ARTICLE SERIES: Imaging
The plasma membrane is the barrier that all molecules must cross
to enter or exit the cell, and a large number of biological processes
occur at or near the plasma membrane. These processes are
difficult to image with traditional epifluorescence or confocal
microscopy techniques, because details near the cell surface are
easily obscured by fluorescence that originates from the bulk of
the cell. Total internal reflection fluorescence (TIRF) microscopy
– also known as evanescent wave or evanescent field microscopy
– provides a means to selectively excite fluorophores near the
adherent cell surface while minimizing fluorescence from
intracellular regions. This serves to reduce cellular photodamage
and increase the signal-to-noise ratio. TIRF primarily illuminates
only fluorophores very close (e.g. within 100 nm) to the cover-
slip–sample interface. The background fluorescence is minimized
because the excitation of fluorophores further away from the
cover slip is reduced. The plasma membrane of an adherent cell
lies well within the region of excitation, allowing imaging of
processes occurring at or near the membrane. On the basis of
these unique features, TIRF has been employed to address
numerous questions in cell biology.
This Commentary details key issues for researchers who are
using, or are considering using, TIRF for live cell imaging. We
begin with a brief selection of specific areas of cell biological
research in which the use of TIRF imaging has made a major
impact. Subsequently, we describe the physical basis of TIRF, and
discuss key issues to consider when setting up and employing
TIRF. Finally, we identify several potential pitfalls and provide
helpful suggestions. A basic knowledge of fluorescence microscopy
is assumed. For general background on fluorescence microscopy,
we refer readers to North (North, 2006) and Waters (Waters, 2009)
For reviews containing an extensive treatment of TIRF theory and
advanced applications, we refer readers to Axelrod (Axelrod, 2003;
Cellular processes visualized with TIRF
TIRF microscopy has been used in many different types of studies
for the visualization of the spatial-temporal dynamics of molecules
at or near the cell surface, particularly in cases in which the signal
would otherwise be obscured by cytosolic fluorescence. Some of
the advantages of TIRF for imaging near the cell surface are
illustrated in Fig. 1. Actin (LifeAct–GFP in Fig. 1A,B), clathrin
(GFP–clathrin light chain in Fig. 1C,D) and caveolin (caveolin-1–
EGFP in Fig. 1E–G) have been imaged by both conventional
epifluorescence (Fig. 1A,C,E) and TIRF (Fig. 1B,D,G). In each
case, it is apparent that TIRF minimizes the out-of-focus
intracellular fluorescence, resulting in images with a much higher
signal-to-noise ratio. Similarly, although confocal microscopy (Fig.
1F) shows a reduced cytosolic signal relative to epifluorescence
(Fig. 1E), the corresponding TIRF image (Fig. 1G) provides the
greatest amount of information for fluorophores associated with
the plasma membrane. The suppression of background fluorescence
is crucial for studying each of the areas of cell biology on which
TIRF has had a major impact.
TIRF has had an impact on many varied areas of cell biology,
including HIV-1 virion assembly (Jouvenet et al., 2006) and
intraflagellar transport in the Chlamydomonas flagella (Engel et
al., 2009), and in single-molecule experiments. Below, we highlight
several areas of cell biology – the cytoskeleton, endocytosis,
exocytosis, cell–substrate contact regions and intracellular signaling
– that have particularly benefited from investigation by TIRF.
The dynamics of the cytoskeleton near the plasma membrane have
been studied with TIRF (Fig. 1A,B), leading to new insights. Before
TIRF was used to study vesicle trafficking, it was not known that a
cortical microtubule network extended immediately adjacent to the
plasma membrane, and that secretory vesicles remained attached to
these microtubules until the moment of vesicle fusion (Schmoranzer
Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly
well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field
decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover
slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF
include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of
the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study
of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.
Key words: Total internal reflection fluorescence microscopy, Evanescent wave microscopy, Evanescent field microscopy, Fluorescence
Imaging with total internal reflection fluorescence
microscopy for the cell biologist
Alexa L. Mattheyses1,*, Sanford M. Simon1and Joshua Z. Rappoport2,‡
1Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
2The University of Birmingham, School of Biosciences, Edgbaston, Birmingham B15 2TT, UK
*Current address: Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
‡Author for correspondence (email@example.com)
Journal of Cell Science 123, 3621-3628
© 2010. Published by The Company of Biologists Ltd
Journal of Cell Science
fluorescence resonance energy transfer (FRET) (Riven et al., 2006;
Wang et al., 2008) or atomic force microscopy (AFM) (Brown et al.,
2009; Kellermayer et al., 2006), will provide a wide variety of data
on molecular dynamics in living cells. The superior background
reduction provided by TIRF has allowed the development of several
super-resolution techniques (Patterson et al., 2010). In the future,
TIRF will continue to provide a unique view of cell biology.
J.Z.R. is funded through BBSRC grant BB/H002308/1. The authors
thank Alexandre Benmerah for GFP-tagged clathrin, Ari Helenius for
GFP-tagged caveolin-1 and Roland Wedlich-Soldner for GFP-tagged
LifeAct. The authors also thank Natalie Poulter for assistance in the
generation of Fig. 1. The TIRF microscope used in this research to
generate Fig. 1 was obtained through the Birmingham Science City
Translational Medicine Clinical Research and Infrastructure Trials
Platform, with support from Advantage West Midlands (AWM). S.S.M.
is funded through NIH grant R01 GM087977. Deposited in PMC for
release after 12 months.
Supplementary material available online at
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