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Most of our research involves intracellular recordings from rat neurons maintained in vitro in acute brain slices. We typically use IR/DIC microscopy to visualize individual neurons prior to making a whole cell patch clamp recording. Thin (~300 um) sections of the hippocampus and olfactory bulb generally remain healthy for 6-10 hours after slicing. All four patch clamp setups are capable of recording in both voltage- and current-clamp modes. In two setups we are able to simultaneously measure intracellular calcium using photometry or a cooled CCD camera. We also employ computer modeling, typically with the Neuron program written by Michael Hines, to test our understanding of our results and to guide future experiments. We also have a custom-built 2-photon microscope designed for rapid (3000-4000 lines per second) scanning in conjuction with patch clamp electrophysiological recordings.
Whole Cell Patch Clamp Recording
All four patch clamp rigs are based on the recently-discontinued Zeiss Axioskop FS upright microscope and use infrared differential interference contrast (IR/DIC) to enhance to normally weak contrast inherent in (healthy) biological membranes. Using this microscope and a conventional CCD camera, we are able to visualize individual cells on a b/w monitor and to identify neuronal type (mossy cell, granule cell, mitral cell, etc.) The entire optical train (microscope and camera) is mounted on a moveable platform supported in the front by a Newport XY translation stage and in the back by two ball transfers. Patch clamp electrodes can then be readily positioned on the cell membrane under visual guidance using a Newport crossed roller bearing XYZ translation stage equipped with three open-loop motorizers. Whole cell recordings are actually made using Axoclamp 2B or Axopatch 1D amplifiers from Axon Instruments. We use ITC-18 analog-to-digital converters (from Instrutech Corp.) connected to Windows NT workstations to record and analyze electrophysiological and photometry data. Most setups also have a LeCroy digital oscilloscope. We also have one interface recording chamber that can be used for either sharp electrode or field potential recording.
Calcium Photometry and Imaging
One setup is equipped for photometric calcium or voltage dye recording. A moveable prism controls whether the video port output goes to a CCD camera (used for making a patch clamp recording under IR/DIC microscopy) or to a condenser lens and a photodiode or photomultiplier tube (PMT). The one "techniques" question that we've been asked most often is how to chose between a photodiode- or PMT-based detection system. Chi-Bin Chien's Ph.D. dissertation is an extremely useful document that has a thorough discussion of this and many other relevant issues. Since our condenser will collect emitted fluorescent light over a large area, we use the epi-illumination field stop (iris) to restrict the photometric measurement to a particular region of the cell. This setup has a 150 watt Xe illuminator made by Opti-Quip connected to the microscope with a Oriel liquid light guide.
We also have a setup for imaging calcium responses. This setup is based on the Quantex 57 camera from Photometrics (now part of Roper Scientific) with an EEV 57 back-illuminated CCD sensor. We use the Poly2 system from TILL as an epi-illumination source on this setup. The camera, illuminator, and ITC-18 A/D are presently controlled by a custom Visual Basic program. We use the IDL language from Research Systems, Inc. for imaging processing and analysis. Since IDL is available as an ActiveX component (more than we can say for the camera controller!), we are able to embed an IDL window in our Visual Basic program for online image analysis.