Tcs spe dmi4000

Images were taken using an inverted confocal microscope (Leica Microsystems TCS SPE DMI4000) attached to an external light source for enhanced fluorescence imaging (Leica EL6000) with a 10x objective. Pinhole settings were chosen in such a way that axially all the cells were entirely present within the confocal volume. For the 2D culture method, one layer was taken. For the 3D culture method, z-stacks were generated (3–4 layers) to visualize the whole 3D network. The maximum intensity projection was then used for analysis that was performed using ImageJ (Neurophology plugin) software to evaluate parameters of neuroplasticity:

Images were captured using an inverted microscope (Leica Microsystems TCS SPE DMI4000, Wetzlar, Germany) attached to an external light source for enhanced fluorescence imaging (Leica EL6000) with Leica LAS AF imaging software. Each dataset was imaged in a single session, with the same imaging settings maintained throughout the session. Image analyses were performed in a user-blinded manner using ImageJ and de-noised using a background subtraction rolling ball radius of 50 pixels. For display, images were adjusted for brightness and contrast, with channel minimum and maximum values kept consistent between images. Maximum projections were used for analysis.
To quantify autophagy, high-resolution z-stacks imaged at 40× resolution with an optimal step size were performed to capture multiple cells across the coverslip. Regions of interest (ROIs) were drawn around single cells in ImageJ and selected in the 568 nm channel containing the LC3 signal. The fluorescence intensity of LC3-RFP was measured above a set threshold to exclude background pixel values. To quantify mitophagy, both LC3-RFP and Mitotracker were measured above a set threshold to exclude background pixel values, and the percentage of LC3 puncta that colocalized with mitochondria was calculated for each cell.