Advances in recording scattered light changes in crustacean nerve with electrical activation

We investigated optical changes associated with crustacean nerve stimulation using birefringent and large angle scattered light. Improved detection schemes disclosed high temporal structure of the optical signals and allowed further investigations of biophysical mechanisms responsible for such changes. Most studies of physiological activity in neuronal tissue use techniques that measure the electrical behavior or ionic permeability of the nerve, such as voltage or ion sensitive dyes injected into cells, or invasive electric recording apparatus. While these techniques provide high resolution, they are detrimental to tissue and do not easily lend themselves to clinical applications in humans. Electrical and chemical components of neural excitation evoke physical responses observed through changes in scattered and absorbed light. This method is suited for in-vivo applications. Intrinsic optical changes have shown themselves to be multifaceted in nature and point to several different physiological processes that occur with different time courses during neural excitation. Fast changes occur concomitantly with electrical events, and slow changes parallel metabolic events including changes in blood flow and oxygenation. Previous experiments with isolated crustacean nerves have been used to study the biophysical mechanisms of fast optical changes. However, they have been confounded by multiple superimposed action potentials which make it difficult to discriminate the temporal signatures of individual optical responses. Often many averages were needed to adequately resolve the signal. More recently, optical signals have been observed in single trials. Initially large angle scattering measurements were used to record these events with much of the signal coming from cellular swelling associated with water influx during activation. By exploiting the birefringent properties derived from the molecular stiucture of nerve membranes, signals appear larger with a greater contrast, but direct comparison of birefringent and 90{sup o} scattering signals has not been reported. New developments in computer and optical technology allow optical recording with higher temporal resolution than could be achieved previously. This has led us to undertake more detailed studies of the biophysical mechanisms underlying these transient changes. Optimization of this technology in conjunction with other technical developments presents a path to noninvasive dynamic clinical observation of optical responses. To conduct these optical recordings, we placed dissected leg, claw and ventral cord nerves from crayfish and lobster in a recording chamber constructed from black Delrin. The chamber consisted of several wells situated perpendicularly to the long axis of the nerve that could beelectrically isolated for stimulating and recording electrical activation, and a window in the center for optical measurements. To measure the birefringence from the nerve, light from a 120W halogen bulb was focused onto the nerve from below the window through a 10X microscope objective and polarized at a 45 degree angle with respect to the long axis of the nerve bundle. A second polarizer turned 90 degrees with respect to the first polarizer was placed on top of the chamber and excluded direct source illumination, passing only birefringent light from the nerve. A large area photodiode placed directly on top of the polarizer detected the magnitude of the birefringent light. To measure light scattered 90 degrees by the nerve, a short length of image conduit placed perpendicularly to the nerve directed large angle scattered light from the nerve to a second photodiode. The output of each photodiode was amplified by a first stage amplifier which produced a DC level output, and was coupled to an AC amplifier (0.3 Hz High Pass) with a gain of 1000 to optimally record changes across time