Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

Abstract

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

1. Cell Culture and Transfection

1. 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.<....

Results

Figure 1. TIRF microscope set up. Schematic view and image (inset) of the TIRF microscope system.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Axio Observer Z1	Zeiss	491912-9850-000	inverted microscopehttp://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction
Multiline Argon Laser Lasos 77	Lasos	00000-1312-752	multi-line (458/488/514 nm), 100mW argon-ion laserhttp://www.lasos.com/products/argon-ion-laser
Laser TIRF slider	Zeiss	423681-9901-000	http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html
100x Objective	Zeiss	421190-9900-000	Oil, NA 1.45 Alpha-Planhttps://www.micro-shop.zeiss.com/?l=en&p=us&f=o&a=v&m=a&id=421190-9900-000&ss=1
CCD Camera RetigaSRV Fast 1394	QImaging		http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf
LAMBDA 10-3 optical filter changer with SmartShutter	Sutter Instrument Company		http://www.sutter.com/IMAGING/lambda103.html
Software
Image ProPlus 6.3 Software	Media Cybernetics		spot selection, ROI selection, fluorescence intensity determinationhttp://www.mediacy.com/index.aspx?page=IPP
Excel	Microsoft		photobleaching correction, whole-cell and single-vesicle analyseshttp://office.microsoft.com/it-it/excel/
GraphPad Prism 4.00	GraphPad Software, Inc.		statistical analysishttp://www.graphpad.com/scientific-software/prism/