Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2001 Apr 2;153(1):177–190. doi: 10.1083/jcb.153.1.177

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2001 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

TIRFM Optics. Cells are cultured on plastic culture dishes in which the bottom has been drilled out and a glass coverslip has been glued. The excitation light is provided by the 488-nm line of the laser focused by a long focal lengths just before the beam enters the microscope base. The beam then reflects up from the microscope's base optics, and TIRFM is created by a trapezoidal glass prism (flint glass, 1.648 refractive index, cut from a triangular prism commercially available from Rolyn Optics) mounted on the condenser mount. A thin layer of immersion oil separates the prism from the glass coverslip. The cells are visualized through a 0.75 NA 40× achromat water immersion objective (160-mm tube length) mounted on a Leitz Ortholux upright microscope. Local perfusion of individual cells can be performed by positioning a quartz pipette within a few hundred microns of the cell of interest.