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00:00:12;13 Hello. I'm Ron Vale,
00:00:14;01 and we're going to discuss
00:00:15;22 how to focus the microscope
00:00:17;23 on your specimen.
00:00:18;26 And we're going to go through a number of tips,
00:00:21;09 but, with practice,
00:00:23;03 many of these steps will become second-nature to you.
00:00:26;13 But whenever you're focusing the microscope,
00:00:29;08 it's always important to do it with
00:00:30;27 a great deal of care,
00:00:33;15 because one's working with an objective
00:00:35;07 that can be quite expensive,
00:00:36;27 often costing as much as $10,000.
00:00:41;12 So, the first thing you want to do is
00:00:43;28 to become familiar with the focusing knobs
00:00:46;06 of your particular microscope.
00:00:48;25 In this microscope, on the right side
00:00:52;16 is a fine focusing knob
00:00:54;27 with a remote focusing device,
00:00:58;05 so that allows us to fine-tune the level of focus,
00:01:03;24 and on the left side is a coarse focusing knob.
00:01:07;05 Now, before I even put the sample on,
00:01:09;24 I want to get used to these focusing knobs,
00:01:12;24 in particular knowing
00:01:15;17 which direction I turn the knob...
00:01:18;13 how that will relate to whether the objective lens
00:01:21;21 moves either up or down.
00:01:24;02 And the direction of focus,
00:01:26;15 with regard to the motion of the objective lens,
00:01:29;12 can be different in different types of microscopes,
00:01:32;22 so it's important to become familiar with this
00:01:34;21 with your particular microscope.
00:01:36;19 So, in this case, I'm turning
00:01:39;25 the focusing knob in both directions,
00:01:41;16 and I can see that when I
00:01:44;11 turn the focusing knob towards me,
00:01:46;09 the objective lens is rising up towards the specimen.
00:01:52;23 So, now, when I become familiar
00:01:55;11 with this objective knob,
00:01:57;03 I now want to become...
00:01:59;13 make sure that I have everything set up properly
00:02:03;16 so that I can see the fluorescence
00:02:07;14 from my specimen.
00:02:08;26 So, before you start focusing,
00:02:11;10 you want to make sure that the light path of the microscope
00:02:14;02 is set up correctly,
00:02:16;02 so that the image is being projected
00:02:18;07 to the eyepiece.
00:02:19;23 In this case, actually, the light path
00:02:22;28 is going to the camera,
00:02:24;14 and so I need to switch it to
00:02:27;19 the light path going to the eyepiece.
00:02:29;03 And now I'm ready to start focusing
00:02:32;02 and looking for fluorescence.
00:02:34;11 In this case, I'm using
00:02:37;01 a green fluorescent protein,
00:02:38;24 and when I open up the shutter
00:02:43;01 I can see that there is this bluish-green light,
00:02:46;07 which is the correct excitation for GFP (green fluorescent protein),
00:02:49;16 and I know also, in advance,
00:02:51;13 that I have the correct emission and excitation filters in here,
00:02:56;04 so I should be able to excite
00:02:59;13 and view the fluorescence of the GFP.
00:03:01;21 Next, we have to make sure we know what objective we're using,
00:03:11;04 whether it's an air objective, a water objective,
00:03:13;16 or an oil-immersion objective.
00:03:14;25 In this case, we have
00:03:16;09 an oil-immersion objective.
00:03:18;23 So, I have to place a drop of oil
00:03:21;17 between the objective lens and the coverslip,
00:03:27;01 so... one drop of oil should be enough,
00:03:31;17 and I'm being careful not to
00:03:34;24 touch the objective lens directly.
00:03:36;24 There we go, we have our oil droplet.
00:03:40;26 Now we can put our specimen on,
00:03:44;05 but one has to be very careful one puts it on
00:03:47;23 with the correct orientation,
00:03:49;06 that being that the coverslip
00:03:51;10 should be the side in contact
00:03:54;25 with the oil on the objective.
00:03:56;12 So, I'm flipping it over on the correct side,
00:03:59;22 over here,
00:04:01;28 and placing it on the stage.
00:04:06;02 Now, before I start focusing with the eyepiece,
00:04:09;27 I want to get the objective lens
00:04:11;13 to approximately the correct position,
00:04:14;20 and I can do that with the coarse focusing knob,
00:04:17;09 and what I'm looking for is
00:04:22;07 the moment when the oil
00:04:24;00 comes in contact with the coverslip,
00:04:27;12 and at that moment...
00:04:29;07 as I'm now focusing up, here,
00:04:31;21 I'm watching for the moment
00:04:36;09 when the contact is made and the oil
00:04:38;18 spreads on the coverslip surface...
00:04:40;28 there... there it goes, right there...
00:04:44;06 and now I know I'm at a good starting point
00:04:46;11 to continue the rest of my focusing
00:04:49;01 by looking through the eyepiece.
00:04:51;27 Now, to do this next step,
00:04:54;22 I am going to go to the eyepiece,
00:04:56;10 but the fluorescence is fairly dim
00:04:58;26 and this room is quite bright,
00:05:02;23 and I would have a very difficult time
00:05:05;06 seeing the dimmer fluorescence
00:05:07;03 with all this background illumination
00:05:09;16 from the room.
00:05:11;03 So, I'm going to turn off the lights now,
00:05:14;04 and I'm going to become
00:05:16;11 dark-adapted with my eye,
00:05:17;25 and then I can do the rest of the focusing
00:05:19;28 on the specimen.
00:05:21;29 Okay, so now I'm dark-adapted,
00:05:23;23 I'm ready to look through the eyepiece
00:05:26;05 and continue focusing up to the specimen,
00:05:30;12 and look for the GFP fluorescence.
00:05:32;13 And, everything looks fairly dim at this point,
00:05:35;14 I can see maybe a little bit of green haze,
00:05:39;19 and I'm turning the coarse focusing knob slowly,
00:05:43;22 while carefully looking through the eyepiece,
00:05:45;13 until I now begin to see
00:05:49;05 fluorescence becoming brighter,
00:05:50;24 and I can see some, kind of,
00:05:55;19 outlines of some green objects.
00:05:59;26 And now I'm going to the fine focusing device,
00:06:03;06 so I can now bring the cells
00:06:09;09 into crisp focus.
00:06:13;03 And that's it.
00:06:14;28 And now I'm ready to do my experiment.
00:06:20;01 So, a few more focusing tips.
00:06:22;16 In some cases, you may have
00:06:24;23 an extremely dim fluorescence sample,
00:06:27;14 for example, fluorescence coming from single molecules,
00:06:32;00 in which can it may be very difficult
00:06:35;02 to see this single-molecule fluorescence
00:06:37;16 with the eyepiece.
00:06:39;08 And in that case, you may have to
00:06:41;26 direct the light to a very sensitive camera
00:06:45;14 and actually undergo
00:06:48;10 the same kind of focusing routine,
00:06:50;02 but now looking at the image
00:06:52;11 from the camera to focus,
00:06:54;21 rather than the eyepiece.
00:06:56;15 Finally, in some cases,
00:06:58;14 you may have a very difficult time
00:07:02;23 focusing on your specimen,
00:07:05;23 and you may think you've gone too far.
00:07:08;15 If that's the case,
00:07:10;13 it's good to stop at that moment
00:07:12;04 and look at the specimen
00:07:13;29 to see if you have indeed have over-focused.
00:07:17;11 You can see this happening right here...
00:07:19;27 when we've gone over-focus
00:07:21;06 and now the coverslip...
00:07:25;10 the objective lens is actually pushing up
00:07:27;08 on the entire slide, lifting it up.
00:07:29;20 Now, this can be, potentially,
00:07:33;28 damaging to your objective lens,
00:07:35;16 and there are a couple ways that you can
00:07:37;10 protect against this.
00:07:38;19 First of all, if you know you have
00:07:40;13 a difficult sample to focus,
00:07:43;06 you may not want to clamp it down
00:07:45;29 rigidly onto the stage,
00:07:47;03 but keep it loose,
00:07:49;06 so that if you go over-focus
00:07:52;24 it's just lightly lifting
00:07:55;06 the slide and coverslip.
00:07:57;13 And, second of all,
00:07:59;06 another thing that helps protect these objective lenses
00:08:01;17 is a spring-load
00:08:05;05 that's built into the objective lens.
00:08:06;10 So, you can see this spring-load, here...
00:08:10;02 so, if it pushes against something
00:08:13;07 rather than just pushing on the objective lens itself,
00:08:16;22 the spring-load will go down.
00:08:19;13 Nevertheless, the best strategy is,
00:08:22;21 if you're concerned that you've gone over-focus,
00:08:26;08 just stop, have a look at where you are,
00:08:30;04 and then put your objective lens down
00:08:35;06 and just start the process all over again,
00:08:37;08 to try to find focus.
00:08:40;17 So, let's now review all the steps
00:08:43;08 in focusing the microscope.
00:08:45;04 First, look at your focusing knobs,
00:08:49;12 know which one is the fine focus,
00:08:50;27 which is the coarse focus.
00:08:52;14 Also, get used to how the direction of
00:08:59;08 turning these knobs
00:09:01;03 relates to the motion of the objective lens.
00:09:04;01 The next step is to make sure that
00:09:07;28 you have everything about your light source
00:09:12;01 properly arranged,
00:09:13;27 the correct dichroic filters
00:09:16;19 for the fluorescence from the object
00:09:19;05 you want to view.
00:09:22;01 Next, look at your specimen.
00:09:25;10 Make sure that you put the coverslip side down,
00:09:28;15 onto the objective.
00:09:31;02 If you're using oil,
00:09:32;20 you just need one drop of oil,
00:09:34;29 placed on top of the objective lens,
00:09:39;20 making sure not to touch it directly.
00:09:42;26 Then, put your sample in place,
00:09:47;06 and look at the sample
00:09:50;09 and look for the moment
00:09:52;02 when the oil just hits the coverslip surface,
00:09:56;07 turn the lights out in the room,
00:09:59;08 make sure that the light path
00:10:03;01 is directed to the eyepiece,
00:10:04;24 if you're using the eyepiece,
00:10:06;10 and then sit down and
00:10:09;29 carefully use the coarse knob,
00:10:12;00 very, very slowly,
00:10:14;18 to find the approximate correct position,
00:10:18;27 and then use the fine focusing knob.
00:10:22;12 And, if there's any doubt,
00:10:24;23 look at the objective,
00:10:26;22 make sure it's not over-focus,
00:10:28;29 and you can always just put your objective
00:10:32;06 back to a starting position
00:10:34;12 and start the whole process again.
00:10:36;13 And, again, just practice,
00:10:39;27 and focusing a sample will become easy for you.
Focusing is the first step in microscopy. This video provides some tips on how to find the right focal plane.
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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