Light, shine inward, and the mind through all her powers irradiate,...--Milton, Paradise Lost
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This is what the set-up in our lab actually looks like from two different views.
The trash on the breadboard and shelves may not be essential :-}.
You may notice that the pictures don't exactly match the schematic. The laser we use is much bigger than the one I've recommended you buy. It costs twice as much and has twice the pulse energy. But since the higher pulse energy isn't really good for anything (oh, the wisdom of hindsight), I recommend you buy the small laser.
This picture shows the small laser on top of the big one, for your comparison.
These pictures show the relationship of the optics once you've got it all set up.
OK, I admit it: I'm not a professional photographer.
Finding the Image
The purpose of all these optical components is to focus the laser beam
to a point in the specimen. (See here for more
explanation.) The lens actually focuses the beam to a point in an
image of the specimen. To find out approximately where that should
be, you need to hold a piece of paper behind the scope and move it
around until you get an image. The microscope isn't really meant to
be a projector, so you'll want to darken the room when you do this,
and you'll want to make the image as bright as possible. Remove the
polarizer and analyzer from the light path. Turn the light up as
bright as it will go. Now swing in the low-power objective and focus
the condensor so that the image-plane iris diaphragm is in focus.
After all this, you should easily see a light coming out the back of
the scope, in the place where the light from an epifluorescense light
source would normally come in.
Your problem now is to find the image. Shut down the image-plane diaphragm as far as it will go. The beam coming out the scope will become narrower. Now move a piece of paper back and forth to find the place where the beam focuses to a point. (Actually, it will focus to a tiny image of the diaphragm.) That's where you want the laser light to focus. The lens should be about 5 cm behind that (because its focal length is 5 cm).
Here's what it actually looks like with our microscope. The first picture shows the focus, the second what it looks like with the paper too close, and the third with the paper too far.
Here's what it would look like if the lights were on. You can barely see the focus this way.
With our Zeiss Axioscope and epifluorescence condenser, the image plane is about 8 inches behind the microscope (or would be, if the mirror weren't reflecting it). I don't know where it will be with other microscopes, or even if all Zeiss axioscopes will form an image at the same point. (Zeiss has a way of changing the internal optics from time to time.) It is in fact entirely possible that there won't be an image behind the scope. What do you do in this case?
Actually, it's pretty simple: you just need to add another lens to form an image. Choosing the right lens is more complicated than I want to go into here. If you run into this problem, feel free to get in touch with me.
After you have the beam focused to a point in the image and lined up, you need to make sure the beam diameter is large enough to fill the entire objective. Unscrew the microscope objective, and hold a piece of paper under the hole to see how big the spot of laser light is. It should be at least as big as the hole in the back of the objective. If it's too small, you'll get poorly localized damage. If it's too big, too little of the light will enter the objective, and there won't be enough to kill cells. The best size is just small enough to bust holes in a glass coverslip. You adjust the beam diameter with the beam expander. This has a lens mounted in a tube that slides in and out, and can be locked in place with a set screw. As you slide it in (towards the laser), the beam will diverge a little bit more, making it a little bigger at the objective. If you can't get the beam big enough, try putting the tube in backward, so that the end holding the lens is towards the laser.
The final adjustment is the focus, which you do by moving the lens along the optical axis: towards the laser is up in the specimen. You can get it approximately right by busting holes in a coverslip, and moving the lens back and forth until the holes seem to be in focus. But for really accurate focus, I've always found it necessary to mount a worm and shoot at cells, moving the lens if the point of damage appeared to be above or below the image.
The first time you set everything up you'll have to do all this several times. For instance, as you move the beam expander lens, everything else will go out of alignment. You can adjust the direction of the beam by loosening the ring that holds the beam expander in place and shoving it around. (Hint: LSI designed the beam expander so that the base is held by two metal-metal contacts. Since there's no give, it's almost impossible to tighten the ring so that the beam expander can be moved easily and yet will stay in place when you let go. You'll find it much easier to work with if you get a sheet of teflon or gasket material from the hardware store, cut out two rings, and stick them on the back and front of the base of the beam expander with a dab of grease.) But this is a crude adjustment. You'll have to adjust the lens and mirror again afterward. Once you have everything lined up, though, it's easy to make small adjustments. For instance, you can vary the beam intensity by moving the beam expander lens in or out slightly.
An even more egregious waste of money is the epifluorescence condensor for the Axioskop. I don't remember how many thousand dollars this cost (I don't think I want to remember), but it could be replaced with a single $50 planoconvex lens. Well, you'd have to buy some positioning hardware, but still, it would save thousands of dollars, be more versatile, and work at least as well. (The epifluorescence condensor has been carefully designed to do something completely different from what we're doing.)
The neutral density filters are interposed between the laser and the lens to cut down the beam. Their positioning doesn't have to be precise. I just use a ring stand. They should be at a slight angle to the beam (i.e., not quite flat on).
You'll probably need to get a few 1/4-20 bolts. Oriel sends you as many as you need, but Newport doesn't. Just take one of the ones you get from Oriel to a hardware store and buy about a dozen more. You'll also need a long hex wrench, to reach down into the rod holders when you screw them down.
That's it. I hope I didn't leave anything out.
Leon Avery, (Leon.Avery@email.swmed.edu)