 micron,
00:13:12.21 then this retardation produced by this specimen
00:13:16.10 is actually quite small.
00:13:19.15 It’s 0.04 nanometers,
00:13:22.27 which is about 1/13 of the wavelength of a green light,
00:13:26.09 so it’s very small.
00:13:29.18 And the retardation is calculated as the thickness
00:13:34.02 times the retardation of the specimen, here,
00:13:37.18 minus the retardation of the media.
00:13:40.27 So, we have a very small retardation of the wavefront.
00:13:43.29 Now, that wavefront is then imaged at the image plane,
00:13:47.15 and that imaging that’s taking place there
00:13:50.17 is the consequence of…
00:13:54.05 produces a wavefront that’s a magnified image by the objective
00:13:58.20 of that wavefront just after the specimen.
00:14:01.29 And so, we have the undiffracted light
00:14:05.10 in the background, being here.
00:14:09.06 We have the light that passed through the specimen
00:14:12.13 being retarded, here.
00:14:16.06 And based on the idea that the specimen light
00:14:19.17 is generated by the interference
00:14:23.00 between the diffracted light and the undiffracted light, alright?
00:14:26.17 If you subtract this from that,
00:14:29.29 one gets the diffracted light.
00:14:33.19 And what Zernike realized is that the diffracted light
00:14:37.12 coming from a thin transparent specimen
00:14:40.25 is approximately a quarter-wavelength out of phase
00:14:44.03 with the undiffracted light hitting the specimen.
00:14:47.19 And it is not of sufficient amplitude
00:14:50.28 to make much difference in the amplitude
00:14:54.09 of the specimen at the image plane.
00:14:57.17 What we have here is Zernike’s solution for phase contrast
00:15:01.12 using that quarter-wavelength information
00:15:05.02 about thin transparent specimens
00:15:09.26 for the diffracted light.
00:15:13.06 So, he set up… he made a circular glass disc,
00:15:18.15 in which in the center he milled or ground
00:15:26.07 — or however he did it —
00:15:29.23 an indentation that made this part of the plate thinner,
00:15:34.14 so that for the undiffracted light
00:15:38.04 coming through the objective back focal plane,
00:15:41.19 at this place here, right?,
00:15:45.22 it received a quarter-wavelength less retardation
00:15:49.06 than for the scattered or diffracted light
00:15:52.24 that is unfocused at this point
00:15:56.13 in the back focal plane.
00:16:00.05 And the sum of those two would produce the half-wavelength
00:16:03.28 he needed in order to get destructive interference contrast
00:16:07.18 at the image plane.
00:16:08.03 And in order to bring the intensity
00:16:09.25 of the undiffracted light
00:16:13.13 down to match the intensity…
00:16:17.04 or to come close to the intensity…
00:16:21.01 of the weak intensity of this… of the diffracted light,
00:16:23.17 this hole, here, was also coated with a material
00:16:27.04 that attenuated the intensity of the illuminating light beam
00:16:32.00 to approximately, oh, 75% or so of what its normal level is.
00:16:41.23 And as a result, at the image plane
00:16:46.08 we now get the interference of the diffracted light…
00:16:52.15 the undiffracted light with the diffracted light,
00:16:57.13 and because they’re now a half-wavelength
00:17:01.09 or 180 degrees out of phase,
00:17:05.00 this produces a dark contrast
00:17:09.09 for the transparent specimen that you saw in our example
00:17:13.05 of my cheek cell.
00:17:16.28 So, pretty simple.
00:17:17.18 Now, the problem with the Zernike test system
00:17:19.11 is that there is no numerical aperture
00:17:23.10 in the illumination from the condenser.
00:17:27.02 And as you probably have learned,
00:17:31.02 the resolution in transmitted light microscopy
00:17:35.04 is equal to the wavelength of light divided
00:17:39.02 by the numerical aperture times point…
00:17:43.29 the wavelength of light divided by the numerical aperture of the objective
00:17:46.15 plus the numerical aperture of the condenser.
00:17:50.10 So, in his system, he was only getting the resolution
00:17:54.16 that was produced by the numerical aperture of the objective.
00:17:58.12 And in phase contrast microscopy,
00:18:02.07 the way it’s implemented now with modern lenses
00:18:06.27 is to use an annular… an annulus of illumination in the condenser
00:18:10.25 so that we have an annular cone of light
00:18:15.21 that illuminates the specimen.
00:18:16.21 That cone of light is collected by the objective
00:18:18.00 and passes through this phase…
00:18:19.16 what’s called the phase plate
00:18:21.01 that’s in the back focal plane
00:18:22.21 or back aperture of the objective.
00:18:25.03 And it’s a ring, now, instead of a spot, right?
00:18:28.10 And then the light coming through this ring,
00:18:30.28 the illuminating beam,
00:18:32.20 then spreads out up here on the ceiling
00:18:35.05 where the image plane is,
00:18:37.13 at the same point where the specimen-diffracted light image
00:18:42.25 comes into focus, right?
00:18:45.14 So, the diameter of this annulus, here,
00:18:50.03 of illumination
00:18:52.02 and the diameter of the phase ring
00:18:54.09 is typically chosen to be about
00:18:56.21 a half of the aperture of the objective,
00:19:01.05 which means the illumination from the condenser
00:19:04.09 is about half of the numerical aperture
00:19:06.09 of the objective.
00:19:09.07 Now, if I take a tel…
00:19:11.23 out the ocular in the microscope
00:19:13.10 and put a telescope in so I can form…
00:19:17.03 focus on the objective back focal plane,
00:19:19.12 I can see for a phase contrast objective
00:19:22.01 the periphery, over here,
00:19:24.21 of the objective aperture…
00:19:27.22 and actually the objective aperture may be a little further out than this,
00:19:31.15 because this is the…
00:19:33.18 this is as much as my condenser,
00:19:36.20 which has a lower NA than the objective,
00:19:38.16 is able to illuminate that aperture.
00:19:40.29 But then right here is the phase ring,
00:19:43.09 and we see it illuminated…
00:19:47.15 we see it in the objective back focal plane
00:19:49.16 because it’s absorbing light
00:19:54.01 for the illumination light that goes through it
00:19:57.00 in order to attenuate that light.
00:19:58.16 So, that’s the phase ring.
00:20:00.05 And so, all phase contrast objectives
00:20:01.28 have this phrase ring built into the objective
00:20:04.11 at their back focal plane or back aperture.
00:20:08.21 Now, in alignment for phase contrast…
00:20:11.26 we mentioned before that the phase ring diameter
00:20:15.07 is about 50% of the numerical aperture
00:20:18.14 of the objective
00:20:20.03 so that the condenser, in combination with the phase annulus,
00:20:24.15 has to produce the proper cone of light
00:20:28.03 with the right numerical aperture
00:20:30.02 in order to become in focus…
00:20:32.16 that cone to become in focus at the position
00:20:35.22 where the phase ring is in the objective aperture.
00:20:38.13 And so, the condenser annulus diameter
00:20:42.08 must be chosen for a particular condenser lens
00:20:46.21 to produce that correct cone of illumination.
00:20:54.00 And phase objectives are classified
00:20:56.06 as phase I, II, III, and IV
00:20:58.13 as they go to higher numerical apertures,
00:21:02.09 or it’s now Ph1, 2, 3, and 4,
00:21:06.12 indicating matching condenser annuli…
00:21:12.00 to be labeled on them.
00:21:13.24 And the condenser annulus
00:21:16.25 must be aligned with the phase ring,
00:21:18.22 and there’s usually adjustment screws
00:21:21.03 that allow this to take place.
00:21:22.24 So, here’s an inverted microscope
00:21:24.28 that we use for…
00:21:26.28 often for tissue culture or microinjection.
00:21:29.06 Here’s the holder for a needle
00:21:33.02 for microinjecting tissue culture cells.
00:21:35.22 And we observe those cells using phase contrast.
00:21:38.02 And in this case, it’s a long working distance
00:21:40.25 condenser lens.
00:21:42.09 And then at the front focal plane of the condenser lens
00:21:46.13 is this turret, and the turret has…
00:21:50.22 it can be rotated,
00:21:52.27 and it has openings
00:21:57.10 for I think three different phase annulus... annuli,
00:22:02.27 as well as one condenser diaphragm,
00:22:05.08 if you want to just use full brightfield illumination.
00:22:08.29 These screws here on the microscope
00:22:12.09 are used to center the condenser lens properly,
00:22:16.13 which is used to adjust the microscope
00:22:18.14 for Kohler illumination
00:22:20.28 and to center the image in the field diaphragm.
00:22:24.13 There’s also screws that you use with an allen wrench
00:22:28.20 to center each of the annuluses
00:22:32.08 that are inside the turret, here.
00:22:34.15 And I think I have a picture of what that looks like.
00:22:37.02 So, if you take that…
00:22:39.01 take this off and look at it,
00:22:41.15 this is the… this is the position of the wheel
00:22:44.08 that contains the diaphragm,
00:22:46.22 and then this is the phase III position, here,
00:22:51.13 and then this is… over here is the phase II,
00:22:56.11 and then down over here is the phase I.
00:22:59.00 Notice as we go to…
00:23:01.07 this is the higher NA
00:23:03.04 and this is the lower NA,
00:23:04.26 and you can see, for the same condenser lens,
00:23:06.23 how much bigger the diameter… whoops…
00:23:09.07 of the higher NA annulus has to be to match
00:23:12.25 the 50% of the numerical aperture
00:23:15.01 of the high NA objective
00:23:17.07 compared to that for the low NA objective.
00:23:20.07 So, you have to choose the correct one.
00:23:23.11 And by the way, if you switch condensers
00:23:26.02 and go to a high NA condenser,
00:23:28.07 there will be a different annulus for that high NA condenser
00:23:30.08 than the one you use for the low NA condenser, right?
00:23:33.26 And so, you need to just make sure with the manufacturer
00:23:37.14 that you have them color-coded properly
00:23:39.24 or something like that.
00:23:41.17 Now, here’s the alignment problem.
00:23:43.09 And so, if you look, as we did before,
00:23:47.05 with a telescope at the objective back aperture,
00:23:49.21 we have no annulus, we can see the phase ring.
00:23:52.22 And I just sort of drew a line where I roughly think,
00:23:57.29 maybe, the actual periphery of the objective aperture is.
00:24:00.24 It’s maybe here, right?
00:24:03.08 And then, if we have an annulus
00:24:05.29 but it’s misaligned,
00:24:08.07 you can see that light is now coming through the aperture
00:24:12.13 in regions that don’t have the phase ring,
00:24:16.14 so the light isn’t being attenuated
00:24:18.22 nor is it being properly phase advanced
00:24:23.26 relative to the diffracted light.
00:24:26.01 And then down here, we’ve…
00:24:28.03 with this one down… way down here at the bottom,
00:24:32.03 now we have the ring centered properly and passing…
00:24:38.01 totally contained within the annulus…
00:24:41.17 the image of the annulus is totally contained within the ring of the objective,
00:24:44.28 and you will get a proper image.
00:24:47.13 So, here we have no phase annulus at all,
00:24:52.10 which is our brightfield image.
00:24:56.22 Here we have a misaligned annulus —
00:25:00.10 ugly picture.
00:25:03.05 And now we have an aligned annulus,
00:25:04.19 and we get a really nice picture.
00:25:06.16 So, once you get phase contrast lined up on the microscope,
00:25:08.18 you usually don’t have to ever change the alignment,
00:25:10.22 and you just simply have to switch the objectives
00:25:13.20 and switch the turrets to make sure
00:25:16.11 that everybody’s lined up and matched.
00:25:18.05 Now, this is a plot that they do for microscope optics.
00:25:23.12 So, essentially the…
00:25:29.00 the contrast that can be generated
00:25:32.06 as a function of the resolution
00:25:39.16 or maximal spatial frequency
00:25:43.10 that can be resolved by an objective-condenser combination.
00:25:50.05 And for this, 100 is the maximum resolution
00:25:52.14 for this particular objective.
00:25:54.20 And if you had brightfield illumination,
00:25:57.00 this is the potential contrast
00:26:00.11 — this solid line curve, here —
00:26:02.12 that you would get, where the…
00:26:04.18 where the numerical aperture of condenser illumination
00:26:07.08 is equal to the numerical aperture of the objective elimination.
00:26:11.24 Now, for that same objective, in phase contrast…
00:26:15.07 the contrast curve is this green line,
00:26:18.06 and you can see that it peaks
00:26:23.01 at a lower spatial frequency, or a lower resolution,
00:26:27.22 and then maximizes out down here
00:26:30.26 at about 75% of what you could get
00:26:33.24 in a fully illuminated objective aperture,
00:26:38.03 if you had the contrast there to see it, right?
00:26:40.28 And that’s… this is because, in fact,
00:26:44.02 the condenser numerical aperture
00:26:49.06 is typically 50% of the objective numerical aperture,
00:26:52.21 so you don’t really expect to get much above 75%.
00:26:56.11 So, that’s a kind of formal way…
00:26:59.13 and so things tend to be more highlighted in phase contrast
00:27:04.00 that aren’t quite as fine in structure
00:27:06.21 as what might…
00:27:09.05 you might consider to be the limit of resolution
00:27:11.12 of a particular microscope objective.
00:27:15.26 Besides not being able to achieve
00:27:17.27 the maximum resolution
00:27:20.24 that objective numerical aperture would allow you,
00:27:25.00 the other problem with phase contrast
00:27:28.11 is that the phase ring is larger than the…
00:27:33.20 than the phase annulus.
00:27:37.00 So, the illumination is…
00:27:39.06 and as a result, lower…
00:27:41.07 low angle diffracted light,
00:27:43.17 which contains information
00:27:46.04 about low spatial frequencies in the specimen,
00:27:48.18 is attenuated by the phase ring
00:27:51.29 and doesn’t get to the image plane,
00:27:54.08 and this produces halos around phase objects
00:27:57.18 like the nucleus, here.
00:28:00.09 And in thicker specimens, those halos
00:28:02.28 propagate up and down through the specimen
00:28:05.05 and cause confusion, much like in darkfield.
00:28:07.14 Phase contrast imaging
00:28:10.02 is one of the more popular optical modes in cell biology
00:28:13.21 because once you have your microscopes aligned properly
00:28:17.18 it’s relatively easy to use,
00:28:19.09 and it provides a…
00:28:22.26 and its impact has been great,
00:28:24.29 because it in particular
00:28:29.03 allows you to look at dynamic behavior of movements in cells
00:28:33.02 that aren’t too thick.
00:28:35.03 And in this case, this is an example.
00:28:37.00 What I have here is a mitotic PtK1 cell,
00:28:40.27 which is an epithelial cell in mitosis.
00:28:44.02 And we… this is part of a time lapse movie,
00:28:51.06 and these are the chromosomes,
00:28:53.12 of which there are 12 or 13,
00:28:55.07 depending on whether it’s male or female.
00:28:58.21 And the mitotic spindle forms
00:29:00.15 between the spindle poles —
00:29:02.11 one is here and another one is up here.
00:29:05.18 And so, this cell has just entered into prometaphase,
00:29:08.25 and the movie will show you, from prometaphase,
00:29:11.02 the alignment on the metaphase plate
00:29:13.13 and then into anaphase and then into cytokinesis.
00:29:17.07 The worm-like structures up here in the cytoplasm
00:29:20.17 are mitochondria,
00:29:22.08 and then the periphery of the cell —
00:29:24.15 here’s one edge here,
00:29:26.02 and you can see the other edge down here,
00:29:28.17 and so forth.
00:29:31.15 And when we do time lapse imaging,
00:29:33.13 we typically use a 100-watt quartz halogen illuminator,
00:29:39.00 a standard white light illuminator,
00:29:41.12 and then a good heat reflection filter
00:29:43.22 and a wideband green light filter
00:29:48.24 with high transmission efficiency.
00:29:50.12 And the cells… this illumination virtually can be…
00:29:55.21 we can film the cells for long periods of time
00:29:57.21 without any damage to the cells.
00:30:01.10 So, here we go.
00:30:04.25 So, here’s metaphase,
00:30:06.13 and then we’re into anaphase,
00:30:08.13 and then we’re into cytokinesis.
00:30:10.24 Yeah.
00:30:13.02 Okay, so this could be routinely used
00:30:15.12 to screen siRNA knockdowns
00:30:18.27 or other things like that,
00:30:21.05 in terms of how they affect chromosome movement
00:30:22.22 or spindle assembly
00:30:24.13 or what have you, fairly easily.
00:30:27.02 The other thing about phase contrast
00:30:30.08 that’s been particularly useful
00:30:34.09 is that it is convenient to combine with epifluorescence microscopy
00:30:37.06 so that you can use the major advantages of fluorescence
00:30:42.18 — and particularly for genetically encoded fluorophores
00:30:46.20 like GFP and its relatives —
00:30:49.04 to view the locations of specific proteins
00:30:53.03 and then use phase contrast
00:30:56.06 to see where those locations are
00:30:58.29 relative to the structural dynamics of cells.
00:31:01.29 And the convenience comes from the fact that you…
00:31:05.15 all you need is two shutters:
00:31:08.22 one shutter to open and close trans-illumination
00:31:13.10 and the other shutter to open and close epi-illumination.
00:31:16.22 And in the illumination that you use for phase contrast,
00:31:19.22 you use the same color light
00:31:22.20 as the fluorescence-emitted light,
00:31:25.08 so you need a filter there
00:31:28.22 that matches the fluorescence-emitted light.
00:31:30.15 And so, you just have to…
00:31:32.16 every time you, let’s say, take time lapse pictures,
00:31:34.17 you first take a phase picture
00:31:36.05 and then open the shutter and close it,
00:31:38.21 and then open the fluorescence shutter and close it,
00:31:42.07 and then wait your delay
00:31:44.28 and take the next one and the next one and the next one.
00:31:48.21 So, the downside is that that phase ring absorbs
00:31:51.28 about 15% of the fluorescence light,
00:31:54.19 and so if your fluorescence objects are really weak,
00:31:57.03 that can be a little bit of trouble.
00:31:59.08 And the phase ring slightly spreads out the Airy disc,
00:32:02.17 reducing slightly the resolution
00:32:04.21 that you get in fluorescence.
00:32:07.08 But for many applications,
00:32:09.09 that’s not really very severe.
00:32:11.24 So, for this inverted scope, which is a different one,
00:32:15.17 we have the shutter up here for the trans-illumination
00:32:18.14 and then we have the shutter right here
00:32:21.28 for the epi-illumination.
00:32:23.29 And then we’ve selected the filter for GFP
00:32:26.08 that’s in here.
00:32:27.25 And then up here,
00:32:30.02 we have both the heat reflection plus we have a green filter
00:32:33.16 for the 510 nm emission
00:32:35.29 that comes from the GFP.
00:32:38.08 And then that we used to make this movie, here,
00:32:40.16 which is again a PtK cell in mitosis,
00:32:42.28 but this cell is now expressing
00:32:46.13 a GFP fused to a kinetochore protein called Cdc20.
00:32:52.15 And so, you can see the kinetochores
00:32:54.17 marked with the green fluorescence of Cdc20,
00:32:57.05 and you can see that cell outlines
00:32:59.15 and the chromosomes by phase contrast.
00:33:06.27 And we’ve pseudo-colored this movie
00:33:08.28 so that the phase contrast image is generated…
00:33:11.10 is in the red channel
00:33:13.24 and the GFP image is in the green channel.
00:33:16.10 And so here it goes.
00:33:18.06 This cell is almost in metaphase,
00:33:20.01 and what it highlights is the oscillatory nature
00:33:22.19 of kinetochore motility.
00:33:24.20 When aligned at metaphase,
00:33:27.19 kinetochores can switch back and forth
00:33:29.24 between polymerizing and depolymerizing
00:33:32.21 their kinetochore microtubules,
00:33:34.12 and then in anaphase they depolymerize them,
00:33:37.03 and that helps carry chromosomes to the poles.
00:33:39.23 And so, that can be used as an assay
00:33:41.20 for studying proteins that are involved
00:33:44.03 in that force-generating mechanism for chromosome movements.

This Talk
Speaker: Ted Salmon
Audience:
  • Researcher
Recorded: July 2012
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Talk Overview

This lecture describe the principles of dark field and phase contrast microscopy, two ways of generating contrast in a specimen which may be hard to see by bright field. The lecture describes how the phase rings work to generate interference between the diffracted and undiffracted light.

Questions

  1. When Zernicke blocked the illuminating light at the back aperture of the objective, he saw
    1. Both the field and the particles (specimen) were bright
    2. The field was dark and the particles were bright.
    3. The field was bright and the particles were dark.
    4. Both the field and the particles (specimen) were dark
  2. True or false: In transmitted light microscopy, an image is formed by undiffracted light interfering with diffracted light from the specimen.
  3. Which of the following might be a good specimen to examine by dark field microscopy?
    1. A mammalian tissue culture cell
    2. A thin histological section of liver
    3. 25 nm microtubules polymerized in vitro
    4. Pollen grains
  4. Which of the following statements is false about dark field microscopy?
    1. Poor depth of field
    2. Is generally a higher resolution technique than phase microscopy
    3. Requires a very bright light source
    4. Can use both dry and oil condensers and objectives
  5. In phase microscopy, the function of the phase ring in the objective lens is to:
    1. Attenuate and produce ¼ l phase retardation of the illuminating light
    2. Increase the relative brightness and produce a ¼ l phase retardation of the illuminating light
    3. Attenuate and produce a ¼ l phase advance of the illuminating light
    4. Attenuate and produce a l phase advance of the illuminating light
  6. True or false. One phase condenser annulus will suffice for 20x, 40x, and 60x objective lenses on most microscopes.
  7. What produces halos in a phase image?
  8. In dark field microscopy, why must the N.A. of the objective be less than the N.A. of the condenser?
  9. If you are just performing fluorescence microscopy, why is a phase objective somewhat less desirable than the comparable non-phase objective (same mag, NA, etc.)?
  10. Which of the following statement is false about phase contrast microscopy?
  1. The phase ring retards or advances the phase of the illuminating light relative to the scattered light by half of a wavelength.
  2. The phase ring reduces the intensity of the illuminating light
  3. Phase rings are built into the objective lens
  4. Phase microscopy requires a matching annulus in the condenser turret.

Answers

View Answers
  1. B
  2. True
  3. C: Dark field, which collects diffracted light and focuses it onto a dark background, produces very nice high contrast images of microtubules in vitro, which are small but simple objects.  The other objects are more complex and thicker and generate a tremendous amount of scattered light which produces a bright but information poor image of cells or tissues.
  4. B: Dark field has limited resolution since the objective NA must be lower than the NA of the condenser.  In practice, this means using objectives with NA below 1.  A phase objective does not have such restrictions and one can use higher NA lens.
  5. C: In phase microscopy, you want the light traveling through the transparent sample to interfere with the illuminating light to generate contrast.  A thin sample typically produces a ¼ l phase retardation of the illuminating light.  If illuminating light is phase advanced by ¼ l, then the specimen and illuminating light will be ½  l out-of-phase, resulting in destructive interference and a darker image than the background.  The illuminating light also is attenuated to make it closer in intensity to the weaker diffracted light from the specimen and thus generate better contrast.
  6. False: You need a specific annulus on the condenser that will match the phase ring on each objective.  Then, the illuminating light will be confined to and be modified by the phase ring.  Furthermore, the condenser annulus must be adjusted (with set screws) so that it is aligned with the objective phase ring in the light path.
  7. Diffracted light from the specimen hitting the annulus of the phase ring.
  8. So that the cone angle of the illuminating light (that angle being determined by the N.A. of condenser) is not captured by the objective. Then the field will appear dark because the illuminating light is not being imaged.  Some of the light diffracted by the specimen, however, will be collected by the objective.
  9. Some of the fluorescence light from the specimen is attenuated by the objective phase ring, resulting in a loss of valuable photons if the image is dim.  The phase ring also slightly spreads out the Airy disk.
  10. A

Speaker Bio

Ted Salmon

Ted Salmon

Ted Salmon is a Distinguished Professor in the Biology Department at the University of North Carolina. His lab has pioneered techniques in video and digital imaging to study the assembly of spindle microtubules and the segregation of chromosomes during mitosis. Continue Reading

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Reader Interactions

Comments

  1. abubakar sulaiman koya says

    October 17, 2021 at 3:23 pm

    compare and contrast between dark field and phase contrast mocroscopy

    Reply

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