An Ode to Patch Clamping. I’ve been a graduate student in bioengineering for quite a while now—let’s call it “more than five years”—but I harbor a far more embarrassing secret (for a bio- centric program) than that. I’ve only known how to pipette for four of them. When I entered graduate school my lab experience had been limited to computer work: mostly data analysis in Matlab. I had never actually gathered my own data.
Perhaps I should’ve directly mentioned this in my application, though at times computational work is all one does to complete a Ph. D in bioengineering (the field is diverse). Manual whole-cell patch-clamping of the HERG cardiac K+ channel. Manual whole-cell patch-clamping provides the most sensitive and accurate way. In practice, current-clamp. Patch clamp recording from cells in sliced. Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. An Introductory Guide To Patch Clamp. The Practice of Patch Clamping (pages 95. There were many times as an undergrad that I was so frustrated troubleshooting code in Matlab I actually yelled at whatever living thing was inside the computer and obviously out to get me. But I eventually figured out how to massage the computer into doing what I wanted. And that particular problem was solved. T 0113/10 (Patch-clamping/IONSCOPE). Patch-clamping and its use in. As a grad student I’ve come to know the pains of experimental work with actual living things. One particular technique deserves the utmost reverence: patch clamping. Less widely practiced than computer programming, patch clamping is one of the most transformative techniques in neuroscience. It’s a delicate process in which you, the experimenter, first bring the tip of a microscopic glass pipette down to a cell membrane ever- so- gently under a microscope. You then physically apply suction with your own mouth on the other end of the pipette, which is archaically connected though a long rubbery tube. By applying suction you draw the cell membrane so close to the pipette tip that it adheres and forms a seal on the rim of the glass. Then with more powerful suction, you break open the bit of membrane that’s stuck to the pipette opening, all while the cell is still alive. A tiny glass pipette (blue) patched onto a neuron in the hippocampus, a region of the brain that processes memory. When this happens, you suddenly have all kinds of access to the inside of a cell (in my case, a neuron). The liquid contents in your pipette diffuse into the cytoplasm, and the small electrical signals generated by ion channels in the cell membrane are detectable by an electrode that sits further up in the pipette (a cell membrane usually filters these signals such that they are undetectable from the outside). You are now part of the living neuron and can inject current or perfuse drugs into the 1. DNA. Patch clamping is a powerful technique that has no alternative. But it takes a special kind of person to perform such a technique regularly. When one troubleshoots a computer program, there is complete control of the system (even if it doesn’t feel that way). Bugs in code, once solved, are solved permanently. They possess no quantum phenomenon like randomly changing or disappearing. Bugs occur in experimental protocols as well, except that they are much less well defined. Patch- clamp bugs require a personality with the highest tolerance for frustration I’ve ever encountered. They reappear with very few hints to a reason. It’s a maniacal game. There are obvious parameters to fiddle with when patch clamping. I can count them on both hands, but beyond that, there are an unknown number of parameters that govern the cell about to be patched. Humans made computers, so we know exactly how every part works, down to the transistor. But humans did not make cells, and we don’t know nearly enough about how they work. When something common goes wrong during a patch clamp experiment—the cell membrane won’t seal onto the pipette, or the cell dies as soon as you break into the cytoplasm, or the patch fails too soon to measure what you wanted—there’s no quick or permanent fix. One can tweak the shape of the pipette to be more amenable to sealing, one can mix up new internal solution (the liquid that goes inside the pipette), one can grow or harvest new cells, trying to be extra careful so as not to hurt them. Any number of these solutions might fix the problem, and each one takes anywhere from an hour to days to try. A computer program contained in a stack of punched cards. The red markings depict where changes have been made from the last iteration through a central computer. If you converge on something that works, you patch a bunch of cells that day and celebrate! But when you come back next day, the same problem happens. Or a different problem happens, and the cycle starts again. Perhaps this was the frustration early programmers experienced when they had to wait in line to try out revised code on punched cards in a centralized computing center. I applaud them for sticking it out! Patch clamping can be incredibly frustrating. In my lab we joke about making sacrificial offerings to the “patch clamp Gods.” The technique is often likened to black magic, but when done by someone with utmost patience, it is a powerful art form. To be able to peer at and control the electrochemical signals that govern neurological processes within single cells was revolutionary over three decades ago and has advanced neuroscience at an incredible rate. We now know that ion channels (the pores in cell membranes that control flow of various chemicals in or out) open in a probabilistic manner. We can study how neurotransmitters like serotonin modulate internal electrical signals, and how a neuron decides whether to fire an action potential (i. I commend those who stick with the technique and accept the frustration in exchange for the rewards it offers.*Though as they improve, optical techniques might one day replace patch clamping. The Practice of Patch Clamping- -. Thepurpose of this is to indicate good practice. Practitioners might find thatthis Chapter helps to increase the success rate, facilitates trouble- shootingand reduces having to . Novices hopefully will be savedmuch time. The method used in this Chapter is to provide a step- by- stepaccount of how to establish a patch clamp configuration, using thebackground information outlined in the previous Chapters. Electrophysiologyis one of the disciplines within biology that allow many instantquality controls; these will be indicated where appropriate. The Practice of Patch Clamping 9. Preparing the Experiment and Making a Seal 9. Setting up 9. 54. Bringing the pipette near the preparation 9. Making the seal 1. Whole- cell Modes 1. Conventional whole- cell recording 1. Perforated patch recording 1. Single- channel Modes 1. General notes 1. 10. Cell- attached patch 1. Excised patches 1.
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