Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. The technique is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.

Introduction and Theoretical Aspects - The basic concept of TIRFM is simple, requiring only an excitation light beam traveling at a high incident angle through the solid glass coverslip or plastic tissue culture container, where the cells adhere. Refractive index differences between the glass and water phases regulate how light is refracted or reflected at the interface as a function of incident angle. At a specific critical angle, the beam of light is totally reflected from the glass/water interface, rather than passing through and refracting in accordance with Snell's Law. The reflection generates a very thin electromagnetic field (usually less than 200 nanometers) in the aqueous medium, which has an identical frequency to that of the incident light.

Basic Microscope Configuration - A wide spectrum of optical configurations was placed under scrutiny during the early stages of instrument development for total internal reflection fluorescence microscopy investigations (TIRFM). The majority of these designs centered on inverted microscopes, primarily due to the convenience of adding TIRFM optics above, rather than below, the bulky microscope stage. Upright microscope configurations can also be utilized, especially when this represents the only practical choice for the experimental conditions or the investigator's budget.

TIRFM - Olympus Application Note - Olympus has designed a new high numerical aperture apochromatic objective specifically matched for total internal reflection fluorescence microscopy at high critical angles. The objective utilizes a specialized immersion oil having a refractive index of 1.78 and the company offers glass coverslips that match this refractive index. The ease of use afforded by the new objective makes TIRFM more easily accessible to microscopists who are investigating surface phenomena in cell biology.

Alignment of Prism-Based TIRF Systems - The guidelines presented in this section offer an outline of the basic requirements for TIRFM microscope configuration using a prism, laser light source, and a focusing lens. They are intended to serve as a starting point for those interested in exploring fluorescence excitation at an interface of dissimilar refractive indices.

Alignment of Objective-Based TIRF Systems - Total internal reflection can be investigated utilizing high numerical aperture objectives (ranging between 1.4 and 1.65 in aperture), preferentially using an inverted tissue culture microscope. The procedure described here involves configuration of a suitable microscope with an external laser source, a collimating lens, and a high numerical aperture objective.

Laser Fundamentals - The behavior of a collimated light beam upon refraction or reflection from a plane surface is fundamental to the understanding of TIRFM. In addition, knowledge of the general pinciples behind continuous and pulsed laser action is necessary in order to fully appreciate many aspects of total internal reflection. This section discusses how basic laser systems operate through stimulated emission, and how they are designed to amplify this form of light to create intense and focused beams. Interactive tutorials explore many of the fundamental concepts in laser technology and demonstrate the properties of selected individual systems.

Laser Systems for Optical Microscopy - The lasers commonly employed in optical microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.

Olympus IX70 Microscope Cutaway Diagram - The Olympus IX70 inverted tissue culture microscope is a research-level instrument capable of imaging specimens in a variety of illumination modes including brightfield, darkfield, phase contrast, Hoffman modulation contrast, fluorescence, and differential interference contrast. A recent accessory for this popular microscope is the TIRFM-IX illuminator, which attaches to the rear lamphouse port and contains an FC connector for an external laser source and a port for a mercury or xenon lamp housing. A view of the microscope from the rear (illustrated in this section) reveals how the total internal reflection illuminator configuration is interfaced to the instrument.

Excitation by an Evanescent Wave - Total internal reflection fluorescence is employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology. This interactive tutorial explores TIRFM excitation of fluorophores residing in the membranes of living cells in tissue culture.

Evanescent Field Penetration Depth - In total internal reflection fluorescence microscopy (TIRFM) a small portion of the reflected light penetrates through the interface and propagates parallel to the surface in the plane of incidence creating an electromagnetic field in the liquid adjacent to the interface. This field is termed the evanescent field, and is capable of exciting fluorophores residing in the immediate region near the interface. This tutorial explores penetration depth of the evanescent field as a function of refractive index differences between the two phases surrounding the interface, the critical angle of incident illumination, and the laser excitation wavelength.

Evanescent Field Polarization and Intensity Profiles - Evanescent wave intensity at the interface surface (I(o)) is a function of both the incident angle and the polarization components of the light beam. The I(o) intensities observed for polarized vibration vectors are discussed in terms of a coordinate system with the plane of incidence (the x-z plane) defined as being parallel to the exciting light beam. This tutorial explores how changes in the incident angle affect evanescent wave intensity and the relationships between the electric field vectors of parallel and perpendicular components of the incident beam.

Polarized Light Evanescent Intensities - The light intensity at a TIRFM interface is a function of the illumination angle of incidence and the polarization of the incident light. This interactive tutorial explores how evanescent field intensities vary as a function of critical angle and the refractive index of the glass medium.

Variable Prism Configurations - A majority of the currently utilized TIRFM configurations rely on an added prism to direct laser illumination toward the interface where total internal reflection occurs, which is in the specimen conjugate plane of the microscope. This interactive Java tutorial explores TIRFM with a variable prism that morphs between a trapezoidal and cubic geometry with adjustable side angles and refractive index.

Trapezoidal Prism Microscope Configuration - The simplest approach to achieve total internal reflection from a culture chamber on an inverted microscope is direct laser illumination through a glass cube, prism, or trapezoidal block positioned on top of the chamber. This tutorial explores the effects of variations in refractive index and prism side angles on the critical angle and resulting incident laser angles.

Olympus TIRFM Fiber Illuminator Alignment - Designed as an accessory for the inverted IX-series of microscopes, the Olympus TIRFM illuminator acts to couple laser emission to the microscope via an external port. This tutorial explores alignment of the input fiber connector with the microscope optical path in order to optimize the incident light angle through high numerical aperture objectives.

High Numerical Aperture Objectives - TIRFM instrument configurations lacking a prism have been developed to take advantage of high numerical aperture immersion objectives to both produce excitation illumination at supercritical angles and to retrieve fluorescence information emitted by the specimen. This tutorial explores the effect of objective numerical aperture on incident angles in TIRFM.

Substage Prism Microscope Configuration - A TIRFM instrument configuration that is compatible with simultaneous microinjection or patch clamp experiments utilizes a substage prism as illustrated in the tutorial window. In order to provide easy and continuous access from above to a tissue culture bathed in buffer, the prism is deployed below stage level to contact the substrate glass. This tutorial explores multiple total internal reflection by the laser illumination source in designs of this type.

Olympus IX70 Microscope Light Pathways - This interactive tutorial explores illumination pathways in the Olympus IX70 research inverted tissue culture microscope. The microscope drawing presented in the applet illustrates a cut-away diagram of the Olympus TIRFM-IX laser illuminator that contains a positioning and focusing system for light input through a FC connector from an external laser source. In addition, the illuminator is equipped with a port for an arc-style lamphouse that can supply light from a mercury or xenon lamp for epi-fluorescence excitation. Also included is the traditional transmitted light pathway, which operates with a tungsten-halogen illuminator positioned on a pillar above the stage.

Selected Literature References - A number of high-quality review articles on total internal reflection fluorescence microscopy have been published by leading researchers in the field. This section contains periodical location information about these articles, as well as providing a listing of selected original research reports from this cutting-edge field of research.

TIRFM Resources on the Web - Although not as plentiful as other techniques, web resources on total internal reflection fluorescence microscopy will probably grow as the field becomes more popular.

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