Methods used in the lab
Fast imaging by line illumination confocal microscopy
Schematic of the microscope setup.
Spatial resolution:
Lateral 0.6-1.8 µm, Axial 1.4-4.2 µm
Temporal resolution:
2D 50-800 Hz, 3D up to 10 Hz
Fluorescent microscopy using functional dyes allows the simultaneous observation of dozens of neurons' activity, yet suffers from a comparably low temporal resolution. To overcome this limitation, we designed and built a confocal microscope that allows higher frame rates, thereby enabling the investigation of spatio-temporal activity patterns of neuronal populations with high resolution in space and time. By focussing the laser into a line instead of a point (see figure), scanning is necessary in only one direction. Using a fast linear CCD array as sensor and a fast acquisition system, we are thus able to achieve high frame rates. By reducing the number of optical elements we were able to increase the efficiency in the emission pathway. The setup is thus optimized for the investigation of biological specimen, were low levels of excitation intensities are necessary to avoid damaging the sensitive samples. Custom written software allows the creation of complex scan protocols, as well as the generation of triggers to synchronize other parts of the setup to the image acqusition, such as a patch clamp unit, odor application devices or a laser for uncaging exeperiments. As the line illumination microscope is an upright microscope, imaging experiments in tissue slices can be combined with electrophysiological recordings.
By using a piezo actuator to control the position of the objective, the microscope is additionally capable of acquiring time series of image stacks, thereby allowing the observation of large fractions of a given tissue containing hundreds of neurons. This capability is additionally useful to generate data for the ACI algorithm.