FRET, FRAP, FISH - Technology & Techniques. The use of fluorescence microscopy in the life sciences runs the gamut from basic photodocumentation to dynamic single-molecule fluorescence (SMF) studies. Recent advances in digital imaging technology are helping to expand the utility and popularity of many fluorescence microscopy techniques, including Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and fluorescence in situ hybridization (FISH). The newly created Microimaging Applications Group, which comprises Photometrics, Media Cybernetics, QImaging, Gatan, and MAG Biosystems, offers a broad range of innovative imaging solutions designed to enhance life science research capabilities.

FRET Imaging

Förster resonance energy transfer is a phenomenon in which nonradiative transfer of energy occurs between donor and acceptor molecules in close proximity (2-7 nm). Since FRET efficiency decays as a function of the inverse sixth power of the distance between the donor and acceptor, this phenomenon can be leveraged to provide solid evidence of an interaction between the donor and acceptor in a FRET pair.

In FRET, the donor molecule is returned to a ground state without fluorescence emission while the acceptor molecule is raised to an excited state. Upon decay of the acceptor's excited state, fluorescence emission may be witnessed. Thus, an increase in FRET between label molecules will result in a decrease in donor emission and a simultaneous increase in acceptor emission. Using FRET detection, interactions between molecules can be monitored in subcellular compartments and tracked as a function of time. FRET applications include evaluating the structure of proteins, determining the spatial distribution and assembly of protein complexes, monitoring receptor/ligand interactions, and sensing the presence of small molecules in living cells. FRET experiments are often performed using standard ratio imaging techniques. Depending on the application, FRET is used to qualitatively or quantitatively investigate experimental phenomena. While qualitative experiments focus on simply determining the presence or absence of FRET as an indicator of interaction, quantitative experiments require a more methodical strategy.

To help ensure accurate results, next-generation Photometrics electronmultiplying CCD (EMCCD) cameras provide exceptionally high quantum efficiency, quantitative stability across 16 bits, and linear EM gain up to 1000x. Even with superior camera performance, sequential imaging techniques (e.g., using an emission filter wheel or switching microscope filter cubes) can make proper data calibration and correction very difficult, if not impossible, when dynamic samples are used. Therefore, many quantitative FRET applications require that the donor and acceptor emissions be simultaneously imaged. To meet this criterion, MAG Biosystems offers easy-to-use instrumentation that splits the incident beam from the microscope into independent beams. Each of the resultant emission channels is projected onto a region of a CCD or EMCCD array. A precision optical and mechanical design allows subpixel image registration and minimizes light loss for simultaneous multichannel acquisition.

FRAP Imaging

Fluorescence recovery after photobleaching is useful for examining intracellular molecular variables such as nuclear protein complex dynamics, diffusional mobility of membrane proteins, and cytoskeletal dynamics. FRAP is a powerful mode of fluorescence light microscopy in which a specialized illumination strategy is implemented in order to permit perturbation of the steady-state fluorescence distribution by bleaching fluorescence in selected regions of a sample. After the bleaching step, researchers can observe and analyze how the fluorescence distribution returns to the steady state. Because the photobleaching of fluorophores is permanent, changes in the fluorescence intensity in both the bleached and unbleached regions are attributable to the exchange of bleached and unbleached fluorescent molecules between those regions. FRAP microscopy is typically geared towards dynamic, lowlight endeavors.

Recently, MAG Biosystems introduced a widefield imaging system designed to study the intracellular dynamics of proteins and other macromolecular complexes via FRAP and iFRAP (inverse FRAP) experiments in 2D plus time and 3D plus time. Photoactivation and photo- conversion studies with fluorescent proteins such as PA-GFP, EOS, KFP, Kaede, and Dronpa can be performed. Several technological innovations, including Burst mode and a custom optical path, provide a combination of speed, sensitivity, and ease of use not found in other FRAP systems. When run in Burst mode, the delay between the end of the bleach pulse and the first recovery image is minimized, enabling fast dynamic analyses. The FRAP-3D system also lets researchers photobleach-on-the-fly by simply clicking within a live image display window to bleach the region appearing under the cursor. FRAP-3D includes a galvanometerbased FRAP head, an advanced laser launch module, high-speed I/O circuitry to control all system components, acquisition and analysis software with an intuitive graphical user interface (GUI), and a configured workstation. The head can be mounted to many inverted microscopes through the epi-illumination port in order to enable simultaneous laser and widefield illumination. A versatile optical design allows researchers to switch seamlessly between FRAP studies and standard widefield applications without reconfiguring the system's hardware. To meet user-specific quantum efficiency, spatial resolution, and frame rate requirements, the FRAP-3D system utilizes high-performance quantitative CCD and EMCCD cameras from Photometrics. To ensure the utmost instrumentation utility, FRAP-3D allows future upgrades for spinning-disk confocal microscopy and other imaging modalities.