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Seeing Further With The
“Up-and-Down” Microscope
by Gina Shaw
Kenneth N. Fish needed a new microscope. The problem was, he needed one that didn’t
exist.
Dr. Fish, an assistant professor of psychiatry at the University of Pittsburgh, studies long-term memory by focusing on molecular signaling among dendrites and the cellular modifications that arise from these signals. To answer a central question about these processes — how proteins that leave the endoplasmic reticulum and dendrites make their way through the neuron’s secretory pathway to the plasma membrane — he needed to image two events simultaneously.
Transmembrane proteins leave the endoplasmic reticulum in dendrites. “About three years ago, we discovered that they could leave the reticulum anywhere, and in fact left it through the whole dendritic arbor,” says Dr. Fish. “But since the next step in the secretory pathway has been identified to be only in the soma or very proximal dendrites, we wondered why proteins would leave the endoplasmic reticulum 300 microns away from where they’d be processed next. We didn’t buy the current dogma that proteins must travel hundreds of microns back to get processed.”
To challenge that dogma, Dr. Fish needed to figure out a way to follow proteins live from their site of translation to endoplasmic reticulum export, to where they entered the plasma membrane. But how?
Serendipitously, around the same time Olympus Corp. (Center Valley, PA) was a developing a new “two-for-one” package, combining an upright and an inverted research microscope — the IX81 and the BX51— into a single instrument that would eventually be called the Up/Down Microscope. They contacted Dr. Fish, who had previously done research and development work with Olympus, and he agreed to work with them on developing the Up/Down, a process that took a couple of years. “We’re just now at the point where we know it all works, and are ready to start the real science.”
The Up/Down microscope captures simultaneous
images from three different cameras: two cameras attached to the upright
microscope and one to the TIRF microscope. One of the biggest advantages
of using the Up/Down microscope to follow ER to PM trafficking, according
to Dr. Kenneth Fish, is that since the cargo is continuously being visualized,
there is no confusion about where receptors that arrive at the PM originated.”
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One challenge that took some time to resolve was ensuring appropriate alignment
of the two objectives. “The objective system on the inverted part is fixed, and
then we use the nose piece on top to move the other objective. Once you grossly
align them, you then have to align them more finely,” says Dr. Fish. After doing
that, he noted a surprising number of chromatic aberrations. “I guess no one had
ever looked at anything by fluorescence this way before, both from the upper and
the bottom part at the same time. So we had to strip down the system and go to
the manufacturer’s specs and work our way back to minimize those aberrations.”
A much bigger problem turned out to lie with the software that runs the system. “No software out there could simultaneously run two Z-motors,” says Dr. Fish. So he set up a master/slave system; one computer runs half the scope, and another runs the other. It makes the system very easy to set up and run, he says, but it also slows things down. The master/slave system has a simultaneous capture rate of about three frames per second, compared to a normal capture rate of 12 frames per second and up. Given that the biological events Dr. Fish is following take one to 1.5 seconds, this speed is “plenty fast enough,” but he’s also working to develop ideas to improve capture rates.
Now that he’s fine-tuned the Up-Down system, Dr. Fish can finally turn to the original questions he set out to answer. He’s just getting started on that — but he’s already discovered some exciting, unexpected advantages. “There are two types of TIRF synergy, objective-based and prism-based. Objective optics have become so advanced, but a problem no one really discusses is that there’s a tremendous amount of scatter within the object itself,” he says. “With this system, we capture with the upper scope, completely eliminating any scatter that’s happening within the TIRF objective itself — and there’s actually a lot more than we had imagined. We’ve increased even more the signal-to-noise ratio, and the amount of information we’re able to see is incredible.”
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