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00:00:11;28 Hello. I'm Ron Vale,
00:00:13;21 and now we're in the laboratory,
00:00:16;10 at a microscope,
00:00:17;16 and we're going to go through the steps
00:00:19;01 of how to set up Koehler illumination.
00:00:20;28 Now, the main goals of Koehler illumination
00:00:22;28 are two-fold.
00:00:24;01 First of all, we want to
00:00:26;14 focus this condenser lens
00:00:28;24 so the image of the field diaphragm
00:00:32;03 is projected on the specimen plane,
00:00:35;04 so that when we open and close this field diaphragm,
00:00:38;10 we can see a clear image of that field diaphragm
00:00:40;28 through the eyepieces of the microscope
00:00:42;23 or through the camera.
00:00:44;02 Now, the other goal of Koehler
00:00:46;10 is to have
00:00:49;18 even illumination of the specimen
00:00:51;21 from the illuminating source,
00:00:54;10 and the way we do that is by
00:00:56;27 projecting the image of the lamp
00:00:59;14 onto the front focal plane of the condenser lens,
00:01:03;23 and, remember, light diverging from a focal plane
00:01:07;16 will emerge from a lens on the other side
00:01:09;20 as parallel light, and that's what we want.
00:01:12;03 We want parallel light bathing the specimen,
00:01:14;13 because that provides the most
00:01:18;19 even illumination and the light source
00:01:20;05 will be completely defocused.
00:01:22;03 Now, in this particular microscope,
00:01:24;25 the light source here is... and the lens,
00:01:29;24 is perfectly positioned
00:01:31;23 so that when we have the field diaphragm
00:01:33;12 in proper focus at the specimen plane,
00:01:35;25 the light source is also
00:01:38;23 correctly imaged onto the front focal plane
00:01:42;26 of the condenser.
00:01:44;20 So, now, let's go through this step-by-step.
00:01:46;27 So, I'm going to be imaging this sample,
00:01:49;09 a stained section of mammalian taste buds,
00:01:52;15 with a 20x objective lens,
00:01:54;04 so I'm going to put the specimen on,
00:01:56;07 I'm going to make sure that the coverslip
00:01:57;22 is facing the right direction,
00:01:59;11 towards the objective lens,
00:02:04;01 and then the next thing is I'm just going to make sure
00:02:07;25 that I look at the system,
00:02:09;14 so that I know that everything
00:02:11;15 is set up properly for brightfield microscopy.
00:02:14;26 First of all, there's an automated...
00:02:16;06 automatic shutter, here,
00:02:18;12 so I'm going to make sure that's open,
00:02:20;00 and I know it's open if I can see light down here.
00:02:23;15 And there are various filters, here,
00:02:26;05 here's a green filter and a neutral-density filter.
00:02:28;25 For the start, I'm just going to take those out of the light path.
00:02:33;00 Then, there's some other things in the system, here.
00:02:36;07 Here's a polarizer,
00:02:38;09 which we'll need for DIC microscopy,
00:02:41;17 but right now, for brightfield, I'm going to make sure
00:02:44;22 that it's out of the system.
00:02:45;28 And this condenser turret has different settings
00:02:49;28 -- some of them are for phase microscopy or DIC --
00:02:53;10 and I'm going to make sure it's in the right setting
00:02:55;29 for brightfield microscopy.
00:02:58;29 So, now everything is set up here.
00:03:01;19 The first step in Koehler illumination
00:03:03;11 is to focus on the specimen.
00:03:05;16 So, to do that,
00:03:07;20 I'm just going to make sure that
00:03:09;17 I have enough light going through the system
00:03:11;16 so that I can see things properly,
00:03:13;11 so I'll open up the field diaphragm --
00:03:17;16 that lets through more light --
00:03:19;27 I'll open up the condenser diaphragm,
00:03:24;14 and this microscope has an adjustable light source
00:03:26;26 on the other side,
00:03:28;25 so I'm going to try to adjust the light,
00:03:30;24 so I can now sit down at the microscope
00:03:33;01 and be able to have
00:03:36;24 the right amount of light to focus.
00:03:40;03 So, now I'm going to focus on the specimen, here,
00:03:42;05 with this focusing knob,
00:03:44;09 until I begin to see
00:03:47;24 the specimen getting clearer and clearer,
00:03:50;09 until... finally, aah,
00:03:55;24 there it is, it finally snapped into focus.
00:03:57;03 So, that's the first step of setting up Koehler,
00:03:59;25 is being in the correct plane,
00:04:02;19 where I can now image the specimen.
00:04:06;05 Now, the next step is I have to
00:04:09;21 adjust the condenser lens to the right position.
00:04:11;24 I know it will be in the right position
00:04:16;22 when the image of the field diaphragm
00:04:19;16 is in clear view in the specimen.
00:04:22;01 So, what I'm going to do here is, first of all,
00:04:24;19 close down the field diaphragm,
00:04:30;03 and initially I can't see it at all,
00:04:33;14 so now I'm going to adjust the condenser lens,
00:04:37;29 I'm going to bring it down,
00:04:39;16 and I can see the image getting brighter and brighter,
00:04:43;08 and I can see the edges of the field diaphragm
00:04:48;12 come into focus.
00:04:49;13 Okay, there it is.
00:04:51;12 So, that's the second step of Koehler illumination.
00:04:56;16 But you can see, here,
00:04:58;01 that the image of the field diaphragm
00:05:00;24 is not perfectly centered
00:05:03;03 -- it's a little bit off to one side --
00:05:05;25 and that's because this lens is not perfectly
00:05:08;07 in alignment in the optical train, here.
00:05:12;00 So, there are a couple adjustment screws, right here,
00:05:16;11 that allow me to center and move the position of that condenser lens.
00:05:20;09 So, I grab both of them,
00:05:23;03 and now I can kind of move them one way or the other
00:05:26;07 until I see the condenser...
00:05:29;29 the image of the field diaphragm
00:05:32;01 come into the center of my field of view.
00:05:35;27 In the next step, then,
00:05:37;28 I'm going to open up the field diaphragm,
00:05:40;25 and the general rule of thumb is
00:05:43;20 to open up the field diaphragm,
00:05:45;26 just past the field of view,
00:05:51;09 either whether the field of view is for your eye,
00:05:53;13 or for the camera,
00:05:55;15 but you don't want to open it up beyond that,
00:05:58;04 because then you just end up
00:06:00;10 not contributing to the image
00:06:01;23 and just getting extra glare
00:06:04;04 from light passing through the microscope
00:06:07;01 -- that degrades the image.
00:06:08;08 Now, the last adjustment that we may want to make
00:06:11;17 is to change the opening of the condenser aperture.
00:06:16;06 So, there's an iris here that an open and close,
00:06:19;28 and in brightfield illumination,
00:06:23;16 we may want to have this open
00:06:26;03 about three-quarters of the full opening,
00:06:30;01 and we can adjust it to some extent,
00:06:32;21 based upon how the image looks,
00:06:35;28 because opening and closing the condenser,
00:06:37;20 as you'll see in another demonstration lab, later in the course,
00:06:42;25 affects both the resolution and contrast of the image,
00:06:47;04 so we may want to adjust that appropriately,
00:06:49;26 so we get the right kind of image of the specimen
00:06:53;15 that we like.
00:06:54;29 In other types of microscopy,
00:06:56;17 like high-resolution,
00:06:59;03 differential-interference contrast microscopy,
00:07:01;03 where we use computer enhancement of contrast,
00:07:05;08 in that case,
00:07:07;04 we may want to put the condenser fully open.
00:07:10;02 In some microscopes,
00:07:12;01 there may be an additional step,
00:07:13;13 which is adjusting the position of a collector lens.
00:07:18;00 In this case, the collector lens
00:07:20;01 is not adjustable,
00:07:21;11 but if you have a microscope
00:07:23;07 that does have an adjustable
00:07:25;28 focus of the collector lens,
00:07:27;24 then we need to adjust that collector lens
00:07:30;11 so that the image of the light source
00:07:34;00 is projected onto the front focal plane
00:07:37;07 of the condenser lens.
00:07:41;03 So, how do we know if it's in focus?
00:07:43;13 Well, we need to image
00:07:46;21 this front focal plane of the condenser lens,
00:07:49;03 and the way we can do that is with...
00:07:53;03 not looking through the eye...
00:07:54;18 regular eyepiece,
00:07:56;01 but flipping in this additional lens,
00:08:01;10 which is the Bertrand lens,
00:08:02;22 and that focuses on the back focal plane of the objective,
00:08:04;13 which is in the same conjugate plane
00:08:05;24 as the front focal plane
00:08:07;28 of the condenser lens.
00:08:09;23 Once we can see
00:08:13;27 the correct front focal plane of the condenser lens
00:08:19;11 -- and we know we're in the correct plane
00:08:20;29 because if we open and close the condenser diaphragm,
00:08:24;26 we should see that iris opening and closing
00:08:28;02 when we have the Bertrand lens in position --
00:08:30;05 and we should also be able to
00:08:32;29 view the image of the light source itself,
00:08:36;09 provided that there's no
00:08:38;10 diffusing glass or something that's blurring the image.
00:08:41;04 And at that step we could then
00:08:43;28 focus the collector lens
00:08:46;16 so that we can now see
00:08:49;09 the image of the light source clearly focused
00:08:52;06 at the front focal plane of the condenser lens,
00:08:55;26 which will also be in view
00:08:59;08 at the back focal plane of the objective.
00:09:01;25 And, if I look through the eyepiece,
00:09:04;18 I can see an image of the light source
00:09:07;27 -- I can see the little filament
00:09:10;01 that's making the light.
00:09:12;23 Now, I can't show that to you,
00:09:16;11 but another way I can show that you to you
00:09:19;17 is if I defocus the condenser lens,
00:09:24;19 so now I'm focusing the image of the light source,
00:09:28;07 instead of on the front focal plane,
00:09:30;27 I'm focusing the image of the light source
00:09:33;00 on the specimen.
00:09:34;20 And if I do that...
00:09:37;28 I take it out of focus...
00:09:40;12 I can now see the image of the light source
00:09:44;22 at the specimen plane on this piece of paper,
00:09:49;05 and this is called critical illumination,
00:09:52;11 which, as you can see,
00:09:53;29 gives very uneven illumination
00:09:55;16 because we can see all the features of the light source.
00:09:58;22 So, in summary, let's go through all the steps again.
00:10:02;24 So, first of all, we put the sample in place,
00:10:05;10 we adjusted the light intensity,
00:10:07;14 and then we focused on the specimen.
00:10:10;22 The next thing we did is
00:10:14;01 we closed down the field diaphragm
00:10:15;16 and we adjusted
00:10:19;04 the position of the condenser lens,
00:10:20;25 looking through the eyepiece,
00:10:22;24 until we see the iris of the field diaphragm
00:10:25;09 clearly in focus on the specimen plane.
00:10:29;06 The third step is we now open up the field diaphragm
00:10:34;05 just past the field of view.
00:10:36;12 Finally, we adjusted the condenser aperture as well.
00:10:41;06 The key thing, now, is
00:10:43;20 not just to watch the lecture,
00:10:45;04 but go find a microscope yourself
00:10:46;17 and go through all of these steps
00:10:48;23 in Koehler illumination,
00:10:50;11 and practice them
00:10:52;16 so that it becomes just second-nature for you.
Koehler illumination is the prefered method for obtaining even illumination of the specimen and is a critical alignment procedure in microscopy. This lab goes through a step-by-step procedure of aligning the lamp and condenser to achieve Koehler illumination.
Professor of the Department of Cellular and Molecular Pharmacology; Investigator in the Howard Hughes Medical Institute
University of California, San Francisco Continue Reading
Jinming Tang says
Dear Ron Vale,
Thanks for your great courses!
A question about Koehler Illumination: if I put the lamp directly and exactly at the front focus plane of the condensor, could I omit the collector and field lenses?
I want to DIY a lightweight microscope in an automatice system, so I want to use LED illumination. In industrial machine vision, LED light source and colliminator can also provide parallel light, can they replace this system?
Thanks!
Jinming Tang