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