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(Cis-)total internal reflection fluorescence microscope (TIRFM) diagram

Specimen
Evanescent wave range
Cover slip
Immersion oil
Objective
Emission beam (signal)
Excitation beam

(Trans-)total internal reflection fluorescence microscope (TIRFM) diagram

Objective
Emission beam (signal)
Immersion oil
Cover slip
Specimen
Evanescent wave range
Excitation beam
Quartz prism

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm can be observed.

Background

In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules.

Overview

The idea of using total internal reflection to illuminate cells contacting the surface of glass was first described by E.J. Ambrose in 1956.^[1] This idea was then extended by Daniel Axelrod^[2] at the University of Michigan, Ann Arbor in the early 1980s as TIRFM. A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. Note, however, that the region visualised is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualised in addition to the plasma membrane during TIRF microscopy. The selective visualisation of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution.

TIRF can also be used to observe the fluorescence of a single molecule,^[3]^[4] making it an important tool of biophysics and quantitative biology.

References

^ Ambrose, EJ (24 Nov 1956). "A surface contact microscope for the study of cell movements.". Nature 178 (4543): 1194. Bibcode:1956Natur.178.1194A. doi:10.1038/1781194a0.
^ Axelrod, D. (1 April 1981). "Cell-substrate contacts illuminated by total internal reflection fluorescence". The Journal of Cell Biology 89 (1): 141–145. doi:10.1083/jcb.89.1.141. PMC 2111781. PMID 7014571. Retrieved 16 January 2012.
^ Yanagida, Toshio; Sako, Yasushi; Minoghchi, Shigeru (10 February 2000). "Single-molecule imaging of EGFR signalling on the surface of living cells". Nature Cell Biology 2 (3): 168–172. doi:10.1038/35004044. PMID 10707088. |access-date= requires |url= (help)
^ Andre et al. Cross-correlated tirf/afm reveals asymmetric distribution of forcegenerating heads along self-assembled, synthetic myosin filaments. Biophysical Journal, 96:1952–1960, 2009.

Axelrod, Daniel (1 November 2001). "Total Internal Reflection Fluorescence Microscopy in Cell Biology". Traffic 2 (11): 764–774. doi:10.1034/j.1600-0854.2001.21104.x. |access-date= requires |url= (help)

External links

Interactive Fluorescence Dye and Filter Database Carl Zeiss Interactive Fluorescence Dye and Filter Database.
TIRF Microscopy: Introduction and Applications TIRF Tutorial from Microscopy U
TIRF Microscopy: Overview TIRF Tutorial from Olympus Microscopy Resource Center
Olympus TIRFM Microscopes commercial TIRF microscope systems
Carl Zeiss Laser TIRF 3 commercial TIRF microscope systems
Lightguide- and prism-based TIRF microscopy Commercial TIRF Microscopy, TIRF Spectroscopy, TIRF ElectroChemistry, and TIRF Dielectrophoresis systems
TIRF FLIM microscopy Lambert Instruments TIRF - FLIM microscopy
Schwartz Research Group, CU-Boulder Single Molecule Imaging Research Group

English Journal

A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

Li G1, Sanchez V1, Nagaraj PC1, Khan S2, Rajpoot N1,3.
Journal of microscopy.J Microsc.2015 Aug 10. doi: 10.1111/jmi.12299. [Epub ahead of print]
We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment
PMID 26259144

Fluorescence Detection of Single DNA Molecules.

Huang W1, Wang Y, Wang Z.
Journal of fluorescence.J Fluoresc.2015 Jul 28. [Epub ahead of print]
Single-molecule detection (SMD) and single-molecule fluorescence resonance energy transfer (smFRET) were conducted using Cy3- and Cy5-labeled single-strand DNAs (ssDNAs) either immobilized on substrates or encapsulated in microdroplets. High-quality fluorescent images were obtained using a total int
PMID 26215080

Nanoemulsion-templated polylelectrolyte multifunctional nanocapsules for DNA entrapment and bioimaging.

Bazylińska U1, Saczko J2.
Colloids and surfaces. B, Biointerfaces.Colloids Surf B Biointerfaces.2015 Jul 26. pii: S0927-7765(15)30090-4. doi: 10.1016/j.colsurfb.2015.07.056. [Epub ahead of print]
The emerging field of bionanotechnology aims at advancing colloidal and biomedical research via introduction of multifunctional nanoparticle-based containers intended for both gene therapy and bioimaging. In the present contribution we entrapped the model genetic material (herring testes DNA) in the
PMID 26260359