WEBVTT 00:00:16.763 --> 00:00:17.430 ADAM MARTIN: OK. 00:00:17.430 --> 00:00:19.980 So I'm going to start out today's lecture 00:00:19.980 --> 00:00:23.190 on the wrong foot by quoting somebody 00:00:23.190 --> 00:00:25.800 that you guys probably don't know 00:00:25.800 --> 00:00:28.590 and who was a New York Yankee. 00:00:28.590 --> 00:00:34.580 So Yogi Berra, the famous Yankee catcher once said, 00:00:34.580 --> 00:00:38.130 "You can observe a lot by watching," OK? 00:00:38.130 --> 00:00:40.560 And that is very appropriate for biology 00:00:40.560 --> 00:00:46.350 because a lot of things in biology 00:00:46.350 --> 00:00:50.940 have been discovered simply by watching for them in cells 00:00:50.940 --> 00:00:55.620 or watching for them to happen at the molecular level. 00:00:55.620 --> 00:01:00.120 And so our ability to visualize and see 00:01:00.120 --> 00:01:04.050 what's going on inside cells and at the molecular level 00:01:04.050 --> 00:01:06.630 is really critical for the process 00:01:06.630 --> 00:01:09.510 of biological discovery. 00:01:09.510 --> 00:01:13.530 So today I'm going to tell you about tools, 00:01:13.530 --> 00:01:17.940 both sort of older tools but also kind of the cutting edge, 00:01:17.940 --> 00:01:21.210 for how biologists are really observing 00:01:21.210 --> 00:01:28.080 what's going on in living cells and in life in general. 00:01:28.080 --> 00:01:28.590 OK. 00:01:28.590 --> 00:01:34.290 So let me start by just having you guys think a little bit. 00:01:34.290 --> 00:01:40.530 What do you require of me to see what I write on the board? 00:01:40.530 --> 00:01:41.240 Yeah, Rachel. 00:01:41.240 --> 00:01:42.090 AUDIENCE: Light. 00:01:42.090 --> 00:01:42.450 ADAM MARTIN: What's that? 00:01:42.450 --> 00:01:43.470 AUDIENCE: Light. 00:01:43.470 --> 00:01:44.770 ADAM MARTIN: You need light. 00:01:44.770 --> 00:01:47.610 And what does the light help you to do? 00:01:52.470 --> 00:01:53.136 What's that? 00:01:53.136 --> 00:01:55.580 AUDIENCE: [INAUDIBLE] 00:01:55.580 --> 00:01:58.820 ADAM MARTIN: You need it to see the board. 00:01:58.820 --> 00:02:01.140 And so let's say the light's on, OK? 00:02:07.020 --> 00:02:11.150 Is that, can you read this? 00:02:11.150 --> 00:02:13.560 No, what's the problem? 00:02:13.560 --> 00:02:14.060 What's that? 00:02:14.060 --> 00:02:16.130 Size, right? 00:02:16.130 --> 00:02:18.440 So Natalie suggested size. 00:02:18.440 --> 00:02:21.020 Right, so one thing that you need 00:02:21.020 --> 00:02:24.230 is some amount of magnification, right? 00:02:28.700 --> 00:02:32.000 But let's take another-- 00:02:32.000 --> 00:02:34.550 let's say I do magnify this. 00:02:34.550 --> 00:02:36.610 What if I magnify it? 00:02:36.610 --> 00:02:40.970 And I'm going to start writing my notes on the board, right? 00:02:40.970 --> 00:02:41.700 How is this? 00:02:41.700 --> 00:02:42.200 Helpful? 00:02:46.310 --> 00:02:47.530 Jeremy, what's wrong? 00:02:47.530 --> 00:02:50.370 AUDIENCE: Differentiate [INAUDIBLE].. 00:02:50.370 --> 00:02:51.120 ADAM MARTIN: Yeah. 00:02:51.120 --> 00:02:52.830 You have to be able to distinguish 00:02:52.830 --> 00:02:56.400 different objects, in this case, these letters, right? 00:02:56.400 --> 00:03:00.600 So in addition to just magnifying it, 00:03:00.600 --> 00:03:04.410 you also need the structures to be far enough apart such 00:03:04.410 --> 00:03:06.190 that you can distinguish them. 00:03:06.190 --> 00:03:09.690 So you need what is known as resolution. 00:03:13.314 --> 00:03:14.220 OK. 00:03:14.220 --> 00:03:16.170 This was resolution. 00:03:16.170 --> 00:03:18.630 But this is resolution where the letters are actually 00:03:18.630 --> 00:03:20.370 resolved, OK? 00:03:20.370 --> 00:03:30.750 So structures need to be far enough apart 00:03:30.750 --> 00:03:31.980 so that you can resolve them. 00:03:36.960 --> 00:03:39.810 Now let's come back to Rachel's point, right? 00:03:39.810 --> 00:03:44.760 Why is it that we need light to see what's on the board? 00:03:47.650 --> 00:03:48.150 Right? 00:03:51.630 --> 00:03:53.145 What if I draw without pressing? 00:03:57.010 --> 00:03:57.510 Right? 00:03:57.510 --> 00:03:58.683 Is that-- yeah, Orey. 00:03:58.683 --> 00:03:59.850 AUDIENCE: You need contrast. 00:03:59.850 --> 00:04:01.142 ADAM MARTIN: You need contrast. 00:04:01.142 --> 00:04:02.090 Exactly, right? 00:04:02.090 --> 00:04:05.550 The light sort of gives you contrast between the chalk 00:04:05.550 --> 00:04:07.920 and the black part of the board. 00:04:07.920 --> 00:04:09.555 So you also need contrast. 00:04:13.440 --> 00:04:17.550 And contrast is the ability to-- 00:04:17.550 --> 00:04:21.750 the structures need to be differentiated 00:04:21.750 --> 00:04:23.090 from the background, OK? 00:04:23.090 --> 00:04:33.700 So structures need to be different from background. 00:04:40.700 --> 00:04:42.590 What else do you need to read my writing? 00:04:51.550 --> 00:04:52.050 Right? 00:04:52.050 --> 00:04:53.905 What if I were just to-- 00:05:04.110 --> 00:05:06.340 everyone can read that? 00:05:06.340 --> 00:05:07.780 What's wrong? 00:05:07.780 --> 00:05:08.290 Carlos? 00:05:08.290 --> 00:05:10.010 AUDIENCE: Needs to be clear and legible-- 00:05:10.010 --> 00:05:10.630 ADAM MARTIN: What's that? 00:05:10.630 --> 00:05:11.130 Yeah. 00:05:11.130 --> 00:05:13.780 I need to have, like, good handwriting, right? 00:05:13.780 --> 00:05:20.680 So I like to think of this as this is an aspect of sample 00:05:20.680 --> 00:05:22.720 preservation, OK? 00:05:22.720 --> 00:05:27.070 So there's a sample preservation issue. 00:05:27.070 --> 00:05:29.840 I can't butcher the letters and the words. 00:05:32.450 --> 00:05:32.950 OK. 00:05:32.950 --> 00:05:37.330 So in the process of doing all these other things, right, 00:05:37.330 --> 00:05:40.480 magnifying your image, resolving things in your image, 00:05:40.480 --> 00:05:45.130 and generating contrast, you can't destroy your sample such 00:05:45.130 --> 00:05:48.550 that it's illegible, basically, OK? 00:05:48.550 --> 00:06:00.880 So in this case, structure must be preserved while doing one 00:06:00.880 --> 00:06:02.620 through three on this list. 00:06:08.830 --> 00:06:10.120 OK. 00:06:10.120 --> 00:06:13.780 So I'm going to start with resolution. 00:06:13.780 --> 00:06:18.790 We'll talk about, what are the limits to resolving things 00:06:18.790 --> 00:06:20.500 in biological specimens. 00:06:26.440 --> 00:06:30.040 And in biology, the one instrument 00:06:30.040 --> 00:06:33.100 we use a lot is a microscope, OK? 00:06:33.100 --> 00:06:35.590 And a microscope is basically a collection 00:06:35.590 --> 00:06:40.210 of lenses that allow you to do many of the things I just 00:06:40.210 --> 00:06:43.240 drew on the board. 00:06:43.240 --> 00:06:46.510 I'll point out a couple sort of broad sort 00:06:46.510 --> 00:06:48.890 of types of microscopy. 00:06:48.890 --> 00:06:52.660 So the human eye up here can resolve up 00:06:52.660 --> 00:06:55.810 to about 100 to 200 microns, if you're 00:06:55.810 --> 00:06:59.230 looking at something at reading distant distance, right? 00:06:59.230 --> 00:07:02.200 But cells are like way smaller than that, right? 00:07:02.200 --> 00:07:05.060 So we need some sort of instrument 00:07:05.060 --> 00:07:07.630 that allows us to see things that 00:07:07.630 --> 00:07:12.340 is lower than the resolution limit of a human eye. 00:07:12.340 --> 00:07:15.760 And so one way is to use a light microscope 00:07:15.760 --> 00:07:20.140 where you're using visible light to observe your sample. 00:07:20.140 --> 00:07:21.730 And many of the images that you're 00:07:21.730 --> 00:07:23.740 seeing that we're showing you are 00:07:23.740 --> 00:07:28.210 from visible light microscopes. 00:07:28.210 --> 00:07:32.170 To see smaller things, type of microscope that's often used 00:07:32.170 --> 00:07:34.500 is an electron microscope. 00:07:34.500 --> 00:07:38.650 And electron microscopy allows us to observe structures 00:07:38.650 --> 00:07:42.880 all the way down to the sub nanometer level of resolution, 00:07:42.880 --> 00:07:44.020 OK? 00:07:44.020 --> 00:07:47.680 Now one limitation to the electron microscope 00:07:47.680 --> 00:07:50.560 is, you have to kill the sample, OK? 00:07:50.560 --> 00:07:55.150 So that can lead to artifacts and problems. 00:07:55.150 --> 00:08:00.100 And we'll discuss away at the end where light microscopy is 00:08:00.100 --> 00:08:02.590 being extended down to the limits 00:08:02.590 --> 00:08:07.430 that approximate that of an electron microscope. 00:08:07.430 --> 00:08:07.930 OK. 00:08:07.930 --> 00:08:12.500 So what determines then, the resolution of a microscope? 00:08:12.500 --> 00:08:16.510 And so I'm going to sort of define 00:08:16.510 --> 00:08:21.680 a measurement of resolution which I'll call the d-min, 00:08:21.680 --> 00:08:23.870 or minimum distance. 00:08:23.870 --> 00:08:33.410 And this will be the minimum distance between two points 00:08:33.410 --> 00:08:34.285 that can be resolved. 00:08:48.450 --> 00:08:48.950 OK. 00:08:48.950 --> 00:08:52.910 And what I showed you on that past slide is basically 00:08:52.910 --> 00:08:56.330 the limit on the right here is the d-min 00:08:56.330 --> 00:09:00.710 for these different types of microscopy techniques, OK? 00:09:00.710 --> 00:09:03.890 And what that means is, so the minimum distance would be, 00:09:03.890 --> 00:09:08.180 if your minimum distance is 200 nanometers, if two points are 00:09:08.180 --> 00:09:11.540 greater than 200 nanometers apart from each other, 00:09:11.540 --> 00:09:15.760 then you can distinguish them as two different objects. 00:09:15.760 --> 00:09:19.880 However, if they are closer than 200 nanometers together, 00:09:19.880 --> 00:09:21.710 you wouldn't be able to see that these 00:09:21.710 --> 00:09:23.180 are two different objects. 00:09:23.180 --> 00:09:26.750 They would be overlapping each other, OK? 00:09:26.750 --> 00:09:31.160 And typically, the d-min for a light microscope 00:09:31.160 --> 00:09:34.670 is around 200 nanometers, OK? 00:09:34.670 --> 00:09:41.075 And the d-min results, if we are to determine-- 00:09:41.075 --> 00:09:44.330 if I'm to tell you what determines 00:09:44.330 --> 00:09:48.350 this minimum distance, we have to think about a microscope. 00:09:48.350 --> 00:09:52.220 So here, I'm drawing a specimen here. 00:09:52.220 --> 00:09:54.140 I've just drawn my specimen. 00:09:54.140 --> 00:09:56.540 It's on a slide or a cover slip. 00:09:56.540 --> 00:09:57.830 Here's your specimen here. 00:10:00.590 --> 00:10:02.990 And you might have a light source 00:10:02.990 --> 00:10:04.070 to generate the contrast. 00:10:06.770 --> 00:10:11.150 And then there'd be some sort of objective lens 00:10:11.150 --> 00:10:15.710 underneath the slide and the specimen. 00:10:15.710 --> 00:10:17.390 So this would be an objective lens. 00:10:22.670 --> 00:10:25.790 Sorry about my sample preservation here. 00:10:25.790 --> 00:10:28.700 And so the light is going to be hitting the sample. 00:10:28.700 --> 00:10:32.120 And the objective lens will be collecting 00:10:32.120 --> 00:10:37.240 a cone of light that's going into the lens here, OK? 00:10:37.240 --> 00:10:41.430 And maybe I'll magnify this a bit so you can see it better. 00:10:41.430 --> 00:10:45.540 So I'm just going to magnify this region over here. 00:10:45.540 --> 00:10:47.840 So if this is my specimen, I'm going 00:10:47.840 --> 00:10:51.410 to draw the objective a little farther away this time. 00:10:51.410 --> 00:10:52.730 This is the objective. 00:10:55.330 --> 00:10:57.320 And the objective is able to capture 00:10:57.320 --> 00:11:00.020 a range of different angles of light 00:11:00.020 --> 00:11:02.690 that come from the specimen, OK? 00:11:02.690 --> 00:11:06.230 So it's collecting angles. 00:11:06.230 --> 00:11:09.030 And I'll just define here an angle theta, 00:11:09.030 --> 00:11:14.240 which is like the 1/2 angle of this whole cone of light, OK? 00:11:14.240 --> 00:11:16.880 So what determines the resolution limit 00:11:16.880 --> 00:11:27.470 in this type of system is first of all, the wavelength 00:11:27.470 --> 00:11:30.950 of the light that's used. 00:11:30.950 --> 00:11:35.150 So if you're using white light, that might be from 400 to 800. 00:11:35.150 --> 00:11:37.430 If you're exciting GFP, you're going 00:11:37.430 --> 00:11:41.780 to excite with a wavelength that's 488 nanometers. 00:11:41.780 --> 00:11:46.310 So it's usually around maybe between 450 and 550 nanometers 00:11:46.310 --> 00:11:48.780 for many different fluorescent proteins. 00:11:48.780 --> 00:11:49.280 OK. 00:11:49.280 --> 00:12:01.580 So lambda here is the wavelength of the excitation 00:12:01.580 --> 00:12:02.885 of the light you're using. 00:12:09.920 --> 00:12:18.080 And this is all then divided by 2 times the NA, which is 00:12:18.080 --> 00:12:20.510 a property of the subjective. 00:12:20.510 --> 00:12:27.890 And what NA is, NA stands for numerical aperture. 00:12:35.090 --> 00:12:38.810 And what that is, is basically the range of angles 00:12:38.810 --> 00:12:42.800 that this objective can collect, OK? 00:12:42.800 --> 00:12:50.330 So it's N sine theta, where theta is this angle here. 00:12:50.330 --> 00:12:53.690 So you get the best performance if the objective 00:12:53.690 --> 00:12:56.720 can collect all of the light that comes 00:12:56.720 --> 00:12:59.530 from this side of the sample. 00:12:59.530 --> 00:13:13.580 OK, and then N refers to the refractive index of the media 00:13:13.580 --> 00:13:15.020 that this light is going through. 00:13:18.300 --> 00:13:18.800 OK. 00:13:18.800 --> 00:13:21.860 And so if you have an objective and you 00:13:21.860 --> 00:13:24.920 have your sample in here and there's 00:13:24.920 --> 00:13:27.390 a slide and a cover slip-- 00:13:27.390 --> 00:13:29.300 I'll extend this out-- 00:13:29.300 --> 00:13:32.850 you often have immersion oil. 00:13:32.850 --> 00:13:35.030 There'd be some sort of immersion media here. 00:13:41.510 --> 00:13:44.000 And I don't know if you've ever used a microscope that's 00:13:44.000 --> 00:13:46.190 meant to be used with immersion media 00:13:46.190 --> 00:13:47.950 and you don't add that immersion media. 00:13:47.950 --> 00:13:51.350 But your image quality, if you don't add that media, 00:13:51.350 --> 00:13:53.510 is like really bad, right? 00:13:53.510 --> 00:13:57.800 And that's because you're affecting 00:13:57.800 --> 00:14:03.830 the numerical aperture of what this lens can collect. 00:14:03.830 --> 00:14:07.760 And therefore, you degrade your image quality, OK? 00:14:07.760 --> 00:14:11.120 But basically, the more light, the more angles of 00:14:11.120 --> 00:14:16.280 light that you collect, the higher the numerical aperture. 00:14:16.280 --> 00:14:19.560 And therefore, the lower this d-min is going to be. 00:14:19.560 --> 00:14:23.990 And so the greater you'll be able to resolve objects that 00:14:23.990 --> 00:14:26.045 are near each other in space. 00:14:29.850 --> 00:14:30.690 OK. 00:14:30.690 --> 00:14:35.190 So the take home message from all of this 00:14:35.190 --> 00:14:38.430 is that you notice that magnification is not 00:14:38.430 --> 00:14:39.600 a part of this. 00:14:39.600 --> 00:14:43.590 But the wavelength of the light is really critical, OK? 00:14:43.590 --> 00:14:46.470 So usually, this minimum distance 00:14:46.470 --> 00:14:48.870 ends up being the wavelength of light 00:14:48.870 --> 00:14:51.870 that you're using divided by 2. 00:14:51.870 --> 00:14:56.940 And this usually ends up being about 200 nanometers. 00:14:56.940 --> 00:15:00.720 So that's the diffraction limit of a light microscope, 00:15:00.720 --> 00:15:02.370 as you see up there. 00:15:02.370 --> 00:15:09.240 And this resolution is basically limited by the diffraction 00:15:09.240 --> 00:15:11.490 or behavior of light, OK? 00:15:11.490 --> 00:15:18.330 So light microscopy is limited by the diffraction of light. 00:15:28.200 --> 00:15:29.705 And it was thought for a long time 00:15:29.705 --> 00:15:31.080 that no matter what you do, you'd 00:15:31.080 --> 00:15:34.782 never be able to break this limit of about 200 nanometers. 00:15:34.782 --> 00:15:36.240 But at the end of the lecture, I'll 00:15:36.240 --> 00:15:39.630 tell you about some very smart people who figured out a way 00:15:39.630 --> 00:15:41.340 to actually break this limit. 00:15:41.340 --> 00:15:44.280 And we'll talk about how they were able to do that. 00:15:46.890 --> 00:15:50.100 Now I want to talk about a few other limitations 00:15:50.100 --> 00:15:54.180 of microscopy. 00:15:54.180 --> 00:15:57.510 And I'm starting by showing you this electron micrograph 00:15:57.510 --> 00:16:00.810 of the endoplasmic reticulum. 00:16:00.810 --> 00:16:04.080 And one important consideration you have to make 00:16:04.080 --> 00:16:10.230 is two dimensional versus three dimensional structure. 00:16:10.230 --> 00:16:13.440 So for electron microscopy, you basically 00:16:13.440 --> 00:16:16.770 cut the sample so you have a very thin slice of it. 00:16:16.770 --> 00:16:20.010 It's like slicing bread except these slices are 00:16:20.010 --> 00:16:24.270 on the order of 30 to 60 nanometers in height. 00:16:24.270 --> 00:16:27.990 And then you pass an electron beam through the sample 00:16:27.990 --> 00:16:32.400 after it's stained in order to visualize your specimen. 00:16:32.400 --> 00:16:34.380 And one thing you have to keep in mind 00:16:34.380 --> 00:16:37.890 is that, you're looking at a slice through this. 00:16:37.890 --> 00:16:41.400 And it doesn't give you three dimensional information, OK? 00:16:41.400 --> 00:16:46.110 So if we were to think about the endoplasmic reticulum, 00:16:46.110 --> 00:16:48.811 you might have an endoplasmic reticulum. 00:16:52.180 --> 00:16:55.900 And if you take an optical slice through this, 00:16:55.900 --> 00:16:58.360 then you would see something like this where you see each 00:16:58.360 --> 00:16:59.920 of the stacks individually. 00:17:09.230 --> 00:17:12.349 And so this might make you to conclude 00:17:12.349 --> 00:17:15.200 that the way that the endoplasmic reticulum is 00:17:15.200 --> 00:17:19.310 structured is it's kind of like a stack of pancakes, where each 00:17:19.310 --> 00:17:22.940 of these, you have a lipid bilayer 00:17:22.940 --> 00:17:25.210 surrounding a lumen of the ER. 00:17:25.210 --> 00:17:26.810 So right, the lumen would be inside 00:17:26.810 --> 00:17:29.303 like this for each of these. 00:17:29.303 --> 00:17:31.220 And they're just stacked on top of each other. 00:17:34.020 --> 00:17:38.000 And this is the textbook model for endoplasmic reticulum 00:17:38.000 --> 00:17:38.990 structure. 00:17:38.990 --> 00:17:44.330 But it was actually, if you don't consider this in 3D, 00:17:44.330 --> 00:17:45.410 you might miss something. 00:17:45.410 --> 00:17:49.850 And what was missed was reported in 2013 00:17:49.850 --> 00:17:52.490 in this paper, where rather than just taking 00:17:52.490 --> 00:17:57.140 a single slice, what they did is they made lots of slices. 00:17:57.140 --> 00:17:59.400 And they kept track of where they are. 00:17:59.400 --> 00:18:03.770 So they basically did a three dimensional reconstruction 00:18:03.770 --> 00:18:06.830 of the endoplasmic reticulum. 00:18:06.830 --> 00:18:10.040 And by imaging this other dimension, 00:18:10.040 --> 00:18:12.350 they came to a very different conclusion 00:18:12.350 --> 00:18:15.860 about how the endoplasmic reticulum is organized. 00:18:15.860 --> 00:18:19.460 And instead of being stacks of membranes 00:18:19.460 --> 00:18:22.430 on top of each other like pancakes, 00:18:22.430 --> 00:18:24.140 instead, it's a helicoid. 00:18:24.140 --> 00:18:28.940 So this is an ER from a professional secretory cell, 00:18:28.940 --> 00:18:30.990 like a salivary gland cell. 00:18:30.990 --> 00:18:34.130 And you can see in 3D, you get a very different picture 00:18:34.130 --> 00:18:36.830 of the organization of this organelle. 00:18:36.830 --> 00:18:42.920 It's actually wrapped around and spiraling membrane stacks, OK? 00:18:42.920 --> 00:18:45.530 So their model is that, basically 00:18:45.530 --> 00:18:48.890 the endoplasmic reticulum in some cell types 00:18:48.890 --> 00:18:54.290 basically has a parking garage like structure, OK? 00:18:54.290 --> 00:18:56.150 So in this case, in these cells, it 00:18:56.150 --> 00:19:01.950 seems like the ER Is basically a parking garage for ribosomes. 00:19:01.950 --> 00:19:02.450 OK. 00:19:02.450 --> 00:19:04.010 And you don't get that information 00:19:04.010 --> 00:19:06.110 unless you consider the three dimensional 00:19:06.110 --> 00:19:11.940 structure of the thing that you're looking at. 00:19:11.940 --> 00:19:15.170 So in addition to electron microscopy, 00:19:15.170 --> 00:19:19.220 there are techniques that involve light microscopy that 00:19:19.220 --> 00:19:21.590 involve optical sectioning. 00:19:21.590 --> 00:19:24.680 And so normally, if you're looking at fluorescence, 00:19:24.680 --> 00:19:26.930 if you're doing fluorescence microscopy, 00:19:26.930 --> 00:19:30.290 you'd be exciting the whole volume of your sample 00:19:30.290 --> 00:19:32.660 and exciting all of the fluorophores 00:19:32.660 --> 00:19:35.630 such that fluorescence from out of the focal plane 00:19:35.630 --> 00:19:37.610 would be getting into your image. 00:19:37.610 --> 00:19:41.360 And that would give you a much more hazy, unclear image. 00:19:41.360 --> 00:19:44.360 But there are techniques such as confocal fluorescence 00:19:44.360 --> 00:19:48.920 microscopy that allow you to exclude the out of focus light 00:19:48.920 --> 00:19:52.130 such that you're basically getting an optical section 00:19:52.130 --> 00:19:53.270 through your sample. 00:19:53.270 --> 00:19:56.570 And that can give you a much cleaner and better resolved 00:19:56.570 --> 00:19:57.700 image, OK? 00:20:00.800 --> 00:20:06.470 Now I want to talk a little bit about another dimension, which 00:20:06.470 --> 00:20:08.870 is time. 00:20:08.870 --> 00:20:12.950 And again, you're seeing images in textbooks. 00:20:12.950 --> 00:20:15.500 And usually, you're just seeing a single image. 00:20:15.500 --> 00:20:17.720 And whenever you see a single image, 00:20:17.720 --> 00:20:19.190 you have to think about how things 00:20:19.190 --> 00:20:23.820 might be changing in time in order to understand the system. 00:20:23.820 --> 00:20:27.800 So one example that I like here is shown here, 00:20:27.800 --> 00:20:29.750 where these are different proteins that 00:20:29.750 --> 00:20:32.300 are labeled in a yeast cell. 00:20:32.300 --> 00:20:34.970 And you see that these proteins form patches 00:20:34.970 --> 00:20:37.010 at the edge of the yeast cell. 00:20:37.010 --> 00:20:40.490 And some of these patches just contain the green protein, 00:20:40.490 --> 00:20:42.260 which is SLA1. 00:20:42.260 --> 00:20:47.300 And other patches contain just the red protein which is ABP1. 00:20:47.300 --> 00:20:52.280 And there's another class of patch which contains both, OK? 00:20:52.280 --> 00:20:58.573 So how might you interpret this fixed image over here? 00:20:58.573 --> 00:21:00.365 What might be one model you would conclude? 00:21:07.180 --> 00:21:10.270 Well, what was initially concluded 00:21:10.270 --> 00:21:14.320 from this type of experiment is you have three different types 00:21:14.320 --> 00:21:17.950 of patches that are distinct from each other in the cell 00:21:17.950 --> 00:21:21.790 because they have different molecular compositions, OK? 00:21:21.790 --> 00:21:23.830 And that was what was initially thought. 00:21:23.830 --> 00:21:26.320 But it was wrong because researchers 00:21:26.320 --> 00:21:31.710 had to really consider the aspect of time in this problem. 00:21:31.710 --> 00:21:34.900 And I'm going to show you a movie now over here 00:21:34.900 --> 00:21:37.930 where you're going to see this yeast cell. 00:21:37.930 --> 00:21:40.360 And now you have these different proteins 00:21:40.360 --> 00:21:44.830 tagged with different fluorescent proteins. 00:21:44.830 --> 00:21:49.000 And we can watch them in time as they progress 00:21:49.000 --> 00:21:53.110 through a stereotypic cycle. 00:21:53.110 --> 00:21:55.480 So what you're going to notice in this movie 00:21:55.480 --> 00:21:59.440 is that you see these green patches appear. 00:21:59.440 --> 00:22:04.240 And every single green patch at some point is joined by red. 00:22:04.240 --> 00:22:07.330 And then the green disappears and the red stays around. 00:22:07.330 --> 00:22:10.570 And then the thing disassembles, OK? 00:22:10.570 --> 00:22:14.980 So what was initially thought to be three different structures 00:22:14.980 --> 00:22:17.410 in the cell, eventually, it was found out 00:22:17.410 --> 00:22:19.900 that there was a dynamic process where 00:22:19.900 --> 00:22:22.870 this patch sort of matured over time 00:22:22.870 --> 00:22:24.970 and eventually disappeared into the cell. 00:22:24.970 --> 00:22:29.170 And what this process is, is actually endocytosis in yeast. 00:22:29.170 --> 00:22:30.910 And you're seeing different proteins 00:22:30.910 --> 00:22:33.670 getting recruited to endocytic vesicles 00:22:33.670 --> 00:22:37.910 as they bud from the plasma membrane of this yeast. 00:22:37.910 --> 00:22:38.410 OK. 00:22:38.410 --> 00:22:42.970 So that's just my caution in interpreting fixed images, 00:22:42.970 --> 00:22:45.040 because you have to think about how 00:22:45.040 --> 00:22:46.750 they might be changing in time. 00:22:49.280 --> 00:22:49.780 All right. 00:22:49.780 --> 00:22:51.715 So now we have to consider contrast. 00:23:00.748 --> 00:23:08.170 And in bright field microscopy, and bright field microscopy 00:23:08.170 --> 00:23:13.390 basically involves white light as your light source. 00:23:16.080 --> 00:23:22.090 And so you'd have a microscope that has a white light source. 00:23:22.090 --> 00:23:24.095 You might have your specimen here. 00:23:24.095 --> 00:23:24.970 Here's your specimen. 00:23:29.660 --> 00:23:32.020 Then you'd have some sort of detector 00:23:32.020 --> 00:23:33.150 at the end of your system. 00:23:38.070 --> 00:23:41.250 And there would be some objective lens in between, 00:23:41.250 --> 00:23:43.680 which I'm going to ignore for now. 00:23:43.680 --> 00:23:47.010 And so, for bright field microscopy, 00:23:47.010 --> 00:23:51.030 you're taking a sample and shining it with light. 00:23:51.030 --> 00:23:53.150 The light that doesn't go through your sample 00:23:53.150 --> 00:23:55.330 will go right through to the detector. 00:23:55.330 --> 00:23:56.760 And that's your background. 00:23:56.760 --> 00:24:00.390 But some of this light, the light that's going and hitting 00:24:00.390 --> 00:24:06.890 your sample, could be absorbed or it could be refracted. 00:24:06.890 --> 00:24:09.690 And it's the refraction or absorption 00:24:09.690 --> 00:24:13.020 of this light which generates the contrast for bright field 00:24:13.020 --> 00:24:14.380 microscopy. 00:24:14.380 --> 00:24:24.270 So in bright field, native structures in the cell 00:24:24.270 --> 00:24:34.290 absorb or refract light. 00:24:34.290 --> 00:24:36.390 And this is what generates the contrast. 00:24:40.300 --> 00:24:40.800 OK. 00:24:40.800 --> 00:24:44.910 So the images shown up here are bright field images of cells. 00:24:44.910 --> 00:24:47.490 And in each of these cases, there's no dye. 00:24:47.490 --> 00:24:49.200 There's no fluorescent protein. 00:24:49.200 --> 00:24:52.020 But you're able to see the outline of the cell. 00:24:52.020 --> 00:24:56.340 And you're able to see even individual organelles 00:24:56.340 --> 00:24:59.160 or structures within the cell that 00:24:59.160 --> 00:25:03.790 are interacting with the white light and generating contrast, 00:25:03.790 --> 00:25:05.040 OK? 00:25:05.040 --> 00:25:07.760 So that's one way to generate contrast 00:25:07.760 --> 00:25:12.600 is just hope that whatever is native in your cell generates 00:25:12.600 --> 00:25:13.500 the contrast. 00:25:13.500 --> 00:25:15.690 But there are also-- 00:25:15.690 --> 00:25:20.415 you can increase contrast in specimens by adding dyes. 00:25:25.380 --> 00:25:28.500 And if these dyes bind to specific structures 00:25:28.500 --> 00:25:32.940 like a membrane, then that will increase your contrast. 00:25:32.940 --> 00:25:37.450 So the electron microscopy images that I showed you-- 00:25:37.450 --> 00:25:41.460 so for electron microscopy, you generate contrast 00:25:41.460 --> 00:25:45.540 by adding a dye that is an electron-dense dye, which 00:25:45.540 --> 00:25:47.560 will bend the electron beam. 00:25:47.560 --> 00:25:53.340 And that's what allows you to get an image from an EM. 00:25:53.340 --> 00:26:04.230 So an EM contrast is from an electron-dense dye 00:26:04.230 --> 00:26:11.880 such as uranyl acetate or some other type of dye. 00:26:11.880 --> 00:26:19.050 Now, fluorescence microscopy, as Professor Imperiali showed you, 00:26:19.050 --> 00:26:21.300 involves taking a fluorescent molecule 00:26:21.300 --> 00:26:25.230 and attaching it to your protein of interest. 00:26:25.230 --> 00:26:29.410 So you're actually getting protein-specific contrast, 00:26:29.410 --> 00:26:30.285 which is very useful. 00:26:37.220 --> 00:26:38.250 OK? 00:26:38.250 --> 00:26:40.530 And the way a fluorescence microscope works 00:26:40.530 --> 00:26:44.550 is just shown up here where you might have a light source that 00:26:44.550 --> 00:26:46.650 has a range of wavelengths. 00:26:46.650 --> 00:26:50.220 And you can use a filter to select one. 00:26:50.220 --> 00:26:54.180 In the case of GFP, it would be blue light or 488 nanometer 00:26:54.180 --> 00:26:55.140 light. 00:26:55.140 --> 00:26:59.190 And that would then be shined onto your specimen. 00:26:59.190 --> 00:27:02.190 And then the light is absorbed by fluorophores 00:27:02.190 --> 00:27:04.050 in your specimen. 00:27:04.050 --> 00:27:07.320 Some energy is lost, such that the light that's 00:27:07.320 --> 00:27:12.570 emitted from GFP is a longer wavelength, in this case, 00:27:12.570 --> 00:27:14.010 green. 00:27:14.010 --> 00:27:16.290 And then you can filter again to make 00:27:16.290 --> 00:27:19.830 sure only the green light is what goes to the detector. 00:27:19.830 --> 00:27:23.490 So this is a very efficient way of generating contrast 00:27:23.490 --> 00:27:26.220 because you can use filters to select 00:27:26.220 --> 00:27:29.070 only the wavelength of light that is emitted 00:27:29.070 --> 00:27:32.410 from your fluorescent molecule. 00:27:32.410 --> 00:27:32.910 OK. 00:27:32.910 --> 00:27:37.470 Any questions about that and about my very short version 00:27:37.470 --> 00:27:41.120 of how fluorescence microscopy works? 00:27:41.120 --> 00:27:41.770 Yeah, Rachel? 00:27:41.770 --> 00:27:45.460 AUDIENCE: [INAUDIBLE] dichroic mirror? 00:27:45.460 --> 00:27:49.200 ADAM MARTIN: The dichroic mirror reflects certain wavelengths 00:27:49.200 --> 00:27:51.120 that are below a certain wavelength. 00:27:51.120 --> 00:27:54.600 And will pass wavelengths that are above a wavelength. 00:27:54.600 --> 00:27:58.800 So it will basically reflect the excitation light. 00:27:58.800 --> 00:28:04.140 But it will pass the emitted light, OK? 00:28:04.140 --> 00:28:08.310 And so, there are tons of these mirrors. 00:28:08.310 --> 00:28:12.180 Some are not dichroic, but they can 00:28:12.180 --> 00:28:13.920 reflect four different wavelengths 00:28:13.920 --> 00:28:16.200 and pass all other wavelengths. 00:28:16.200 --> 00:28:19.350 And so, this allows you to image multiple fluorophores 00:28:19.350 --> 00:28:21.810 at the same time, OK? 00:28:21.810 --> 00:28:24.000 The specifics aren't as important 00:28:24.000 --> 00:28:28.450 as the general concept of how this works. 00:28:28.450 --> 00:28:28.950 OK. 00:28:28.950 --> 00:28:32.880 Now I want to come back to the resolution limit. 00:28:32.880 --> 00:28:36.600 And I want to tell you about how we can beat it. 00:28:36.600 --> 00:28:39.240 So, beating. 00:28:39.240 --> 00:28:40.650 We all like winning. 00:28:40.650 --> 00:28:43.640 So beating the diffraction limit. 00:28:49.530 --> 00:28:56.940 And this is going to involve a type of microscopy that's 00:28:56.940 --> 00:29:00.870 really sort of been developed in the past decade, which is known 00:29:00.870 --> 00:29:02.910 as super resolution microscopy. 00:29:05.820 --> 00:29:11.070 So super resolution microscopy. 00:29:11.070 --> 00:29:12.690 And remember, I mentioned for you 00:29:12.690 --> 00:29:17.340 before that yes, electron microscopy can 00:29:17.340 --> 00:29:20.310 get you nanometer resolution. 00:29:20.310 --> 00:29:24.000 But you have to kill the cell. 00:29:24.000 --> 00:29:28.080 And also, it's hard to get protein specific contrast, 00:29:28.080 --> 00:29:28.830 right? 00:29:28.830 --> 00:29:31.050 So that kind of sucks because as biologists, 00:29:31.050 --> 00:29:33.630 usually we're interested in how things are 00:29:33.630 --> 00:29:36.660 functioning to stay, to live. 00:29:36.660 --> 00:29:39.570 So wouldn't it be great if we could somehow 00:29:39.570 --> 00:29:45.000 use light microscopy to get down into this nanometer range 00:29:45.000 --> 00:29:48.690 so that we can see how individual proteins are 00:29:48.690 --> 00:29:51.420 interacting with each other and organized 00:29:51.420 --> 00:29:53.310 at the nanometer level, OK? 00:29:53.310 --> 00:29:54.990 And so in the past decade, there's 00:29:54.990 --> 00:29:58.080 really been a revolution that's enabled 00:29:58.080 --> 00:30:02.700 us to do light microscopy with a resolution that gets down 00:30:02.700 --> 00:30:06.120 to the 10 or even single nanometer resolution. 00:30:10.970 --> 00:30:14.330 And there's a number of different super resolution 00:30:14.330 --> 00:30:15.530 techniques. 00:30:15.530 --> 00:30:19.680 I'm going to talk about just one of them. 00:30:19.680 --> 00:30:22.130 But both these techniques basically 00:30:22.130 --> 00:30:27.770 use the same concept, which is that they enable whoever's 00:30:27.770 --> 00:30:31.910 doing it to identify single molecules 00:30:31.910 --> 00:30:35.990 and define where those molecules are very precisely. 00:30:35.990 --> 00:30:39.410 And to turn fluorescent molecules on and off 00:30:39.410 --> 00:30:42.170 so that you can select individual molecules such 00:30:42.170 --> 00:30:44.360 that you can see them. 00:30:44.360 --> 00:30:48.410 So these are two different techniques. 00:30:48.410 --> 00:30:50.790 They're conceptually very similar. 00:30:50.790 --> 00:30:53.030 I'm going to focus on this one here. 00:30:53.030 --> 00:30:57.100 But it's pretty much similar to this one up here. 00:30:59.830 --> 00:31:05.300 And I just want to point out that one of our colleagues 00:31:05.300 --> 00:31:10.250 here at MIT, Ibrahim Cisse who's in the physics department, 00:31:10.250 --> 00:31:14.870 his lab builds these super resolution microscopes. 00:31:14.870 --> 00:31:17.810 And they're using super resolution microscopy 00:31:17.810 --> 00:31:21.470 to study the collective behaviors of proteins, 00:31:21.470 --> 00:31:26.650 in his case, during the function of gene expression. 00:31:26.650 --> 00:31:30.665 OK, so this is research that's actively being pursued at MIT. 00:31:33.740 --> 00:31:36.710 So let's just do a thought experiment again. 00:31:43.710 --> 00:31:44.210 OK. 00:31:44.210 --> 00:31:48.530 I'm drawing a single molecule or what you would see in an image 00:31:48.530 --> 00:31:50.840 if you were looking at a single molecule GFP. 00:31:54.550 --> 00:31:55.050 Great. 00:31:55.050 --> 00:31:56.070 Where is GFP here? 00:32:01.460 --> 00:32:02.293 Carmen? 00:32:02.293 --> 00:32:03.960 AUDIENCE: It's right there on the board. 00:32:03.960 --> 00:32:06.043 ADAM MARTIN: It's right there on the board, right? 00:32:06.043 --> 00:32:08.540 Is it here? 00:32:08.540 --> 00:32:09.664 What's that? 00:32:09.664 --> 00:32:11.110 AUDIENCE: I don't know. 00:32:11.110 --> 00:32:15.160 ADAM MARTIN: Who thinks GFP is right here? 00:32:15.160 --> 00:32:16.990 Who does not think GFP is right there? 00:32:21.550 --> 00:32:23.640 You have to be thinking one or the other. 00:32:23.640 --> 00:32:24.722 Yeah, Rachel. 00:32:24.722 --> 00:32:26.410 AUDIENCE: [INAUDIBLE] 00:32:26.410 --> 00:32:27.340 ADAM MARTIN: OK. 00:32:27.340 --> 00:32:30.920 So what Rachel says is that it's probably not right here. 00:32:30.920 --> 00:32:34.180 It's probably in the middle of this thing, right? 00:32:34.180 --> 00:32:38.140 And so if you're seeing a diffraction limited spot, 00:32:38.140 --> 00:32:40.760 you're going to get some sort of Gaussian of intensity, 00:32:40.760 --> 00:32:42.010 which I didn't draw well here. 00:32:42.010 --> 00:32:44.230 But it might be a little bit brighter in the center 00:32:44.230 --> 00:32:48.010 and drop off as you go towards the edge, right? 00:32:48.010 --> 00:32:50.440 So if I were to take a image intensity 00:32:50.440 --> 00:32:53.920 profile along the line here, you'd 00:32:53.920 --> 00:32:57.520 see something that kind of looked like a Gaussian, OK? 00:32:57.520 --> 00:33:02.500 And GFP, if there's a single molecule that you're imaging, 00:33:02.500 --> 00:33:06.520 should be right in the center of this Gaussian, OK? 00:33:06.520 --> 00:33:10.930 And so even though we're not seeing a spot, 00:33:10.930 --> 00:33:14.440 we're seeing a spot that its width here 00:33:14.440 --> 00:33:17.170 is diffraction limited. 00:33:17.170 --> 00:33:19.900 So this width is 200 nanometers. 00:33:19.900 --> 00:33:24.370 But if we can estimate where the molecule is 00:33:24.370 --> 00:33:27.250 in this region with nanometer precision, 00:33:27.250 --> 00:33:29.560 we could get a very accurate view 00:33:29.560 --> 00:33:33.190 of where this fluorescent molecule is, OK? 00:33:37.890 --> 00:33:40.720 So it relies on certain assumptions. 00:33:40.720 --> 00:33:42.210 The first assumption is that you're 00:33:42.210 --> 00:33:47.820 assuming we can see single fluorescent molecules. 00:33:47.820 --> 00:33:55.230 So that we visualize single fluorescent molecules. 00:33:58.530 --> 00:34:01.680 And that we can then estimate with some amount of precision 00:34:01.680 --> 00:34:05.610 the location of the molecule based 00:34:05.610 --> 00:34:09.389 on this diffraction limited sort of image that we get. 00:34:09.389 --> 00:34:20.190 So then we have to estimate the location based on the image. 00:34:24.360 --> 00:34:25.199 OK. 00:34:25.199 --> 00:34:28.500 And then our resolution is basically the error 00:34:28.500 --> 00:34:31.830 in fitting this curve, OK? 00:34:31.830 --> 00:34:42.810 So the error in the fitted position 00:34:42.810 --> 00:34:47.649 is equal to the standard deviation of this Gaussian. 00:34:47.649 --> 00:34:51.989 The standard deviation divided by the square root 00:34:51.989 --> 00:34:53.850 of the number of photons that you 00:34:53.850 --> 00:34:56.280 collected to get that image. 00:34:56.280 --> 00:34:59.445 So the square root of the number of photons. 00:35:02.420 --> 00:35:03.150 OK. 00:35:03.150 --> 00:35:06.330 And I just told you in the beginning of the lecture 00:35:06.330 --> 00:35:08.700 that this standard deviation is limited 00:35:08.700 --> 00:35:10.500 by the diffraction of light. 00:35:10.500 --> 00:35:12.870 So the standard deviation is going 00:35:12.870 --> 00:35:16.050 to be around 200 nanometers, right? 00:35:16.050 --> 00:35:18.330 But if you collect a lot of photons, 00:35:18.330 --> 00:35:23.760 you can accurately figure out where the fluorescent molecule 00:35:23.760 --> 00:35:27.110 is here if you know that it's a single molecule. 00:35:27.110 --> 00:35:27.610 OK? 00:35:27.610 --> 00:35:34.440 So the number of photons in a typical experiment 00:35:34.440 --> 00:35:37.990 is going to be around 10 to the fourth, OK? 00:35:37.990 --> 00:35:41.880 And so if 200 nanometers by 10 to the fourth, 00:35:41.880 --> 00:35:45.270 you're going to have sub nanometer resolution 00:35:45.270 --> 00:35:48.010 if you do the experiment right. 00:35:48.010 --> 00:35:49.350 OK? 00:35:49.350 --> 00:35:52.230 So you really need to see fluorescent molecules, however, 00:35:52.230 --> 00:35:54.420 in order to do this, OK? 00:35:54.420 --> 00:35:58.140 And the real breakthrough came with the realization 00:35:58.140 --> 00:36:01.530 that you could combine this type of fitting 00:36:01.530 --> 00:36:05.160 to estimate the position of single molecules 00:36:05.160 --> 00:36:08.700 with a certain type of fluorescent protein 00:36:08.700 --> 00:36:13.690 where you can turn the protein on and off stochastically, OK? 00:36:13.690 --> 00:36:14.190 OK. 00:36:14.190 --> 00:36:16.710 So we need one more component which 00:36:16.710 --> 00:36:25.470 is a photo-activatable fluorescent protein, 00:36:25.470 --> 00:36:31.170 in this case, the first one was photo-activatable GFP PA-GFP. 00:36:35.730 --> 00:36:39.900 And PA-GFP is a fluorescent protein like GPF. 00:36:39.900 --> 00:36:41.760 It's genetically encoded. 00:36:41.760 --> 00:36:44.370 But when it matures, it's not fluorescent. 00:36:44.370 --> 00:36:46.530 It's in a dark state, OK? 00:36:46.530 --> 00:36:50.240 So when it matures, it's dark. 00:36:50.240 --> 00:36:53.280 It has a dark state. 00:36:53.280 --> 00:36:55.920 And it starts out in this dark state. 00:36:55.920 --> 00:36:57.810 But you can turn it on. 00:36:57.810 --> 00:37:00.850 And you can turn it on with light. 00:37:00.850 --> 00:37:02.850 So that's where the photo activation 00:37:02.850 --> 00:37:04.830 is, because you're able to photo activate 00:37:04.830 --> 00:37:06.810 this fluorescent molecule. 00:37:06.810 --> 00:37:13.110 And you can photo activate with sort of UV light or 405 00:37:13.110 --> 00:37:16.260 nanometer light. 00:37:16.260 --> 00:37:20.190 And so that's not normally the excitation wavelength. 00:37:20.190 --> 00:37:26.250 But if you shine your sample with 405 nanometer light, 00:37:26.250 --> 00:37:29.910 it will convert some set of your molecules 00:37:29.910 --> 00:37:33.440 into the now fluorescent state, OK? 00:37:33.440 --> 00:37:39.060 So this then causes it to be fluorescent. 00:37:42.288 --> 00:37:43.830 And now it's going to be lighting up. 00:37:51.160 --> 00:37:51.660 OK. 00:37:51.660 --> 00:37:54.690 And I want to thank Professor Cisse because he gave me 00:37:54.690 --> 00:37:56.640 the next slide which I think nicely 00:37:56.640 --> 00:37:58.535 shows how this technique works. 00:38:01.050 --> 00:38:06.270 So the way you can get super resolution 00:38:06.270 --> 00:38:08.580 is you can't be looking at all your fluorophores 00:38:08.580 --> 00:38:11.130 at once because they're not far enough apart 00:38:11.130 --> 00:38:13.170 and they'll all bleed together so that you 00:38:13.170 --> 00:38:15.390 get a bad image, right? 00:38:15.390 --> 00:38:17.670 So this would be your conventional diffraction 00:38:17.670 --> 00:38:20.280 limited image where all the fluorophores-- there's 00:38:20.280 --> 00:38:21.790 about 20 fluorophores here. 00:38:21.790 --> 00:38:24.270 And you can see, you can't see individual fluorophores 00:38:24.270 --> 00:38:27.930 and you can't see what that says, OK? 00:38:27.930 --> 00:38:31.290 But if you take a divide and conquer approach 00:38:31.290 --> 00:38:34.320 with this, if you have a photo-activatable GFP, 00:38:34.320 --> 00:38:36.910 you don't need to look at them all at once. 00:38:36.910 --> 00:38:40.710 You can just look at three to start, OK? 00:38:40.710 --> 00:38:43.860 So now if you only activate a small subset 00:38:43.860 --> 00:38:47.580 and you ensure that you're activating it at a frequency 00:38:47.580 --> 00:38:50.370 such that they're well resolved from each other, 00:38:50.370 --> 00:38:52.920 then you can distinguish that there are 00:38:52.920 --> 00:38:55.150 three single molecules here. 00:38:55.150 --> 00:38:57.330 You can fit where they are. 00:38:57.330 --> 00:39:00.330 And now you know where they are with nanometer precision. 00:39:00.330 --> 00:39:02.820 So you know where those are. 00:39:02.820 --> 00:39:05.400 And then you want to look at other molecules. 00:39:05.400 --> 00:39:07.050 And so you have to get rid of these. 00:39:07.050 --> 00:39:11.190 And so what you would do is to bleach them. 00:39:11.190 --> 00:39:14.820 And bleaching is to use light to basically damage 00:39:14.820 --> 00:39:20.190 the fluorophores and get it to no longer fluoresce, OK? 00:39:20.190 --> 00:39:25.230 So this process is going to involve an iterative photo 00:39:25.230 --> 00:39:42.270 activation followed by measuring and fitting the image 00:39:42.270 --> 00:39:44.790 so that you can basically determine 00:39:44.790 --> 00:39:48.720 where each single molecule is in your image. 00:39:48.720 --> 00:39:53.400 And then ending with bleaching to get rid of the fluorophores 00:39:53.400 --> 00:39:57.060 you just turned on so that everything is now dark again. 00:39:57.060 --> 00:40:00.390 And then you repeat this process iteratively 00:40:00.390 --> 00:40:06.030 to collect all of the single molecules that you can, OK? 00:40:06.030 --> 00:40:10.140 So in this case, we just got these three molecules. 00:40:10.140 --> 00:40:12.180 We would then want to bleach them 00:40:12.180 --> 00:40:14.940 so that we're now going to look at different fluorescent 00:40:14.940 --> 00:40:16.013 molecules. 00:40:16.013 --> 00:40:17.430 And we'll turn on a certain number 00:40:17.430 --> 00:40:19.920 of other fluorescent molecules. 00:40:19.920 --> 00:40:21.570 Here you see four. 00:40:21.570 --> 00:40:22.200 Here are two. 00:40:22.200 --> 00:40:23.867 They're a little close together, but you 00:40:23.867 --> 00:40:25.050 can see that there are two. 00:40:25.050 --> 00:40:25.980 Here are another two. 00:40:25.980 --> 00:40:30.360 They're close together, but you see two clear intensity peaks. 00:40:30.360 --> 00:40:31.850 And so you can fit those four. 00:40:31.850 --> 00:40:35.880 Now you have four more molecules to make up your image. 00:40:35.880 --> 00:40:39.480 You bleach them, excite or activate five more. 00:40:39.480 --> 00:40:41.640 Here are five fluorescent molecules. 00:40:41.640 --> 00:40:42.870 You can fit those. 00:40:42.870 --> 00:40:44.700 Determine their positions. 00:40:44.700 --> 00:40:47.130 And you just do this iteratively over and over 00:40:47.130 --> 00:40:51.130 again till you get as many molecules as you can. 00:40:51.130 --> 00:40:55.440 And at the end, you basically add all these images together 00:40:55.440 --> 00:41:00.840 to get the final super resolution image, OK? 00:41:00.840 --> 00:41:04.560 So this is an iterative process where the photo activation 00:41:04.560 --> 00:41:08.220 allows you to image single molecules such 00:41:08.220 --> 00:41:13.043 that you can see where they are with nanometer precision. 00:41:13.043 --> 00:41:14.460 And then you add them all together 00:41:14.460 --> 00:41:17.680 to get a super resolution image. 00:41:17.680 --> 00:41:20.890 Here is an example of this in practice. 00:41:20.890 --> 00:41:22.680 And this is the storm technique which 00:41:22.680 --> 00:41:25.170 doesn't involve a photo activatable fluorescent 00:41:25.170 --> 00:41:28.110 protein, but involves organic dyes blinking. 00:41:28.110 --> 00:41:32.160 And the concept is basically the same. 00:41:32.160 --> 00:41:35.430 And here you see a conventional image of an axon. 00:41:35.430 --> 00:41:37.830 And it's labeled with this beta-II spectrin. 00:41:37.830 --> 00:41:41.430 And you see beta-II spectrin is continuous. 00:41:41.430 --> 00:41:43.620 And it's staining in this axon. 00:41:43.620 --> 00:41:46.700 But if you look at the super resolution image, what 00:41:46.700 --> 00:41:49.080 you see is the beta spectrin actually 00:41:49.080 --> 00:41:53.190 has this repeated periodic pattern along the axon. 00:41:53.190 --> 00:41:55.770 And this is a cytoskeletal element 00:41:55.770 --> 00:41:58.260 that's basically present in rings up 00:41:58.260 --> 00:42:01.595 and down the axons of neurons. 00:42:01.595 --> 00:42:02.970 And you can kind of think of this 00:42:02.970 --> 00:42:05.760 as like the axon is a vacuum hose, where 00:42:05.760 --> 00:42:08.130 you have these rigid sort of rings that 00:42:08.130 --> 00:42:10.710 are aligned all along the axon. 00:42:10.710 --> 00:42:13.980 But because it's repeated and you have intervening areas 00:42:13.980 --> 00:42:16.680 without much cytoskeleton, you can kind of 00:42:16.680 --> 00:42:18.300 think of it as a way for the axon 00:42:18.300 --> 00:42:24.150 to be both rigid but also flexible and maneuverable. 00:42:24.150 --> 00:42:29.010 I just wanted to point out that several super resolution 00:42:29.010 --> 00:42:34.350 techniques were recognized in the 2014 Nobel 00:42:34.350 --> 00:42:36.750 Prize in chemistry. 00:42:36.750 --> 00:42:41.490 Eric Betzig on the left here developed the approach 00:42:41.490 --> 00:42:46.200 using the photo activatable GFP that I described to you. 00:42:46.200 --> 00:42:50.000 And two others, Stefan Hell and W.E. Moerner 00:42:50.000 --> 00:42:54.050 were awarded it for other types of super resolution technique. 00:42:54.050 --> 00:42:57.920 If you get a chance, you should go to the Nobel Prize website 00:42:57.920 --> 00:43:04.160 and listen to Eric Betzig's Nobel lecture. 00:43:04.160 --> 00:43:06.170 He has a very interesting story. 00:43:06.170 --> 00:43:09.230 And part of it involved how he managed 00:43:09.230 --> 00:43:11.280 to develop this technique. 00:43:11.280 --> 00:43:15.360 And he actually developed it in the living room of his friend. 00:43:15.360 --> 00:43:17.810 So this is actually one of the first super resolution 00:43:17.810 --> 00:43:19.520 microscopes. 00:43:19.520 --> 00:43:21.240 Here's the microscope and here you see-- 00:43:21.240 --> 00:43:22.820 I love this chair. 00:43:22.820 --> 00:43:25.790 But you see, you basically have this microscope 00:43:25.790 --> 00:43:28.410 in this guy's living room. 00:43:28.410 --> 00:43:30.290 So if you want to hear more about this story, 00:43:30.290 --> 00:43:31.770 listen to his Nobel lecture. 00:43:31.770 --> 00:43:33.950 He's a really funny guy and you get 00:43:33.950 --> 00:43:36.440 a sense of how science really works 00:43:36.440 --> 00:43:39.080 where you get this unemployed guy like building 00:43:39.080 --> 00:43:41.180 a microscope in his friend's living room 00:43:41.180 --> 00:43:42.500 and then wins a Nobel Prize. 00:43:45.230 --> 00:43:49.640 And just one reminder to end today, remember your news brief 00:43:49.640 --> 00:43:52.670 is due this Friday, November 30th. 00:43:52.670 --> 00:43:54.830 If you need help on selecting a topic, 00:43:54.830 --> 00:43:58.020 please see a member of our staff, 00:43:58.020 --> 00:44:01.400 including Professor Imperiali or myself. 00:44:01.400 --> 00:44:03.380 And so, good luck with that. 00:44:03.380 --> 00:44:04.040 Thank you. 00:44:04.040 --> 00:44:05.920 I'm all set.