Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

In This Article

Abstract

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Source: Daniele, F., et. al TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis. J. Vis. Exp. (2015).

This video demonstrates the imaging of human neuroblastoma cells using a total internal reflection fluorescence (TIRF) microscope to visualize fluorophore-tagged synaptic vesicles. The cells are first focused in epifluorescence mode. Adjustments are then made to achieve total internal reflection, generating evanescent waves that selectively excite fluorophores near the membrane. Before TIRF imaging, acquisition parameters are configured, and the sampling frequency is set.

Protocol

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1. Cell Culture and Transfection

SH-SY5Y cell culture
NOTE: The experiments have been performed using the human neuroblastoma SH-SY5Y (ATCC# CRL-2266). SH-SY5Y cells grow as a mixture of floating clusters and adherent cells. Follow the instructions reported in the protocol (cell density, splitting ratio, etc.) to have cells that grow firmly attached to the glass cover, which is crucial for total internal reflection fluorescence microscopy (TIRFM).
1. Before starting, under the laminar flow biosafety cabinet, make the opportune volume of sterile phosphate buffer saline (PBS) solution and culture medium.<....

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Results

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