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Dark Field, Phase Contrast, Polarization, and Differential Interference Contrast (DIC) Microscopy

00:00:11.04 Hi, I’m Ted Salmon,
00:00:13.01 and I want to provide, today,
00:00:14.25 an introduction to four modes of transmitted light microscopy
00:00:22.17 that give contrast to transparent specimens
00:00:26.23 which are very important in cell biology and other biomedical fields,
00:00:32.01 particularly for live cell imaging,
00:00:35.07 and these are darkfield, phase contrast,
00:00:36.24 polarization, and differential interference contrast,
00:00:39.25 which is also called DIC microscopy.
00:00:44.11 Now, microscopes are designed with three essential functions.
00:00:47.29 One is the magnification,
00:00:50.26 and that is… to make structural detail visible to the detector,
00:00:54.24 which is the eye or the camera,
00:00:58.02 we need sufficient magnification not to be limited
00:01:00.26 by the resolution of the detectors.
00:01:02.21 The second is microscopes are designed
00:01:05.26 to provide resolution that is only limited by the wavelength of light
00:01:09.11 and not by lens… optical aberrations,
00:01:13.29 and these are mostly taken care of by the manufacturer of microscopes.
00:01:19.01 And the third is contrast.
00:01:22.03 Most of our biological specimens are transparent at visible wavelengths,
00:01:26.05 and some means of generating contrast
00:01:29.25 for a specimen’s structural detail
00:01:32.00 is required.
00:01:33.21 Contrast in light microscopy falls into two categories.
00:01:36.25 One is absorption contr…
00:01:39.10 absorption-based mechanisms,
00:01:41.07 and for over 100 years, or 150 years,
00:01:46.26 color dyes were used to give contrast
00:01:50.18 to specimen structural detail —
00:01:52.27 this is often called histology.
00:01:55.00 And the modern way is to use epifluorescence
00:01:56.20 with fluorescent probes,
00:01:58.13 and genetically fluorescent probes,
00:02:00.02 that generate contrast.
00:02:02.07 But equally important are mechanisms
00:02:05.13 for generating contrast,
00:02:07.00 particularly for live cell imaging,
00:02:09.19 that are based on refractive index differences
00:02:12.04 in specimen structural detail…
00:02:14.08 between that structural detail and the background,
00:02:17.25 whether it be the media or the cytosol.
00:02:20.15 And please note that refractive index, “little n”,
00:02:26.14 controls the speed of light.
00:02:28.20 That is, the velocity of light is equal to the speed of light
00:02:31.27 that it would have in a vacuum
00:02:33.19 divided by the refractive index of the particular media,
00:02:36.28 or that part of the media or that part of the specimen,
00:02:40.14 that the light is moving through.
00:02:42.03 So, the higher the refractive index,
00:02:43.22 the slower is the speed of light.
00:02:46.23 Now, specimen details with higher refractive index than the background
00:02:52.29 produce a retardation of transmitted light.
00:02:56.00 And this is shown in this drawing, here,
00:02:57.26 where we have a plane wave illuminating front
00:03:02.04 coming along and hitting our little transparent specimen…
00:03:04.17 very thin specimen…
00:03:06.06 which has a refractive index bigger than the background.
00:03:08.20 And if we look at that wavefront
00:03:10.26 just past that specimen,
00:03:12.24 it has been delayed by the fact that the higher refractive index
00:03:16.08 has slowed down that wavefront, right?,
00:03:18.25 relative to the wavefront in the background medum.
00:03:22.05 And this retardation of the wavefront
00:03:24.16 is given by the thickness of the specimen
00:03:27.28 times the refractive index of the specimen
00:03:30.01 minus the refractive index of the media in the background.
00:03:33.24 And for an example organelle, up here,
00:03:37.02 if it has a refractive index of 1.4
00:03:39.11 — pure protein is about 1.5,
00:03:41.09 but let’s say this organelle is 1.4 —
00:03:44.07 and the cytosol is about 1.36-1.38,
00:03:47.13 and the thickness is 1 micron,
00:03:50.17 then the retardation is 0.04 nm,
00:03:52.13 which is about 1/13 of the wavelength of green light,
00:03:55.15 so it isn’t very much, alright?
00:03:57.18 Now, if we illuminate our transparent specimen
00:04:01.23 in transillumination in the microscope,
00:04:07.10 even though this is transparent,
00:04:09.01 the differences in the refractive indices
00:04:11.22 of the specimen’s structural detail
00:04:15.05 cause diffraction of light,
00:04:18.07 and the diffraction of light…
00:04:20.28 diffracted light, or scattered light,
00:04:23.02 is collected by the objective aperture
00:04:25.04 and focused up here onto the image plane
00:04:28.12 to make an image of the specimen itself.
00:04:35.12 Our illuminating beam is actually out of focus on the image plane,
00:04:40.23 and it’s the interference of the illumination beam
00:04:44.19 and transmitted light
00:04:47.09 with this scattered or diffracted light
00:04:49.26 that actually generates our specimen image.
00:04:52.18 And for transparent specimens,
00:04:55.05 this interaction generates a transparent image
00:04:59.10 from the transparent specimen
00:05:01.29 if not otherwise modified.
00:05:04.28 Now, in darkfield microscopy,
00:05:07.14 one way to get contrast for that transparent specimen
00:05:09.24 is to illuminate the specimen
00:05:12.28 with a cone of light whose aperture
00:05:16.12 —or the angle of the cone of light —
00:05:19.01 is bigger than the angle of the scattered light
00:05:22.25 that could be collected by the objective,
00:05:25.13 the so-called objective aperture.
00:05:30.14 And this is done with condensers,
00:05:32.16 often specialized condensers called cardioid condensers,
00:05:37.06 so that the numerical aperture of the illumination, here,
00:05:41.25 of the specimen produces this cone of light
00:05:44.25 that can’t enter into the objective.
00:05:47.01 The objective collects just the scattered light
00:05:48.27 and generates a bright image
00:05:52.05 of the transparent specimen from that scattered light.
00:05:55.24 Now, the advantage of darkfield
00:05:57.27 is that you can produce a high sensitivity
00:06:00.01 because you have a very black background,
00:06:02.11 and in fact you can…
00:06:04.05 if you have a bright enough light source,
00:06:06.00 you can see the very weakly scattered light
00:06:09.08 that comes from very small objects
00:06:11.13 like 25-nm diameter microtubules,
00:06:14.07 or the flagella of a bacteria
00:06:17.08 can be imaged in darkfield.
00:06:19.11 It’s excellent for magnification…
00:06:21.21 low magnification outlines of individual cells
00:06:25.02 such as sperm and flagella… and their flagella.
00:06:29.00 And I have a movie, here,
00:06:32.11 showing the beating of a sea urchin sperm flagella.
00:06:36.18 This is the head of the sperm with the compacted chromatin and DNA from…
00:06:45.08 and this is the long flagella, here,
00:06:48.08 which is about 250 nm in diameter.
00:06:53.00 And this shows you very nicely in darkfield,
00:06:55.11 which is slightly pseudocolored in this image here,
00:06:58.04 the beating of the flagella.
00:07:02.13 It’s been slowed down in real time.
00:07:05.10 I think they beat at about 30 Hz,
00:07:08.00 so it’s quite fast,
00:07:09.27 so it’s been slowed down,
00:07:12.02 but it’s very clearly seen in darkfield microscopy.
00:07:15.06 Now, the disadvantage of darkfield
00:07:17.22 is that the resolution in the image
00:07:20.28 is limited by the need for the numerical aperture of the condenser
00:07:24.03 to be greater than the numerical aperture of the objectives,
00:07:27.02 and so the objective numerical apertures
00:07:29.04 are typically about 0.8-1 at best,
00:07:32.28 and not the 1.4 which is typical
00:07:36.07 of the high-resolution objectives
00:07:38.21 often used in our microscopy.
00:07:43.03 Specimen… scattered light in thick specimens
00:07:47.15 sort of obscures structural detail.
00:07:50.11 Particularly for thicker specimens,
00:07:52.19 there’s poor depth of field,
00:07:55.01 and images of intracellular structures are often inaccurate,
00:07:57.24 because you’re missing information
00:08:00.16 that comes from the actual interference
00:08:03.06 with the undiffracted light.
00:08:05.29 And it often needs very bright light sources to be used properly.
00:08:09.08 So, the invention… the discovery and invention
00:08:12.23 of the phase contrast technique by Zernike in the 1930’s,
00:08:15.27 and it’s implementation in light microscopy
00:08:18.27 in the 1940’s,
00:08:21.10 was a major advance
00:08:24.12 because it allowed you to get very nice dark contrast
00:08:27.00 for transparent structural detail,
00:08:30.04 like this squamous epithelial cell from my cheek, here,
00:08:33.15 and observed with a full objective numerical aperture
00:08:38.20 so that you can begin to see the fine ridges
00:08:41.28 along the edges of my cell, here.
00:08:44.17 But this is the same cell observed just with…
00:08:48.19 not in the phase contrast mode
00:08:51.11 but just in the widefield mode,
00:08:53.17 and I think you can just barely make out the nucleus,
00:08:56.00 so you can see how transparent our cells actually are
00:08:59.27 to green light.
00:09:02.03 So, how does phase contrast work?
00:09:04.19 So, phase contrast works by putting in the back focal plane of the objective
00:09:10.27 a phase plate,
00:09:14.19 which is a glass disc in which there’s a ring
00:09:17.26 that’s etched out or cut into the disc
00:09:21.14 so that the thickness at the base of the ring
00:09:24.00 is thinner than for the rest of the disc
00:09:27.09 that covers the back aperture
00:09:32.15 or the back focal plane of the objective.
00:09:36.21 And the specimen is illuminated by a cone of light
00:09:39.01 generated by an annulus
00:09:41.11 that’s down near the condenser diaphragm plane.
00:09:43.20 This illuminates the specimen,
00:09:46.14 but the objective collects this cone of light
00:09:49.10 and focuses this light onto this phase ring, right?…
00:09:53.20 so, these are conjugate planes
00:09:56.07 in the microscope path,
00:09:59.05 and the light is focused right on the phase ring,
00:10:01.22 and in the base of the phase ring
00:10:04.04 there’s some aluminum or other materials
00:10:06.17 that attenuate this illuminating beam
00:10:07.26 so that by the time it comes up to the image plane,
00:10:10.21 which is up toward the ceiling here,
00:10:13.08 the intensity of the illumination beam is much weaker
00:10:16.06 than what it was when it hit the specimen down here.
00:10:19.27 In addition, what this phase ring does is
00:10:24.23 advance the phase of the illuminating beam by a quarter-wavelength
00:10:27.23 such that when it adds to the quarter-wavelength retardation
00:10:31.10 that occurs for scattered light from a specimen
00:10:34.19 produces at the image plane a half-wavelength difference
00:10:39.10 between the scattered light and the illuminating beam
00:10:42.21 so that you can get destructive interference
00:10:46.12 and therefore produce dark contrast
00:10:49.00 that we see in phase contrast.
00:10:52.09 So, phase contrast is fairly simple.
00:10:55.06 You have to buy objectives that have these phase plates and rings
00:10:58.07 built into their back focal plane
00:11:03.00 and have condensers with turrets
00:11:04.15 so you can select the proper diameter phase annulus,
00:11:07.16 and this is all that’s necessary to get phase contrast,
00:11:10.21 and so it’s implemented from the simplest microscopes used for tissue culture
00:11:14.06 all the way to the most sophisticated microscopes
00:11:18.11 used for detailed cell analysis.
00:11:21.19 I’d like to first show you this fun example of phase contrast microscopy
00:11:24.27 in this blood smear of a human white blood cell
00:11:28.20 that’s chasing a bacterium,
00:11:32.14 and I’ll step over here so you can see it.
00:11:35.21 So, here’s the polymorphonucleocyte,
00:11:39.01 and it’s chasing this bacterium,
00:11:40.26 which you can see right here.
00:11:44.00 There we got. So, you can now watch it,
00:11:46.27 and I’ll come along with you.
00:11:50.01 It’s been montaged so you can just kind of follow it as it goes.
00:11:52.28 And now we’re moving over into here,
00:11:56.11 and now it’s right there.
00:11:59.20 The bacterium is still away.
00:12:01.25 Finally, up here, right up in there…
00:12:04.26 you can see that phagocytosis has just taken place,
00:12:08.14 and it’s eaten the bacterium.
00:12:11.19 Heh heh heh… I like that.
00:12:15.05 Another routine use of phase contrast
00:12:18.12 is assaying the problem of wound healing,
00:12:21.28 and this is studied in tissue culture,
00:12:25.04 where cells are grown out to confluency on the surface of a dish,
00:12:28.19 a coverslip surface or a dish.
00:12:31.26 And then a little rubber rod is used to create a wound,
00:12:36.04 and then timelapse phase microscopy is used to study
00:12:40.13 how cells move in to seal up this wound
00:12:42.28 as well as how they’re activated to go through cell divisions
00:12:47.02 to replace the cells that are needed
00:12:50.24 and, eventually, how cells contact each other
00:12:54.07 and use that contact information to stop further cell motility
00:12:57.10 and make a nice confluent layer again.
00:13:01.06 If you’re a cancer cell line, you may not have that contact inhibition
00:13:04.24 and therefore get overgrowth of cells and so forth,
00:13:08.17 and it becomes an assay for factor
00:13:11.23 s involved with normal wound repair.
00:13:14.26 Now, here’s a nice movie of mitosis
00:13:18.02 where we get to see the chromosome segregation
00:13:19.00 and the dynamics of chromosome segregation,
00:13:21.04 and phase contrast is used a lot to look at this type of activity,
00:13:24.20 for people studying how cells
00:13:29.19 segregate the duplicated genome into daughter cells.
00:13:33.18 Now, a third form of transmitted light microscopy
00:13:41.11 that takes advantage of refractive index properties of the specimen
00:13:47.16 is polarization microscopy.
00:13:51.01 But here, rather than… like in phase contrast,
00:13:54.10 looking at the difference between the refractive index in the background
00:13:58.06 giving, in this case, a dark contrast for this clumps of ribosomes, alright?,
00:14:02.08 in polarization microscopy,
00:14:05.14 it’s the difference in refractive index in different directions in the specimen
00:14:08.22 — that is, an optical anisotropy represented by two different refractive indices,
00:14:12.25 one for light that’s vibrating with its electric vector this way
00:14:18.23 and one for light vibrating with its electric vector in a perpendicular direction.
00:14:23.27 And for this isolated mitotic spindle, here,
00:14:27.22 viewed both in phase contrast and polarized light,
00:14:31.16 the birefringence — that is, the two difference refractive indices —
00:14:34.27 arise from a form anisotropy due to the microtubules,
00:14:38.18 the 25-nm diameter microtubules that are all lined up
00:14:42.04 roughly parallel to each other
00:14:45.13 that make up the spindle fibers within this central spindle.
00:14:50.04 They also make up the fibers of the aster arrays,
00:14:54.02 and these aster arrays are arranged in the same direction
00:14:58.02 as those in the central spindle,
00:14:59.00 and they have the same contrast in this polarized micrograph.
00:15:03.08 Those aligned in a perpendicular direction have a dark contrast,
00:15:04.16 and I’ll explain where that comes from shortly.
00:15:06.15 Let me begin by reminding you that light propagates,
00:15:11.06 in this case in the Z axis direction,
00:15:14.16 as a transverse vibrating electrical and magnetic wave.
00:15:23.27 The vibrations are perpendicular
00:15:25.08 to the direction of propagation.
00:15:29.04 The velocity of propagation, v,
00:15:31.16 is given by c divided by n,
00:15:35.18 where n in the refractive index and c is the velocity that light
00:15:38.17 would have in a vacuum,
00:15:41.21 or it’s very similar in air.
00:15:43.18 And n is the refractive index of the medium
00:15:45.19 through which the light is propagating.
00:15:50.08 Now, plane polarized light has its electric field vector
00:15:52.18 oscillating within a plane, and if we look…
00:15:55.16 I can’t do this here well,
00:15:57.16 but if we look down the Z axis, as shown here,
00:16:00.13 if it’s plane polarized light, then this oscillation stays
00:16:04.09 with its vector in this plane
00:16:06.21 and it can be defined by a given azimuth angle, theta in this case,
00:16:10.16 relative to let’s say an X-Y coordinate system that’s orthogonal
00:16:14.15 to the Z axis of propagation.
00:16:17.23 Lasers, for example, emit polarized light
00:16:20.18 from coherent sources within the laser.
00:16:23.02 On the other hand,
00:16:25.12 if a tungsten filament has many molecular oscillators
00:16:27.24 that emit light asynchronously with different orientations,
00:16:31.22 one gets as a result unpolarized light
00:16:34.13 with electric vectors in many different directions,
00:16:37.15 and this is typical of most of the light sources
00:16:40.08 that we encounter,
00:16:43.26 including arc lamps and tungsten filaments
00:16:47.10 used in our light microscopes.
00:16:49.29 Now, a polarization microscope
00:16:51.28 has a polarizer, then the condenser,
00:16:56.02 then an objective, another birefringent material,
00:17:00.18 which is used as a compensator,
00:17:03.15 and then an analyzer.
00:17:05.03 And the polarizer produces plane polarized light
00:17:07.18 with its electric field vibrating in one direction,
00:17:10.28 and that’s aligned east-west,
00:17:14.10 and the analyzer is made out of the same kind of polarizing material
00:17:19.12 and has its electric vibration direction
00:17:23.26 lined up in a perpendicular direction
00:17:27.09 — we call it north-south, but it’s in and out of the board, here —
00:17:30.23 and it extinguishes the light that comes from the polarizer.
00:17:35.08 So, the light that can get through the analyzer
00:17:39.06 is generated by the modification of the plane polarized light
00:17:42.18 by the specimen or by the compensator.
00:17:44.26 And this works in…
00:17:47.01 simply in the following way,
00:17:49.07 that if a plane polarized light hits the specimen,
00:17:51.20 the birefringent specimen generates two orthogonally polarized light waves;
00:17:55.19 they move through the specimen at different velocities,
00:17:58.15 dependent on n-sub-e and n-sub-o;
00:18:01.05 and when they exit the specimen,
00:18:03.00 because of these different velocities,
00:18:04.18 there’s a phase difference between the two light waves,
00:18:09.26 and this phase difference is called the retardation.
00:18:13.28 And retardation can be measured in nanometers
00:18:17.05 — it’s n-sub-e minus n-sub-o
00:18:20.06 times the thickness, d,
00:18:22.25 or in radians, it’s just two pi times retardation
00:18:28.00 divided by the wavelength of light.
00:18:30.26 Now, this retardation can’t be seen by eye
00:18:33.17 — the light intensity still looks the same to your eye —
00:18:37.08 but if you put an analyzer in the light path,
00:18:40.01 then what comes through the light path
00:18:43.01 has this equation of the form sin-squared of the retardation
00:18:46.13 divided by two.
00:18:48.24 And if we plot this out as a function of specimen retardation,
00:18:51.21 we start with zero, here,
00:18:53.09 with no birefringence or no thickness,
00:18:55.00 and we get maximum brightness
00:18:57.13 coming through at a particular wavelength
00:18:59.22 for a half-wavelength retardation
00:19:01.18 between the e and the o waves, right?
00:19:05.24 So, this is a quantitative technique.
00:19:09.07 It measures the product of birefringence time thickness,
00:19:12.11 which is retardation,
00:19:14.15 and for something like the spindle,
00:19:16.20 the actual measurement of retardation
00:19:18.25 tells us something about the number density
00:19:21.18 in a cross-section of the spindle,
00:19:23.15 of how many microtubules we have there, for example.
00:19:26.02 And if we look at our spindle, here,
00:19:28.04 in the presence of a compensator,
00:19:30.05 another birefringent object, that has its n-sub-e this way
00:19:33.24 and n-sub-o axis that way
00:19:36.08 — the larger refractive index in this case being the n-sub-e axis —
00:19:39.06 and if we’ve aligned our specimen with that compensator,
00:19:43.23 then these two retardations add together
00:19:45.29 to make a larger retardation,
00:19:47.28 which makes a brighter light,
00:19:49.22 and that brightens the central spindle and it brightens the aster.
00:19:53.07 For the astral fibers going this way,
00:19:58.06 their axes are in opposite directions,
00:20:01.05 and so we get a negative compensation,
00:20:04.13 which actually results in a lower total retardation.
00:20:11.02 And since the background is produced by the compensator retardation,
00:20:14.25 this is lower than that produced by the compensator retardation,
00:20:18.00 so it appears darker than the background,
00:20:20.16 whereas this part appears much brighter than the background,
00:20:23.12 and that’s how compensators help both brighten the image in a specimen
00:20:30.05 but also can give you a dark contrast for oppositely oriented fibers,
00:20:33.26 in this case.
00:20:36.14 So, one of the important examples
00:20:38.16 for the use of polarization microscopy
00:20:41.09 was performed by Shinya Inoue
00:20:44.15 in the early 1950’s,
00:20:45.29 where he developed a polarization microscope
00:20:48.11 of sufficient sensitivity
00:20:51.09 to image living cells in mitosis,
00:20:54.09 and the results of this were very important
00:20:57.10 in the field of cell biology
00:21:00.10 because it was the first proof that spindle fibers
00:21:02.23 were not a fixation artifact.
00:21:05.01 And so, this movie of the lily pollen mother cell…
00:21:08.16 and the brightness here is the brightness of the spindle fibers
00:21:15.12 that shorten and pull the chromosomes up to the poles
00:21:18.03 until you make the nucleus,
00:21:20.01 and then another brighter ray forms, here,
00:21:21.28 at the position where the cell plate is formed,
00:21:24.21 and we now know that this is a midzone microtubule complex
00:21:27.17 that’s used to support microtubule motor trafficking
00:21:31.05 that brings vesicles that build the cell wall
00:21:33.28 and the new plasma membrane.
00:21:37.03 Now, here we are 60 years later,
00:21:39.14 and now in polarization microscopy
00:21:44.29 electronic compensators can be produced
00:21:49.06 by using liquid crystals
00:21:51.07 and controlled by computers,
00:21:52.27 and Rudolf Oldenbourg and his colleagues
00:21:54.24 at the Marine Biological Labs
00:21:56.17 have built a microscope called the LC-PolScope,
00:21:58.27 in which images can be obtained
00:22:01.01 in which the magnitude of the intensity is…
00:22:07.29 for each pixel in the image,
00:22:10.29 carries information about the retardation independent of the orientation.
00:22:14.03 And this is a movie of that.
00:22:17.04 So, there’s no orientation dependence anymore,
00:22:20.14 and so the brightness of this image
00:22:24.04 is directly proportional to the microtubule number
00:22:28.14 and the thickness of the fibers at this stage,
00:22:31.29 and this is true for the plasma membrane cortex,
00:22:35.12 or for the mitochondria that are in here, alright?,
00:22:38.18 and these are mitochondrial bundles that are coming in here at the end, alright?
00:22:44.19 And so, a great deal of quantitative information
00:22:47.22 can be gotten from these sorts of information, now,
00:22:50.21 as well as just being able to see structure in transparent cells
00:22:54.18 by this type of technique.
00:22:58.03 Now, the last type of transmitted image contrast mode
00:23:03.04 that I like to talk about
00:23:05.05 is differential interference contrast,
00:23:06.19 and people were very frustrated with phase contrast,
00:23:09.14 particularly in trying to image thicker specimens,
00:23:12.01 because the organelles like this nucleus, here,
00:23:18.10 some of these mitochondria…
00:23:21.14 although nice contrast was produced in phase,
00:23:25.28 around the edges there was a white halo
00:23:28.15 that’s typical of phase contrast images.
00:23:31.15 You can see it here for the nucleus.
00:23:33.12 You can see it down here for the edge of the cell.
00:23:35.28 And in thicker specimens,
00:23:37.24 these halos acted like the edges in darkfield,
00:23:40.17 and they accumulated through the depths of the specimen
00:23:43.23 and made optical sectioning very poor.
00:23:47.27 And so, the invention of differential interference contrast
00:23:52.25 by Nomarski and Smith in the ’60’s,
00:23:56.02 and its implementation, initially by Zeiss and Leica,
00:24:00.24 was quickly accepted in the cell biology field
00:24:06.27 because this technique highlighted edges
00:24:09.22 and was less sensitive to the absolute mass of objects.
00:24:13.29 And so here we can see the typical image for the same cell.
00:24:17.23 This is the DIC image,
00:24:20.08 and this is the phase image,
00:24:21.22 and there are no halos at the edges.
00:24:23.17 The edges are very sharply defined,
00:24:25.05 and in fact the thickness of the specimen
00:24:28.05 gives the same contrast as the background,
00:24:31.03 and therefore doesn’t carry through
00:24:34.04 as you do optical sectioning through the material.
00:24:38.00 So, how do we get this?
00:24:40.04 Well, DIC is actually a polarization microscope,
00:24:42.28 and it has two additional birefringent components
00:24:46.15 — a condenser DIC prism
00:24:49.23 and an objective DIC prism —
00:24:53.02 which together make a dual-beam interferometer
00:24:57.00 that functions as defined here.
00:25:00.02 So, the lower DIC prism
00:25:02.25 splits the incoming polarized light beam into two equal amplitude,
00:25:07.05 orthogonally polarized light beams
00:25:11.03 that can’t interfere with each other,
00:25:13.07 and these pass up through the condenser and then through the specimen.
00:25:17.16 And one beam coming through the middle of the lower beam
00:25:20.18 is shown here.
00:25:22.00 And as they pass through the specimen,
00:25:24.08 these two light beams are sheared apart
00:25:27.14 — that is, they’re separated —
00:25:29.21 by a very small distance,
00:25:32.29 which is called the shear distance,
00:25:34.26 and this is set by the manufacturer to be about half the resol…
00:25:38.09 the limit of resolution of the microscope
00:25:40.23 and condenser optics, right?
00:25:42.16 And so, at the specimen,
00:25:45.18 these two wavefronts,
00:25:47.11 the e and the o wavefronts,
00:25:48.29 are lined up with each other,
00:25:50.24 but when we move through out transparent specimen,
00:25:53.12 we now have this retardation that takes place
00:25:56.13 for both wavefronts.
00:25:58.06 Then we pass through the condenser
00:26:00.16 and we also pass through a birefringent compensator,
00:26:04.08 and the compensator advances one wavefront over the other,
00:26:09.04 and the objective prism realigns the two beams
00:26:14.24 so their edges come back together,
00:26:17.11 and that process of realignment
00:26:19.26 causes a shift in these two edges
00:26:22.11 so that when this…
00:26:24.21 these two wavefronts pass through the analyzer,
00:26:29.17 they’re no longer lined up with each other,
00:26:32.18 no longer in phase,
00:26:34.11 and we have a phase difference.
00:26:36.23 And because of this offset induced by the compensator,
00:26:40.04 on one side the phase difference
00:26:43.08 goes negative
00:26:46.08 and on the other side it goes positive,
00:26:49.01 elative to the phase difference in the background. So, this produces the…
00:26:55.15 one edge being darker and one edge being brighter than the background
00:26:59.13 that we normally use for compensation,
00:27:01.06 and you can switch the compensator the other way
00:27:03.10 and advance the other beam over the other one
00:27:05.14 and shift the direction you see for the edges.
00:27:08.19 So, that’s how DIC is produced conceptually.
00:27:12.05 And we usually adjust this compensation
00:27:15.20 to be about 1/10 of the wavelength,
00:27:17.29 and that makes the highest contrast for the edges.
00:27:22.04 And I’m gonna show you some pictures of embryos,
00:27:25.10 and in particular these are early embryos
00:27:28.14 of the model genetic organism C elegans.
00:27:32.03 That’s a worm.
00:27:34.04 And the picture…
00:27:37.25 movie that we’re gonna begin with, the sperm has entered here on the righthand side,
00:27:43.17 maybe a short time previously, and the sperm nucleus has decondensed
00:27:51.07 and formed the sperm pronucleus, here,
00:27:54.00 when the movie starts.
00:27:56.04 And so, we’re gonna look at the first four cell divisions.
00:27:58.23 And over here, on the other side of the cell,
00:28:00.29 this is the female pronucleus that’s formed after going
00:28:05.21 through its meiosis I and meiosis II divisions.
00:28:08.02 And so, you’re gonna see pronuclear movement
00:28:11.04 — that is, the movement of the two nuclei that come together —
00:28:13.21 and that’s because the centrosome that the sperm brings in
00:28:16.23 makes a microtubule aster, here,
00:28:19.20 that actually reaches out and grabs this other nucleus
00:28:23.18 and then pulls them both together
00:28:26.17 as well as moving them into the center of the cell.
00:28:30.10 Now, DIC can show you this aster a little bit,
00:28:33.22 but not too much,
00:28:35.22 but there’s the spindle, there’s first division,
00:28:38.07 and then now we have interphase,
00:28:40.21 and now we have second mitosis and division,
00:28:44.21 and into the third.
00:28:47.01 So, you get very nice, clear optical sectioning
00:28:49.02 with this DIC, which would not be possible with phase contrast.
00:28:54.14 Now, here is another important feature of using transmitted light microscopy,
00:28:57.19 which is…
00:29:00.24 this worm has a small number of total cells
00:29:04.10 that make up the organism
00:29:07.01 when it’s finally a mature organism.
00:29:09.06 And investigators have been able to use DIC microscopy
00:29:11.18 to actually follow the development of all of these cells
00:29:15.01 and their relationships to each other during morphogenesis…
00:29:19.14 cell divisions and morphogenesis in this process.
00:29:22.02 And I’ll show you… the first example from Bob Goldstein’s lab.
00:29:27.17 This is just the beginning of first mitosis,
00:29:31.06 and… first division, second, third, fourth, fifth.
00:29:38.08 So, this is all for one focal plane,
00:29:41.18 and what you mainly see are the nuclei,
00:29:43.20 or the big round objects here, right?
00:29:46.11 And the beauty of this is
00:29:52.01 they’re able to image this organism using a green filter
00:29:55.00 and a very good heat reflection filter
00:29:59.07 almost all the way through to the formation of the adult worm
00:30:02.14 and before hatching.
00:30:05.05 And they’ve even developed techniques in DIC
00:30:09.11 so that it can take four different optical planes at the same time
00:30:12.18 and then timelapse all four of them…
00:30:16.28 and so use this information actually to develop…
00:30:19.15 to build this developmental map
00:30:22.10 of where all the cells are relative to each other
00:30:24.15 in the developing organisms
00:30:29.29 throughout these many stages of development in making the final mature organism.
00:30:38.16 This would be impossible to do in fluorescence microscopy
00:30:41.23 because absorption of light inevitably hurts the specimen,
00:30:45.10 whereas here we do not have absorption of light.
00:30:48.21 So, I hope you’ve enjoyed these examples
00:30:50.29 of these four modes of transmitted light imaging,
00:30:56.25 and I hope you have a chance to get in the lab
00:30:59.07 and give them a try.

This Talk
Speaker: Ted Salmon
Audience:
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: July 2012
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Talk Overview

This lecture describes various methods of generating contrast in biological specimens using transmitted light (i.e. without staining or fluorescence). Principles of dark field, phase contrast, polarization and differential interference contrast (DIC) microscopy are described and examples of how these techniques are used for imaging are described.

Questions

  1. Phase contrast and differential interference contrast microscopy generate contrast based upon:
    1. Light absorbance of molecules in the specimen
    2. Refractive index differences in the specimen
    3. A shift in wavelength between the illuminating and scattered light
    4. Modulating the frequency of the emitted light
  2. True or False. Light will travel slower in an organelle, such as a mitochondria, with a refractive index of ~1.5 than the cytoplasm with a refractive index of ~1.35
  3. In the above example, if the mitochondria is 0.5 microns thick and the specimen is illuminated with 500 nm light, what is the phase shift (in nm) between a light wave traveling through the organelle and another light wave traveling through the cytoplasm.
  4. Which of the following statements is false about dark field microscopy?
    1. Poorer depth of field than phase contrast microscopy
    2. Is generally a lower resolution technique than phase microscopy
    3. Is a higher sensitivity technique than phase microscopy for detecting scattered light from small objects
    4. Requires that the N.A. of the objective is higher than the NA of the condenser
  5. Which of the following statement is false about Phase 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.
  6. Which of the following statements is true for Polarization Microscopy?
    1. The polarizer and analyzer are oriented so that they have directions of transmission that are parallel to one another.
    2. Plane polarized light can split in two different propagating waves that travel with different speeds through a birefringement material.
    3. The compensator is usually placed after the analyzer and before the eyepiece/camera.
    4. A compensator makes all parts of the specimen brighter than the background.
  7. True or False. Differential interference contrast microscope consists of a polarizer and analyzer and a single DIC prism located above the objective.
  8. Match the statements below to one of these microscopy techniques
    1. Dark field microscopy
    2. Phase contrast microscopy
    3. Polarization microscopy
    4. Differential interference contrast microscope
      i) Which technique can produce halos as an artifact?
      ii) Which technique tends to produce a shadow effect across edges (dark on one side and bright on the other)?
      iii) Which technique splits the incoming light into two orthogonal polarized beams that act as an interferometer to interrogate refractive index differences in the specimen.
      iv) Which technique produces the best optical sectioning?

Answers

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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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