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Choosing the Right Microscopy Technique

00:00:11.19 Hello, I'm Ron Vale and in this lecture, I'd like to discuss
00:00:15.28 with you how to choose the right microscope technique. So, if you're
00:00:20.21 a newcomer to microscopy, it may seem like there's a jungle
00:00:24.04 of different choices out there. All kinds of techniques and various
00:00:28.24 acronyms. And it may seem quite daunting to decide which
00:00:33.17 type of microscopy technique to use. Now, in this course, we've
00:00:37.26 discussed these different techniques one by one, and then
00:00:41.21 illustrate in detail how they work. And what I'd like to do today is
00:00:45.28 really just give you a broad overview of how you go about
00:00:50.08 choosing between the different microscopy techniques available
00:00:54.02 to you. Now, in making that choice, the first thing you have to ask
00:00:59.22 yourself is, what kind of biological information are you trying to
00:01:03.27 gather? What question are you trying to answer? And based upon
00:01:09.02 that, you can think about what kind of image data do I need to
00:01:13.24 answer that question. And that will impart be the most important decision
00:01:18.22 in dictating what microscopy technique will be the most relevant
00:01:22.16 for answering your problem. The second issue is pragmatics,
00:01:27.17 what do you have available? What's in your lab or department?
00:01:30.21 Or in your building? And you may have to make some adjustments
00:01:34.23 based upon what kind of equipment is available. And guide your
00:01:42.13 research based upon that. In some cases, you may have a very
00:01:46.16 specific need and no equipment in your particular institution. In that
00:01:52.14 case, you may consider even setting up a collaboration or visiting
00:01:56.09 a lab to do the kind of microscopy that you need to do.
00:01:59.29 And thirdly, you also should think about in advance, how you
00:02:06.06 might potentially analyze your image, because that will also
00:02:10.05 may influence how you collect the data. And microscopy's changing
00:02:15.21 dramatically in recent years. It's not just a technique to
00:02:19.19 generate beautiful images, but those images contain a lot of
00:02:24.21 quantitative information that can be extremely important for
00:02:28.11 you being able to answer a particular biological question.
00:02:31.05 So, you should think about quantitation even at the very beginning
00:02:36.09 of your thinking of your choices and your project.
00:02:41.22 Now I also want to say, that you don't always need to have the
00:02:45.10 most expensive microscope out there to answer a biological problem.
00:02:49.29 And in fact, when I started my lab at UCSF, this was the workhorse
00:02:56.14 microscope of our laboratory. It was a very simple darkfield
00:03:01.04 microscope. Just a simple condenser, one objective lens, and there was
00:03:07.09 a camera on top. But a simple microscope you could pick up with one
00:03:10.24 hand. And this microscope was in fact, a very good microscope
00:03:17.06 and technique for imaging microtubules and how they were moved by
00:03:23.01 motor proteins. And in fact, this particular image here on the cover of Cell was
00:03:28.17 generated with this microscope here. So, first decision you may want
00:03:39.07 to think about is whether you want to use transmitted light
00:03:42.27 or fluorescence. And this is perhaps a little bit of a gross generalization.
00:03:48.17 But transmitted light, on one hand, can be a very good technique
00:03:53.12 for measuring cell behavior. It's very non-perturbing, can be
00:03:58.11 done with long term time lapse imaging. And a very simple uncomplicated
00:04:05.09 technique. On the other hand, if you want to measure a specific
00:04:11.02 molecule in the cell or specific organelle, then you need a
00:04:16.08 molecular marker. And for that, you have to go to fluorescence.
00:04:19.16 But let's discuss transmitted light first. So as I already mentioned, the
00:04:25.19 big advantage of transmitted light is that there's no special preparation
00:04:29.10 needed. You could just take your cells and image them right away.
00:04:32.28 You don't need to encode or do any molecular biology or genetics
00:04:37.18 to encode GFP on a particular gene. And also, in general, transmitted
00:04:46.06 light is less damaging to the specimen than fluorescence excitation.
00:04:51.11 So it can be very good for long term observation of cells and keeping
00:04:57.01 them happy without any damage. And this just shows an example
00:05:01.23 of a time lapse movie of cells in phase contrast microscopy, of cells
00:05:08.04 closing in on a wound, a scratch made in a tissue culture cell.
00:05:13.11 And this time lapse movie provides a lot of information on the cell
00:05:17.26 behavior and how they migrate in this special circumstance.
00:05:23.23 And this shows another case of dark field microscopy, which I alluded
00:05:28.23 to before. This is work by Hotani in the 1980's, where he used this to image
00:05:35.10 the growth of individual and shrinkage of individual microtubules.
00:05:41.13 And this was the first time that microtubule dynamic instability was
00:05:47.21 imaged live under a microscope. So you can see in a very simple
00:05:51.22 sample like this, transmitted light dark field can be very powerful.
00:05:57.13 So, as I said, in many cases you have to go to fluorescence to see
00:06:03.05 a specific marker or a protein in the cell. And then there are many
00:06:08.24 choices of different kinds of fluorescence microscopy.
00:06:11.04 And in terms of the decision tree here, you first have to think
00:06:15.00 about what kind of specimen you have. Do you have a thick specimen?
00:06:19.05 Or a thin specimen? Because different types of microscopy techniques
00:06:24.09 are going to be appropriate depending on the depth of penetration
00:06:27.29 that you have to go to see your objects, your fluorescence.
00:06:34.18 And the other question you might want to ask is, are you looking
00:06:37.24 at a living sample or fixed sample? If you're looking at something fixed, you don't
00:06:42.22 have to worry about damage, you don't have to worry about time of
00:06:46.12 acquisition. But that's very different if you're looking at dynamics in
00:06:49.22 living cells. So you have to think about how fast you acquire
00:06:52.22 the images, so you can see the dynamics in the cell. And you do have
00:06:57.04 to be potentially cautious about cell damage through fluorescence
00:07:02.21 excitation. So the easiest type of fluorescence is epifluorescence.
00:07:09.04 So, let's consider that first. Now, the advantages of epifluorescence
00:07:13.14 are that it's the very simplest technique out there. It's low cost,
00:07:18.02 you don't need any lasers. It's a very simple technique to use.
00:07:23.15 So, if you can use epifluorescence, this is a very good solution. Now
00:07:29.21 there are some disadvantages, because epifluorescence
00:07:34.13 -- in epifluorescence, you have out of focus fluorescence.
00:07:39.01 And that contributes to kind of this background haze in your
00:07:42.12 image, and therefore in your plane of focus, you may have kind of
00:07:48.16 -- your signal may not be that good because of all the unwanted
00:07:52.10 out of focus background fluorescence. And second of all,
00:07:55.19 the epifluorescence penetrates through the entire specimen.
00:07:59.29 And again, that fluorescence excitation can damage the cell,
00:08:04.29 not only in the plane of focus, but in out of focus planes, as well.
00:08:08.19 But in many cases, it's perfectly good. So here's an example
00:08:13.18 of an application that we used epifluorescence, which was
00:08:18.00 to image the mitotic spindle. And in this particular case, we wanted
00:08:22.27 to image many, many samples. In fact, this was an RNAi screen
00:08:28.26 of the entire drosophila genome, where we knocked down
00:08:31.22 individual genes one by one in 96-well plates, and looked
00:08:37.02 whether that gene knockdown affected the shape of the spindle.
00:08:41.02 But in this case, we could use fixed cells. These are fixed drosophila
00:08:45.27 cells. We could stain them with antibodies for tubulin in green,
00:08:51.00 the centrosome in red, and DNA in blue. We could get all the
00:08:55.26 information that we needed to see if that gene knockdown affected
00:09:01.13 mitotic spindle shape. And this was a pretty simple technique
00:09:05.10 that allowed us to get all the information we wanted. Now,
00:09:08.28 in some cases, you may also want to get more information or
00:09:12.26 better clarity out of your epifluorescent image. And in that case,
00:09:17.04 you may want to think about a technique which is a computational
00:09:20.11 technique really, which is deconvolution microscopy.
00:09:23.24 And this is a way of computationally removing kind of the
00:09:30.10 contributions of out of focus fluorescent light. And this involves taking
00:09:35.07 a z-stack of images from your sample and then doing a computational
00:09:41.05 correction to remove the out of focus light. And one advantage
00:09:45.26 here is that hardware-wise, it's actually quite simple. Just requires
00:09:50.07 an epifluorescent microscope, and the only special piece of
00:09:54.01 hardware that's required is a motorized z-axis stepper. So you can
00:09:59.12 take different z sections, images of different z sections through your
00:10:04.08 specimen, which is needed to do deconvolution microscopy.
00:10:08.18 But the one thing you will need which is special is the software
00:10:12.18 that can do this computational correction and sharpen the in focus
00:10:17.28 light computationally. So this may involve purchasing a
00:10:24.02 commercial software package to do that. And this just shows an example
00:10:29.20 of an epifluorescent image of a cell, where you can see it's
00:10:33.14 a little bit hazy and not that crisp. And then after deconvolution
00:10:39.02 you can see that the image is much sharper and crisper
00:10:46.00 after this computational method. So another class of getting good
00:10:51.28 optical sections, or good sectioning, is using optics. And not a
00:10:58.18 computer. And there are several ways in which one can do optical
00:11:02.23 sectioning. Most of them really pragmatically are done using lasers.
00:11:06.25 So that adds some extra costs to the microscopy. And there are
00:11:12.04 several different ways you can use lasers to do sectioning.
00:11:16.09 And it depends upon the depth of your specimen. So in some
00:11:21.26 cases, you may have an object or specimen where what you want
00:11:26.17 to image is right at the coverslip surface. And for this, total internal
00:11:31.18 reflection fluorescence microscopy is a very powerful technique.
00:11:37.09 It doesn't allow you to penetrate very far, it's good to within 200nm
00:11:43.13 or so of the coverslip surface. But if your object is in that regime, it's very good.
00:11:48.22 But if you have a thicker sample, like a cell or even a section
00:11:53.25 of a tissue, you then may have to go to pin hole microscopy.
00:11:57.27 So these techniques are for example, point scanning confocal
00:12:04.28 microscopy or line scanning confocal microscopy or another
00:12:10.24 technique, which is spinning disk microscopy. And if you have
00:12:14.17 a still thicker sample, such as a piece of a tissue, a piece of
00:12:20.11 brain section for example, in this case, you would have to think
00:12:24.18 about another technique, which is two photon microscopy.
00:12:28.02 And in some cases, if you have an extremely thick specimen,
00:12:32.26 for example an entire brain of a mouse, there is a method for doing
00:12:38.10 that that I'll mention later, which is actually a physical sectioning technique,
00:12:41.16 which is called array tomography. Now first let's talk about
00:12:46.15 TIRF microscopy. So this just shows an example of how powerful
00:12:52.29 TIRF can be. In this case, we're looking at microtubules in a
00:12:58.04 xenopus egg extract by epifluorescence. And you can see
00:13:01.15 everything is very fuzzy, its very hard to see anything. There's
00:13:06.11 tremendous amount of just general blur from out of focus fluorescence here.
00:13:10.21 And now if we look at the same specimen by TIRF microscopy,
00:13:14.21 we can look at the behavior of microtubules right at the coverslip
00:13:18.28 surface in much more detail, much better signal than noise. And we can
00:13:23.00 see individual microtubule behavior here. So this just shows, if
00:13:29.02 you're in that regime, how powerful TIRF microscopy can be.
00:13:34.04 Now, in a way, TIRF is not only good at looking at cells or extracts,
00:13:40.08 but you really can think of it as a very general tool for any kind
00:13:44.12 of biochemistry that can be visualized on a glass surface.
00:13:50.04 And there are all kinds of creative ways now of doing biochemistry
00:13:54.17 using microscopy techniques and TIRF. So even classical biochemical
00:14:00.07 techniques, such as immunoprecipitations, which are done in
00:14:04.20 bulk solution, recently have even been done now at the single
00:14:09.10 molecule level, or single immunoprecipitation level using TIRF
00:14:14.00 microscopy. And in this case, an antibody that's used for the
00:14:19.09 immunoprecipitation is attached to the glass coverslip, and
00:14:23.00 used to pull out a molecular complex from a cell or an extract
00:14:28.23 with, at some point, a fluorescent tag on one of the molecules.
00:14:33.19 And in this case, each of these individual spots that you see here
00:14:38.16 corresponds to an individual immunoprecipitation reaction.
00:14:43.10 And you can even look at the type of complexes that are formed
00:14:48.14 at a single molecule level using this particular technique.
00:14:53.17 And this is just a control here without any antibody on the surface
00:14:58.01 for the immunoprecipitation. Even the new revolution of DNA
00:15:02.00 sequencing is also effectively involving imaging and TIRF
00:15:07.09 microscopy. Many of the more recent next generation sequencing
00:15:13.12 techniques, such as the ones recently developed by Illumina,
00:15:17.15 which allow you to do very high throughput sequencing,
00:15:21.19 involve hybridizing fluorescent DNA to oligos that are attached
00:15:28.05 to the surface and then using a visual microscopy readout
00:15:32.19 as a way of getting the DNA sequence information. So,
00:15:40.02 now let's go to the methods of confocal microscopy.
00:15:46.00 And a decision point here may be whether to use spinning disk
00:15:50.06 or point scanning confocal microscopy. So spinning disk is
00:15:55.04 a particularly powerful technique for live cell imaging, and fast
00:16:01.00 acquisition. The reason is the images here are acquired by a
00:16:05.14 camera. And the camera, you can run the camera as fast as your
00:16:10.06 camera can run, and you can use very sensitive cameras to detect
00:16:13.13 very low level fluorescence. So if you have a relatively thin
00:16:18.14 sample under 30 microns, typical of most cells, and you want
00:16:22.25 to measure something live, dynamic, fast, and have very little
00:16:27.00 also cellular damage, then spinning disk microscopy could maybe
00:16:31.05 a very good technique. If you have a slightly thicker specimen
00:16:36.15 however, point scanning confocal microscopy may be better.
00:16:41.20 In general also, point scanning confocal microscopy tends to be better for
00:16:46.28 fixed tissue. It tends to be slower than spinning disk. But if you
00:16:52.06 have a fixed specimen, there's really no limitation on time of acquiring
00:16:57.11 the whole field of view. It also tends to be -- because it focuses a very intense
00:17:03.26 laser at an individual point, it tends to be more photo damaging than
00:17:08.04 spinning disk confocal. So again, you might have to think about that.
00:17:11.25 Again, less of a concern for a fixed sample. And it also has some
00:17:16.15 advantages, such as measuring dynamics of molecules in cells
00:17:20.22 by the technique called fluorescence recovery after
00:17:24.21 photobleaching. And you can combine that by bleaching a
00:17:29.12 very defined point in your sample, using the point scanning microscope.
00:17:33.23 And after the bleach, then go back and acquire an image of the
00:17:39.02 specimen to see how fast that photobleach has recovered.
00:17:43.09 So that's a particularly good use of point scanning confocal.
00:17:46.02 There are other ways of generating optical sectioning, and
00:17:53.02 kind of an interesting recent technique is light sheet microscopy.
00:17:57.19 Where instead of having the light travel through the objective
00:18:03.06 lens, you actually have the light traveling perpendicular to the optical
00:18:08.11 axis. Now, this is very good because it doesn't illuminate, it only
00:18:12.17 illuminates the specimen in one focal plane. It doesn't illuminate
00:18:16.18 it below or above that sectioning plane. So that means, you don't
00:18:22.15 have light damage above or below the field of observation.
00:18:29.19 And so, because it has very little photodamage, it's made it a very
00:18:34.24 good technique for imaging processes that occur in complex
00:18:40.10 systems, such as embryos for example. And it allows for very
00:18:45.20 fast acquisition as well. So I'll just show you an example of
00:18:51.15 a beautiful movie done with this technique, of a zebrafish
00:18:56.02 embryo here. And this is from the Stelzer group, labeling
00:19:02.24 DNA here with a GFP-histone, so you can watch individual
00:19:09.02 cells in this zebrafish embryo over time as this embryo
00:19:14.19 develops. And you have this beautiful single cell clarity in this
00:19:20.02 still fairly big embryo. You can see all the details of embryogenesis
00:19:25.29 here. So in some cases, however, you may need to go even deeper
00:19:33.01 into your specimen. And in this case, a particularly powerful technique
00:19:38.06 is multi-photon microscopy. Or most frequently, employed as 2-photon
00:19:44.10 microscopy. And this just shows a nice comparison of the two
00:19:49.19 done many years ago by John White and his colleague. And it
00:19:56.03 shows comparison of the same sample in confocal microscopy
00:20:02.19 using a pinhole or 2-photon microscopy, and you can see you get
00:20:06.23 much better penetration depth with 2-photon. Now, the exact
00:20:12.03 depth of penetration is not solely dependent upon the optics,
00:20:17.02 it also depends quite a bit on the particular tissue, and how
00:20:21.19 much that tissue will scatter light. So that may dictate how
00:20:26.04 far you could go. But in all cases, 2-photon is a preferable
00:20:31.22 technique for imaging deeper into tissues. Now, in some cases,
00:20:37.23 maybe 2-photon is not even enough. You'd like to create, for example,
00:20:42.00 a whole image of a big piece of tissue, part of a brain, or even
00:20:49.15 maybe an entire brain, like what Stephen Smith and his colleagues
00:20:54.14 were interested in doing. And they solved this problem not by using
00:20:58.19 optical sectioning, but using physical sectioning. So to take
00:21:02.18 a big tissue like a brain, and optically sectioning it into very
00:21:06.27 small slivers, even down to like 50nm in size. And these slivers
00:21:13.06 of tissue, you can now stain for various cellular markers. And
00:21:20.18 you can just image those slivers by conventional epifluorescence
00:21:26.02 microscopy. And then you can just put all of these slices together
00:21:31.22 computationally, to create this beautiful movie that you see here.
00:21:37.03 Penetrating deeper and deeper into a mouse brain, all derived from
00:21:43.29 physical sections that have been intercalated together.
00:21:50.17 So, finally, we also discussed in this course super resolution microscopy.
00:21:55.02 Of course, this is a very new exciting technique in light microscopy.
00:21:59.29 There are several different methods that are out there.
00:22:04.04 And while you shouldn't take these numbers as absolute,
00:22:07.11 this gives you a kind of rough guideline of the resolution
00:22:11.10 improvement for some of these super-resolution techniques.
00:22:15.23 And they're still not quite at the real molecular level. Of course for that,
00:22:20.28 one has to go to cryo electron microscopy. But still, an enormous
00:22:26.29 improvement. But I think a major decision, because these techniques
00:22:31.21 are still, they're more expensive, they're still harder to implement,
00:22:35.04 it's important to think, if you have a problem that is really
00:22:40.04 well-suited to the resolution improvement that you see here
00:22:45.06 from these techniques. So I'll give you one example of an
00:22:48.28 interesting biological question that did require an improvement of
00:22:53.05 resolution, but was still within the boundary of what super-resolution
00:22:58.00 could do. And this is analysis of the centrosome. So this is an organelle
00:23:04.07 in cells that nucleates microtubules. So they're nucleating proteins and the
00:23:10.00 microtubules grow off of the centrosomes. And these are important for
00:23:15.16 organizing the microtubule cytoskeleton. And if you put a fluorescent
00:23:20.06 marker on a GFP-tagged centrosomal protein, and you look at it by
00:23:26.10 wide-field, it's not very impressive. It's just a little round blob.
00:23:31.09 But if one looks at it by structured illumination microscopy,
00:23:35.13 which you know, can give you a 2-3 fold improvement in the
00:23:39.26 resolution, as was done in this study by David Agard's lab.
00:23:43.29 They could now see interesting structure from this GFP-tagged
00:23:47.15 protein. It's not just a ball, it actually has this donut-shaped
00:23:51.13 structure. And when they looked at actually several different
00:23:55.10 centrosomal proteins using GFP-tagging and structured
00:23:59.03 illumination, you now get molecular details showing that they're not
00:24:04.04 all in identical locations. And they could begin to define different
00:24:10.02 molecular localizations for centrosomal proteins that were never
00:24:16.23 known before. So, in addition to optical techniques that we've just
00:24:24.05 talked about, I think another important thing that's been an enormous
00:24:29.06 advance in microscopy and biology is coming up with new creative
00:24:33.28 ways of measuring activities, biochemistry that's going on in
00:24:39.07 cells. And measuring these events with light. So in this case, you need to have
00:24:44.04 some kind of reporter that you can introduce into the cell that
00:24:49.15 in response to some kind of change in the environment of the cell,
00:24:54.09 or biochemical activity, will produce a readout of light.
00:24:58.20 So this may require both designing new molecules or sensors, or
00:25:05.06 genetic proteins. And there are so many examples of this, of different
00:25:10.27 kinds of sensors that are being designed. But you may want to think
00:25:13.25 about designing your own sensor or implementing a specific sensor that someone else
00:25:18.22 has made. So this just shows work from Adam Cohen's lab
00:25:22.21 of a new reporter that they made, which was based upon a
00:25:27.21 microbial rhodopsin. And in this particular case, they engineered
00:25:31.21 it so that it would be a reporter of voltage inside a cell. And
00:25:36.08 what you're seeing here is a rat hippocampal neuron, and this
00:25:40.02 bright flash is the readout of light when there was an action
00:25:46.21 potential in this neuron that changed the voltage in this cell.
00:25:50.13 And the sensor reports on this voltage change, as you see
00:25:55.24 here. And this could be an incredibly exciting technology
00:25:59.06 now, to put inside a mouse and get readout of how neurons
00:26:05.27 behave in their natural environment. And actually watch the
00:26:11.11 action potentials as they're firing, not with electrophysiology,
00:26:15.09 but with light. So, finally, I'd like to talk about how to analyze images.
00:26:25.07 Because in addition to the optics, I think it's very important that
00:26:29.09 if you're getting into microscopy, that you not only learn about
00:26:33.19 the optics, but you also learn to use image analysis software.
00:26:37.16 And we've discussed ImageJ in this course. And because
00:26:43.23 -- the reason for this is that in addition to just producing a
00:26:48.07 nice image, it's very important to extract as much quantitative
00:26:52.14 information out of that image as possible. And this is becoming easier
00:26:57.15 and easier to do, as well. This just shows the ImageJ program
00:27:01.24 that we used. And here, we recorded a movie of a single kinesin
00:27:08.07 molecule moving along a microtubule track. The software now
00:27:12.06 is able to identify in this image the single points of light that represent
00:27:17.25 the molecule. And now we can ask the program here to track
00:27:22.28 that molecule position over time, so we can get an x-y position
00:27:31.13 readout. And from this, we can extract a lot of quantitative
00:27:36.05 information, like how fast that kinesin molecule is moving,
00:27:39.06 or if it changes its behavior in motion. And the main thing to say is
00:27:43.26 that every kind of question that you want to answer with microscopy
00:27:48.23 will probably have a different type of way of approaching it using
00:27:53.23 analysis. So there's not one analysis that will fit all kinds of
00:27:57.23 images, and you'll have to go out there and do some research
00:28:01.00 about what kind of image analysis best suits the kind of quantitation
00:28:05.21 that you want to do. So, in conclusion, yes, it's a very exciting time
00:28:12.07 in microscopy today. There are so many different techniques that are
00:28:16.10 out there. But all these techniques are really to your benefit of
00:28:21.04 being able to look at living samples in many different ways.
00:28:26.18 And if you get to know these different techniques, it won't
00:28:30.14 seem like a jungle to you anymore. And rather, it will allow
00:28:34.11 you to produce fascinating images and get information on how
00:28:40.21 living systems work. Thank you very much.

This Talk
Speaker: Ron Vale
Audience:
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: February 2013
More Talks in Short Microscopy Series
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Talk Overview

In this concluding lecture about choosing the right microscopy technique, Ron Vale summarizes the main techniques covered in the short course and provides an overview of when a specific microscopy method might be advantageous for a particular sample or biological experiment.

Questions

  1. Which of the following methods is best for fluorescence microscopy of thicker samples?
    1. Total internal reflection fluorescence
    2. Multi-photon
    3. Spinning disk confocal
    4. Point scanning confocal
    5. Epifluorescence
  2. Which of the following methods might be good for measuring biochemical reactions at a surface using fluorescence?
    1. Total internal reflection fluorescence
    2. Multi-photon
    3. Spinning disk confocal
    4. Point scanning confocal
    5. Epifluorescence
  3. Which of the following techniques might be well suited for examining mitochondrial dynamics in a living cell in culture with minimal photodamage?
    1. Total internal reflection fluorescence
    2. Multi-photon
    3. Spinning disk confocal
    4. Point scanning confocal
    5. Epifluorescence
  4. Which of the following techniques can be used to measure the dynamics of a chromosomal protein using FRAP (fluorescence recovery after photobleaching)?
    1. Total internal reflection fluorescence
    2. Multi-photon
    3. Spinning disk confocal
    4. Point scanning confocal
    5. Epifluorescence
  5. You are trying to resolve details of protein complexes that you suspect are spaced ~50 nm apart on the mitochondrial membrane of a Hela cell in culture. Which technique might be best suited for this project?
    1. STORM
    2. 2-Photon
    3. Structured illumination
    4. Spinning disk confocal
    5. Dark field microscopy
  6. Which of the following forms of microscopy routinely involves laser illumination?
    1. Phase contrast
    2. Spinning disk confocal
    3. Deconvolution
    4. Array Tomography
    5. Dark field
  7. True or False: Spinning disk confocal has an equivalent depth of imaging as point scanning confocal microscopy.

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Speaker Bio

Ron Vale

Ron Vale

Professor of the Department of Cellular and Molecular Pharmacology; Investigator in the Howard Hughes Medical Institute
University of California, San Francisco Continue Reading

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