Microscope Imaging and Koehler Illumination

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00:00:11.20 00:00:12.27 Hello I'm Ron Vale and I'm gonna follow up from the last lecture
00:00:12.28 00:00:17.18 where we talked about how lenses work.
00:00:17.19 00:00:20.27 And now discuss how lenses are used in a microscope
00:00:20.28 00:00:26.00 specifically how they're used to focus light from the specimen
00:00:26.01 00:00:29.18 so that you can see it either with your eye or the camera.
00:00:29.19 00:00:34.16 And also how we use lenses to collect light from a light source
00:00:34.17 00:00:40.13 and focus it on the specimen using a technique called Köhler illumination.
00:00:40.14 00:00:46.08 And finally I'm gonna discuss the conjugate image planes of the microscope.
00:00:46.09 00:00:49.05 Well before we start discussing the lenses in the microscope,
00:00:49.06 00:00:52.12 let's just review some of the features of lenses
00:00:52.13 00:00:54.18 that we learned about from the first lecture.
00:00:54.19 00:00:57.25 So here we have an object. This object is an arrow.
00:00:57.26 00:00:59.11 And we are gonna place that object
00:00:59.12 00:01:05.04 to the left side of the focal plane of this particular lens here.
00:01:05.05 00:01:08.19 And the object is going to be emitting light here.
00:01:08.20 00:01:14.02 Some of that light is collected by this lens and bent.
00:01:14.03 00:01:19.07 And it comes to a focus here using principles of ray tracing
00:01:19.08 00:01:20.19 that we learned from the first lecture.
00:01:20.20 00:01:25.00 And here is just another point in the middle of the object
00:01:25.01 00:01:30.02 that again is being focused and the bottom of this arrow again being focused
00:01:30.03 00:01:33.25 to create an image on the other side of the lens.
00:01:33.26 00:01:38.07 Now we also learn from the lens makers equation
00:01:38.08 00:01:41.18 that if you know the focal length of this lens,
00:01:41.19 00:01:45.02 in the distance of this object away from the lens,
00:01:45.03 00:01:50.01 you can predict where the image will form on the right side of the lens.
00:01:50.02 00:01:53.22 And the magnification that is created by the lens
00:01:53.23 00:02:00.07 well by definition is the size of the image, divided by the size of the object
00:02:00.08 00:02:04.28 which is also proportional to the distance of this image away from this lens
00:02:04.29 00:02:09.10 divided by the distance of the object from the lens.
00:02:09.11 00:02:13.17 Now just as a review there are two other key points about lenses
00:02:13.18 00:02:16.20 that I would just like to cover again
00:02:16.21 00:02:19.02 because they're very important for understanding
00:02:19.03 00:02:21.09 how these lenses work in the microscope.
00:02:21.10 00:02:25.13 So one key point about how lenses work is that
00:02:25.14 00:02:28.26 parallel apparel beam of light coming into the lens
00:02:28.27 00:02:36.05 as you see here comes to a focus at the focal plane of this particular lens
00:02:36.06 00:02:38.17 in fact that's the definition of the focal plane.
00:02:38.18 00:02:42.27 So parallel light comes to focus on the other side
00:02:42.28 00:02:51.10 and conversely light that is diverging from this focal plane over here,
00:02:51.11 00:02:54.26 passes through the lens on the other side is parallel light.
00:02:54.27 00:02:59.17 And we'll see how this comes into play in the way the optics work in the microscope.
00:02:59.18 00:03:03.28 So the most important lens in your microscope
00:03:03.29 00:03:08.03 is the objective lens which I am I showing over here.
00:03:08.04 00:03:14.08 I'm depicting it here as a pretty simple thin lens in reality
00:03:14.09 00:03:19.01 as you'll hear from the next lecture which is devoted to objective lenses
00:03:19.02 00:03:23.13 it is actually quite a complex series of lenses that are put together.
00:03:23.14 00:03:27.28 Up here I'm just simplifying that into a simple lens.
00:03:27.29 00:03:33.17 And if you bought an objective lens or have a microscope that is
00:03:33.18 00:03:40.20 from the nineteen seventies or eighties, it works by a principle of finite imaging.
00:03:40.21 00:03:44.19 In other words, this lens works pretty much like
00:03:44.20 00:03:47.07 the lens I just showed you a couple slides ago,
00:03:47.08 00:03:53.08 where the specimen or your object is placed a short distance away
00:03:53.09 00:03:56.06 from the focal point of this objective.
00:03:56.07 00:04:00.00 And then light travels through this objective lens here,
00:04:00.01 00:04:03.03 here is the tip of the arrow coming into a focus,
00:04:03.04 00:04:05.13 the middle and the bottom of the arrow
00:04:05.14 00:04:10.15 which is all focused by this objective lens to create an image on the other side.
00:04:10.16 00:04:13.27 At a fixed distance away from that objective lens.
00:04:13.28 00:04:17.10 And for many microscopes are built in that era
00:04:17.11 00:04:21.15 that distance is about 160 millimeters.
00:04:21.16 00:04:27.24 Now if you have a microscope that you purchased more recently
00:04:27.25 00:04:32.16 like in the last twenty years those objective lenses in those microscopes work
00:04:32.17 00:04:37.14 using a different optical system, using infinity objectives.
00:04:37.15 00:04:42.02 And the way this works is now the object, your specimen,
00:04:42.03 00:04:47.11 is placed exactly at the focal plane of this objective lens.
00:04:47.12 00:04:55.21 And as we just discussed light coming from the focal plane from your specimen,
00:04:55.22 00:05:00.08 will travel through this objective lens on the other side as parallel light.
00:05:00.09 00:05:04.05 So here is a light coming from the top of this arrow head
00:05:04.06 00:05:07.29 the middle and the bottom all emerging on the other side
00:05:08.00 00:05:11.14 is parallel light, which obviously is diffuse light.
00:05:11.15 00:05:16.10 It's not going to create an image. So you need to have another lens in this system
00:05:16.11 00:05:22.14 which is the tube lens such as shown here which now takes this parallel light
00:05:22.15 00:05:26.29 and refocuses it to create an image on the other side.
00:05:27.00 00:05:35.15 So the one reason why manufacturers have turned to this is it offers more flexibility
00:05:35.16 00:05:38.25 in the design of the microscope and where you place the tube lens.
00:05:38.26 00:05:44.10 Because this all this light is a traveling parallel
00:05:44.11 00:05:50.01 so you could place this tube lens in different points of the light path.
00:05:50.02 00:05:54.04 And that allows you also flexibility for putting more types
00:05:54.05 00:06:00.13 of devices such as dichroic lenses,or other modifiers to to the light in the optical path
00:06:00.14 00:06:02.28 between the objective lens and the tube lens.
00:06:02.29 00:06:09.20 Now in this case, the magnification of the combination of these two lenses is
00:06:09.21 00:06:14.22 given by this formula here, which is the focal length of the tube lens,
00:06:14.23 00:06:20.23 which is usually a big number are divided by the focal length of the objective lens
00:06:20.24 00:06:22.27 which is usually a much smaller number,
00:06:22.28 00:06:27.06 now that gives you the degree of magnification of the system.
00:06:27.07 00:06:31.18 Okay so then how do we actually see this image?
00:06:31.19 00:06:34.13 Well. They're two ways.
00:06:34.14 00:06:38.14 One is to view it with your eye through an eyepiece.
00:06:38.15 00:06:43.11 The way this is typically done is light comes from your specimen here.
00:06:43.12 00:06:46.16 It's focused through these two lenses that we just talked about,
00:06:46.17 00:06:52.16 the objective lens and the tube lens to come and produce an intermediate image.
00:06:52.17 00:06:56.25 But we're now gonna put another lens system here
00:06:56.26 00:07:00.06 that can further magnify this intermediate image,
00:07:00.07 00:07:04.16 and produce greater magnification by going through an eyepiece.
00:07:04.17 00:07:07.03 And we're gonna have a short lecture
00:07:07.04 00:07:10.00 where you can learn more about how these eyepieces work.
00:07:10.01 00:07:13.11 And then through another lens which is actually the lens in your eye
00:07:13.12 00:07:19.09 to create a final image on your retina which is your detector.
00:07:19.10 00:07:23.19 So here we have different points of your specimen
00:07:23.20 00:07:29.08 all traveling through this optical system to be focused on your retina.
00:07:29.09 00:07:33.23 to create you know this final image that you can see
00:07:33.24 00:07:36.04 on your retina and process by your brain.
00:07:36.05 00:07:39.04 Now the other way to to capture your image
00:07:39.05 00:07:42.29 is by using a camera and the simplest approach for that,
00:07:43.00 00:07:47.14 is just to place the camera at the intermediate image plane.
00:07:47.15 00:07:49.25 So again here we just learned that
00:07:49.26 00:07:53.05 light coming from your object travels through the objective
00:07:53.06 00:07:55.15 in tube lens and comes into focus.
00:07:55.16 00:08:01.24 Here form different points in the object in this is the intermediate image plane.
00:08:01.25 00:08:06.14 And now we can just place a camera right at that image intermediate image plane
00:08:06.15 00:08:12.01 and the detector now will be able to record this in focus image.
00:08:12.02 00:08:19.00 And for many cases the magnification that you achieve
00:08:19.01 00:08:22.07 through the objective in tube lens is efficient
00:08:22.08 00:08:26.29 and we will talk about how to optimize the proper magnification.
00:08:27.00 00:08:32.21 For particular cameras later on in this lecture series.
00:08:32.22 00:08:37.10 In some cases you may want to magnify the image more.
00:08:37.11 00:08:41.21 In which case, you can now introduce a projection eyepiece
00:08:41.22 00:08:47.17 that again collects the light that's coming through this optical system.
00:08:47.18 00:08:50.12 from this intermediate image plane is then,
00:08:50.13 00:08:54.02 it diverges, it's re-collected by this eyepiece,
00:08:54.03 00:09:01.10 which gives you additional magnification and then ultimately focus on the camera.
00:09:01.11 00:09:05.29 So we've just covered now how lenses are used from the specimen
00:09:06.00 00:09:12.05 to create an image either on a camera or through the eyepiece onto your retina.
00:09:12.06 00:09:18.10 Now lenses are also used to illuminate the sample as well
00:09:18.11 00:09:20.19 to collect and focus light from a lamp.
00:09:20.20 00:09:23.18 And you'll hear additional special lecture
00:09:23.19 00:09:26.18 on different kinds of lens that are used in microscopy
00:09:26.19 00:09:31.21 but we need to collect and focus that light onto the specimen.
00:09:31.22 00:09:38.17 And for transmitted light, the best way to eliminate the specimen
00:09:38.18 00:09:41.20 is using a technique called Koehler illumination.
00:09:41.21 00:09:47.02 And the way this works is that you have your light source over here.
00:09:47.03 00:09:53.11 And the light source is first, goes through a collector lens,
00:09:53.12 00:09:56.20 which is usually, the lens is usually placed very close to the light source.
00:09:56.21 00:10:00.15 Many microscopes also have an additional lens called a field lens.
00:10:00.16 00:10:04.12 But the combined goal of the collector in the field lens
00:10:04.13 00:10:08.24 is to focus light coming from the lamp.
00:10:08.25 00:10:13.15 From point sources on the lamp the light is focused,
00:10:13.16 00:10:19.28 to a plane which is the front focal plane of the condenser lance.
00:10:19.29 00:10:22.04 So here's a condenser lens over here.
00:10:22.05 00:10:29.16 The front focal plane is usually right at the bottom of this condenser lens
00:10:29.17 00:10:31.08 at an aperture that I'll describe later.
00:10:31.09 00:10:40.11 And we just learned that if light is emanating of from this focal point,
00:10:40.12 00:10:46.00 at the focal plane of this lens over here it's then going to travel through
00:10:46.01 00:10:49.05 the lens on the other side as parallel light.
00:10:49.06 00:10:53.11 So here we have parallel light illuminating the specimen.
00:10:53.12 00:10:56.20 And we're going to have a special laboratory,
00:10:56.21 00:10:59.19 where we are going to discuss in fact how to adjust
00:10:59.20 00:11:04.03 all of these lenses to achieve Köhler illumination.
00:11:04.04 00:11:12.08 But the contrasting way of illuminating the specimen is called critical illumination.
00:11:12.09 00:11:18.08 And in this case instead of having defocused parallel light at the specimen.
00:11:18.09 00:11:25.16 Critical illumination focuses light from the lamp directly onto the specimen.
00:11:25.17 00:11:30.02 So an image of the light source is projected onto the sample itself.
00:11:30.03 00:11:36.25 Now this can focus and create a very intense illumination at the sample plane.
00:11:36.26 00:11:44.08 But because it creates an image of the lamp, the lamp may be have very uneven,
00:11:44.09 00:11:48.04 may have a filament, may be very uneven
00:11:48.05 00:11:52.13 and that creates uneven illumination of the sample.
00:11:52.14 00:11:55.09 So the main reason for using Köhler illumination
00:11:55.10 00:11:59.13 is to generate uniform light intensity at the specimen
00:11:59.14 00:12:05.03 even if the light source itself may be somewhat non uniform.
00:12:05.04 00:12:08.09 By defocusing into having parallel light you can
00:12:08.10 00:12:13.06 generate the best type of even illumination possible.
00:12:13.07 00:12:16.07 So anyway let's go to the microscope now
00:12:16.08 00:12:21.17 and have a look where all of these lenses are in a real microscope.
00:12:21.18 00:12:27.24 Okay so now let's look at where the lenses are in a real microscope over here.
00:12:27.25 00:12:31.08 So let's first of all start from the lamp.
00:12:31.09 00:12:36.22 So here we have the illuminating source it's this filament here.
00:12:36.23 00:12:43.02 And then we have a lens in front of that which is the collecting lens for the light
00:12:43.03 00:12:46.16 and that light then travels through this part of the microscope.
00:12:46.17 00:12:52.11 And in the top part of the microscope there's a second lens which is our field lens.
00:12:52.12 00:12:55.14 And if I turn it upside down you can then see
00:12:55.15 00:12:58.14 this piece of glass which is of the field lens.
00:12:58.15 00:13:04.13 And the combination of the collector lens and the field lens
00:13:04.14 00:13:12.24 are used to focus the image of the lamp onto the front focal plane of the condenser.
00:13:12.25 00:13:16.21 So that's the next lens in the system so let's look at that one.
00:13:16.22 00:13:22.00 Let's take our our collector lens over here. We can slide it out.
00:13:22.01 00:13:31.05 And so if we look at the front part this is the condenser lens itself over here.
00:13:31.06 00:13:37.24 And the front focal plane of this lens is where an Iris's placed
00:13:37.25 00:13:39.23 this is the condenser diaphragm.
00:13:39.24 00:13:44.12 And we can open and close this diaphragm so the image of the light source is
00:13:44.13 00:13:48.05 focused on onto this part over here.
00:13:48.06 00:13:54.29 And light coming from the front focal plane of the lens like the condenser lens
00:13:55.00 00:14:00.16 will then travel on the other side as parallel light which is illuminating this specimen.
00:14:00.17 00:14:06.17 So then the next lens in the system here is objective lens
00:14:06.18 00:14:09.01 and we will have a whole lecture on objective lenses.
00:14:09.02 00:14:14.25 But this just shows the objective lens over here and
00:14:14.26 00:14:21.22 then the objective lens is used in conjunction with a tube lens that we talked about.
00:14:21.23 00:14:25.15 The tube lens is kind of buried in the body the microscopes I can't show it to you.
00:14:25.16 00:14:31.18 But light collected from the objective in the tube lens is then either
00:14:31.19 00:14:36.02 focused onto the CCD chip of the camera over here,
00:14:36.03 00:14:42.11 or there's an intermediate image created just in front of the eyepiece.
00:14:42.12 00:14:46.15 And the combination of lenses in the eyepiece with the lens of your eye
00:14:46.16 00:14:52.13 is then used to create an image of the specimen on your retina.
00:14:52.14 00:14:58.06 Okay so the last point I'd like to cover in these lectures are conjugate planes in a microscope.
00:14:58.07 00:15:04.18 And conjugate planes are different planes or locations in this microscope system.
00:15:04.19 00:15:07.11 that are in simultaneous focus
00:15:07.12 00:15:11.22 and when you view these planes through the microscope system
00:15:11.23 00:15:16.23 they appear to be superimposed and in focus with one another.
00:15:16.24 00:15:22.29 And let me illustrate this with some examples here.
00:15:23.00 00:15:27.03 So first of all we have image forming planes.
00:15:27.04 00:15:31.19 So we already discussed that we can view the specimen
00:15:31.20 00:15:36.03 through this combination of the objective lens and the tube lens.
00:15:36.04 00:15:43.05 And this creates a focus and an image on a detector such as the camera.
00:15:43.06 00:15:52.04 Now below the sample manufacturers have also placed a field diaphragm,
00:15:52.05 00:15:56.03 which if the system is set up in proper Köhler illumination,
00:15:56.04 00:16:03.27 that field diaphragm gets focused onto the plane of the sample.
00:16:03.28 00:16:09.01 So there be an image this field diaphragm is actually a physical entity.
00:16:09.02 00:16:14.26 It has an iris as I'll show you in a second and it gets focused onto the sample.
00:16:14.27 00:16:20.20 And in turn it goes through the objective lens and the tube lens
00:16:20.21 00:16:25.17 and the image of this field diaphragm is also imaged on the detector as well.
00:16:25.18 00:16:33.04 So if we're looking at this aligned microscope through a camera.
00:16:33.05 00:16:36.21 What we see is the following if we closed down the field diaphragm
00:16:36.22 00:16:40.07 we see not only image of the specimen over here
00:16:40.08 00:16:46.17 but we also actually see the image of the Iris of this field diaphragm
00:16:46.18 00:16:51.19 superimposed and in focus with the specimen itself.
00:16:51.20 00:16:58.11 And by opening and closing the field diaphragm as you'll see in a second
00:16:58.12 00:17:02.28 we can restrict or enlarge the field of view.
00:17:02.29 00:17:07.06 So now let's talk about the conjugate image planes of the microscope
00:17:07.07 00:17:09.11 and show you where they are.
00:17:09.12 00:17:16.00 So first of all let's discuss the image forming planes starting with the field diaphragm.
00:17:16.01 00:17:21.25 So the field diaphragm is an Iris that's in front of the lamp.
00:17:21.26 00:17:30.20 And in fact if we take this unit apart here we can see this Iris
00:17:30.21 00:17:36.12 in the body of the microscope and we can see it open and close.
00:17:36.13 00:17:46.16 And if we adjust the position of this condenser lens appropriately on this rail
00:17:46.17 00:17:53.17 we can focus the image of that iris onto the specimen plane.
00:17:53.18 00:17:57.26 So the image of the Iris will be superimposed on the specimen.
00:17:57.27 00:18:03.06 And the specimen and the field diaphragm,
00:18:03.07 00:18:10.08 those images will then be projected by the objective lens and the tube lens
00:18:10.09 00:18:16.27 either directly onto the CCD chip or through the combination of the eyepiece
00:18:16.28 00:18:21.12 and your lens onto your retina.
00:18:21.13 00:18:25.07 There is also a separate group of planes
00:18:25.08 00:18:28.24 that are involved in illumination and Köhler illumination.
00:18:28.25 00:18:33.26 So again we talked about how the light source is focused
00:18:33.27 00:18:39.21 through the set of lenses on to the front focal plane of the condenser lens.
00:18:39.22 00:18:46.04 This is also the location of an iris diaphragm at this front focal plane.
00:18:46.05 00:18:52.27 And then it comes out as parallel light bathing the specimen
00:18:52.28 00:18:58.19 which gets refocused to the back focal plane of the objective lens.
00:18:58.20 00:19:02.08 And then it travels through and becomes diffused again
00:19:02.09 00:19:05.04 at the detector so we can't see an image of the light source.
00:19:05.05 00:19:12.09 But we can see an image of the light source here at the front focal plane of the condenser.
00:19:12.10 00:19:15.00 And there's also an image of the light source
00:19:15.01 00:19:17.18 at the back focal plane of the condenser as well.
00:19:17.19 00:19:22.27 Now normally we can't see the image of the light source on the camera with your eyepiece
00:19:22.28 00:19:25.02 because we're looking at the wrong focal plane.
00:19:25.03 00:19:31.02 But if you put another lens in the system which is often called a Bertrand lens
00:19:31.03 00:19:38.12 or also an eyepiece telescope instead of focusing on the specimen like we normally do
00:19:38.13 00:19:42.10 we can focus on the back focal plane of the objective
00:19:42.11 00:19:45.16 and now see an image of the light source.
00:19:45.17 00:19:51.15 And if fact that's a technique that we can use to center our light source
00:19:51.16 00:19:54.16 and center a light source
00:19:54.17 00:19:58.04 and make sure that everything's aligned properly for Köhler illumination.
00:19:58.05 00:20:03.00 So again we will go through the steps in our at the microscope
00:20:03.01 00:20:10.00 and show how you can use the Bertrand lens to image the light source.
00:20:10.01 00:20:16.02 So there's a separate set of planes for imaging the illuminating source.
00:20:16.03 00:20:23.06 So the light source starts off with a physical object of filament in the bulb.
00:20:23.07 00:20:29.12 And the image of that light is then focused in Köhler illumination
00:20:29.13 00:20:36.07 through the collector lens, field lens onto the front focal plane of the condenser lens.
00:20:36.08 00:20:41.07 Where there happens to be an iris that opens and closes.
00:20:41.08 00:20:45.16 Now light from the front focal plane emerges from the condenser lens
00:20:45.17 00:20:47.08 as parallel light through the specimen
00:20:47.09 00:20:51.07 and that parallel light travels through the objective lens,
00:20:51.08 00:20:56.03 and is refocused on the back focal plane of the of the objective
00:20:56.04 00:21:01.26 which is somewhat usually buried somewhere inside the body of the objective lens.
00:21:01.27 00:21:08.18 Now and then the image of the light source when we look at it with a camera or eye
00:21:08.19 00:21:14.01 is out of focus because we're focusing on the other conjugate planes
00:21:14.02 00:21:15.16 correspond to the specimen.
00:21:15.17 00:21:20.28 But if we want to see the illuminating image planes,
00:21:20.29 00:21:26.26 we can flip in, many microscopes have us and called Bertrand lens
00:21:26.27 00:21:29.13 that are built into the body the microscope
00:21:29.14 00:21:32.14 and that allows us instead of focusing on the specimen,
00:21:32.15 00:21:36.26 to focus on the back focal plane of the objective lens.
00:21:36.27 00:21:40.18 And because all of these are in conjugate image plans
00:21:40.19 00:21:43.28 when we focus on the back focal plane of the objective,
00:21:43.29 00:21:47.12 we should also see in the same conjugate image plane
00:21:47.13 00:21:54.11 the front focal plane of the condenser, and that's where this iris is located.
00:21:54.12 00:21:58.24 So with the Bertrand lens in place if I open and close this Irish of the condenser,
00:21:58.25 00:22:04.11 I should be able to see the physical leaves of the iris opening and closing.
00:22:04.12 00:22:13.28 And the light, the image of the light is also projected or imaged onto that front focal plane.
00:22:13.29 00:22:17.18 So once again I look at the back of the plane of the objective lens
00:22:17.19 00:22:45.16 I should be able to see the image of the light sources well.

This Talk

Speaker: Ron Vale

Audience:

Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: March 2012

Talk Overview

This lecture covers the lenses of the microscope and how they are used to focus light onto a specimen and how light from the specimen is focused onto a camera or the retina of an eye. Koehler illumination, which provides even illumination of a sample, is described.

Questions

List three planes in the microscope that are conjugate to image forming planes.
List three planes in the microscope that are conjugate to the illumination (light source) plane
Why do you want to set up Koehler illumination?

Answers

View Answers

Speaker Bio

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