Total internal reflection fluorescence microscope

Fig. 1 Real-time tracking of cell adhesion [42]. (a) Components of a total internal reflection fluorescent microscope (TIRFM). (b) The cell adhesion process (7) a cell approaches the surface, (2) the cell lands, (3) the cell attaches, and (4) the cell spreads out on the surface. The evanescent field was generated by total internal reflection of a laser beam at the glass-water interface. Cells with fluorescently labeled membranes (dashed lines) were plated on SAMs. Cell membranes within the evanescent field (solid line) were observed by TIRFM. Corresponding TIRFM images are shown below...

Figure 2.6 Application of splinted RNA ligation procedure single molecule FRET. (A) Diagram of prism-type total internal reflection fluorescence microscope (TIRFM) for single molecule FRET measurements. (B) Distribution of single-molecule FRET values for dye-labeled telomerase RNA molecules generated by splinted RNA ligation. (C) Dye intensity and FRET traces of a single telomerase RNA molecule Cy3 emission (green), Cy5 emission (red), FRET ratio (blue).

Nishida, S., Funabashi, Y, and Ikai, A. 2002. Combination of AFM with an objective-type total internal reflection fluorescence microscope (TIRFM) for nanomanipulation of single cells. 

Design for a total internal reflection fluorescence microscope... 

Figure 3.23 shows the layout of a simplified total internal reflection fluorescence microscope (TIRFM). We briefly tour the configmation here, before expanding on experimental details such as alignment, laser power, and optomechanical considerations. The instrument we describe is suitable for single molecule... 

Fig. 15.1. Schematic representation of amyloid fibrils revealed by total internal reflection fluorescence microscopy, (a) The penetration depth of the evanescent field formed by the total internal reflection of laser light is 150nm for a laser light at 455 nm, so only amyloid fibrils lying parallel to the slide glass surface were observed. (b) Schematic diagram of a prism-type TIRFM system on an inverted microscope. ISIT image-intensifier-coupled silicone intensified target camera, CCD charge-coupled device camera...

TIRFM total internal reflection fluorescence microscopy/ microscope 3.7c.iv... 

On the other hand, optical microscopy, confocal microscopy, ellipsometry, scanning electron microscopy (SEM), scanning tunneling microscopy (STM), atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) are the main microscopic methods for imaging the surface structure. There are many good books and reviews on spectroscopic and chemical surface analysis methods and microscopy of surfaces description of the principles and application details of these advanced instrumental methods is beyond the scope of this book. 

During the last few years optical visualization techniques have also been introduced. Among them the total internal reflection fluorescence excitation (TIFR) microscopy [4] and optical interference-enhanced reflection microscopy [5] appear to be the most promising nonintrusive techniques. Their resolution, however, does not even approach the resolution of atomic force microscope and optical techniques may thus serve as an image survey of nanobubbles at 300 nm level (diameter) which is so far their resolution limit. 

Total internal reflection fluorescence (TIRF) microscopes are employed to study a diverse phenomena, including cell transport, signaUng, replication, motility, adhesion, and migration cell membranes and transport the structure of ribonucleic acid (RNA) neurotransmitters and virology. 

Leutenegger M, Ringemann C, Lasser T, Hell SW, Eggeling C (2012) Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS). Opt Express 20(5) 5243-5263... 

Standard fluorescence microscope equipped with high NA (-1.4) objective for efficient photon collection. An inverted microscope is more commonly used for this type of experiments because of possibility to illuminate and image in total internal reflection fluorescence (TIRF) mode but it is possible to use upright microscope as well. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

It is well known that both nanometre and nanosecond-picosecond resolutions at an interface can be achieved by total internal reflection (TIR) fluorescence spectroscopy. Unlike steady-state fluorescence spectroscopy, fluorescence dynamics is highly sensitive to microscopic environments, so that time-resolved TIR fluorometry at water/oil interfaces is worth exploring to obtain a clearer picture of the interfacial phenomena [1]. One of the interesting targets to be studied is the characteristics of dynamic motions of a molecule adsorbed on a water/oil interface. Dynamic molecular motions at a liquid/liquid interface are considered to be influenced by subtle changes in the chemical/physical properties of the interface, particularly in a nanosecond-picosecond time regime. Therefore, time-resolved spectroscopy is expected to be useful to study the nature of a water/oil interface. 

But also other materials with lower refractive indices enabling total internal reflectance like glass slides can be applied as transducer. Then, an expanded laser beam can be launched into the edge of a microscope slide, thus providing even fluorescence excitation within the evanescent field [45]. 

Figure 3.14 Illustration of the arrangement for prism-based evanescent wave excitation.A collimated laser beam (black) is totally internally reflected off the interface formed by the prism/substrate and water. A microscope objective is used to collect the fluorescence generated by the evanescent field at the surface of the substrate in wide field mode.

FRET fluorescence resonance energy transfer FCS fluorescence correlation spectroscopy TIRF total internal reflection fiuorescence PCFI photon counting histogram ICCD intensified charge coupled device EMCCD eiectron muitipiying charge coupled device CMOS complimentary metal oxide semiconductor AFM atomic force microscope. 

One of the challenges to investigate interfaces by FCS is the selective collection of the fluorescence emitted in the vicinity of the surface. The axial resolution of a confocal microscope results in averaging of the fluorescence emission within approx. 1 pm. As a consequence, surface effects are in most cases obscured in normal FCS measurements. With the evanescent wave of a laser beam totally reflected at the solid-liquid interface, however, the excitation can be restricted to a ca. 100 nm thin layer at the interface. This so-called total internal reflection FCS... 

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