Calcium Imaging and Optical Manipulation of Neuronal Activity in Axolotl using a LED-Based Microscope

Here we introduce a novel fluorescence microscope concept which expands the application of functional fluorescence imaging and further enables optical manipulation of biological samples. Taking advantage of the LED technique allows a space saving housing of multiple light sources and illumination at various wavelengths. Furthermore, the microscope provides an optical separation into two independently controllable excitation pathways enabling a simultaneous illumination of different regions which are adjustable in size. Image detection is realized via a super resolution high speed camera that allows high resolution image capturing (2560x2160) at rates up to 100 Hz. Moreover, we implemented a voice-coil driven high NA objective to ensure z-movement at maximum speed and precision. The multiple LED arrangement and the separation of excitation pathways therefore facilitate the usage of multiple imaging approaches at the same time.To test the performance of the microscope we used in vitro whole head preparations of Ambystoma mexicanum tadpoles. This preparation allows studying neuronal systems with all sensory pathways intact and can be maintained up to one week. Thus, we were able to use calcium imaging to record sensory evoked neuronal responses of central vestibular neurons elicited by electric stimulation of specific semicircular canals. Further, to quantify the glutamate uncaging efficiency we calculated the required light intensity and duration to optically evoke action potentials by patching those neurons. Finally, we could show that by means of spatially separating calcium imaging and glutamate uncaging we were able to manipulate ipsilateral semicircular canal evoked calcium responses by optically activating contralateral inhibitory/excitatory pathways.