In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

[Instructor] To begin, prepare 3% low melting point agros in embryo medium, heat the agros to a temperature safe for manipulating zebra fish without causing damage. Before the agros cools and solidifies, position larvae that are two to seven days post fertilization dorsal side up in a petri dish with a glass bottom. Once the agros has solidified with the larvae in place, add five milliliters of embryo medium to the dish. Using a scalpel, cut the agros as required around the larvae. Transfer the petri dish with the zebra fish onto the stage of the confocal microscope. After acclimatizing for 20 minutes, use brightfield imaging to center the fish under the 40 times water immersion objective at room temperature. Set the confocal microscope acquisition parameters based on the qualities and expression levels of the fluorescent molecule, as well as the depth and optical properties of the tissue. Adjust the laser power and master gain settings to capture fluorescent light properly in the optimal range and prevent unnecessary loss of G camp success dependent fluorescence. Then center the field of view on a neuronal cluster located coddle to the in the rostral portion of the spinal cord. Perform a time series scan with a field of view of 79.86 square micrometers at a frame rate of 1.20 frames per second with a spatial resolution of 0.119 square micrometers per pixel. Adjust the field of view, size, and acquisition speed based on the experiment's objectives. Two minutes after the onset of imaging, add 41.67 microliters of either aloe isothiocyanate stock solution or embryo medium to the dish, and continue recording for approximately 30 seconds to observe changes in G Camp 6's activity in the region of interest through time-lapse imaging. If the recorded output is not in the dot TIF file format, export the image files as dot TIF files from the microscope software suite for downstream Fiji analysis. After downloading Fiji software for neural trace analysis, open Fiji, import the dot CZI files into the program. Use the circle or freehand tools in Fiji to highlight an area or neuron of interest by clicking on the respective icons. After selecting the region of interest or ROI, click analyze, tools, and ROI manager from the Fiji toolbar. Click add T to add the selected ROI to the ROI manager window. In the ROI manager, click more and multi measure to analyze the ROI. Leave the settings at default and click okay to generate raw data. Then save the output as a CSV file for further manipulation using Python or spreadsheets. Now normalize the raw data to represent it as a neural trace by averaging the last 30 seconds of the two minute prestimulus and generate normalized fluorescence intensity using the given formula and plot the normalized data as neural traces. Administration of aloe isothiocyanate caused an increase in G CAMP 6S fluorescence within a localized brain region of larval zebrafish indicating enhanced neural activity. At a slow capture speed of 0.10 frames per second, neurons in the hind brain and spinal cord were clearly visible with high resolution. Increasing the capture speed to 0.79 frames per second resulted in a slight loss of spatial resolution, but improved temporal resolution. At 3.16 frames per second neurons appeared less distinct while capturing more temporal dynamics.