Gerald W. Gordon

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

Time‐resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system

The design and implementation of a time‐resolved fluorescence lifetime imaging microscope (TRFLIM) for the biomedical sciences are described. The measurement of fluorescence lifetimes offers many benefits, among which is that they are independent of local signal intensity and concentration of the fluorophore and they provide visualization of the molecular environment in a single living cell. Unlike single photon counting, which employs a photomultiplier as the detector, TRFLIM uses a nanosecond‐gated multichannel plate image intensifier providing a two‐dimensional map of the spatial distribution of fluorescent lifetime in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to the fluorophore inside living cells on the stage of a fluorescent microscope. Images of the fluorescence emission at various times during the decay of the fluorescence are collected using a high‐speed gated image intensifier and the lifetimes are calculated on a pixel‐by‐pixel basis. Lifetimes measured by TRFLIM are compared with those measured by conventional methods.

Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components

Fluorescence recovery after photobleaching has been a popular technique to quantify the lateral mobility of membrane components. A variety of analysis methods have been used to determine the lateral diffusional mobility, D. However, many of these methods suffer from the drawbacks that they are not able to discern two-component diffusion (i.e., three-point fit), cannot solve for two components (linearization procedures), and do not perform well at low signal-to-noise. To overcome these limitations, we have adopted the approach of fitting fluorescence recovery after photobleaching curves by the full series solution using a Marquardt algorithm. Using simulated data of one or two diffusing components, determinations of the accuracy and reliability of the method with regard to extraction of diffusion parameters and the differentiation of one- versus two-component recovery curves were made under a variety of conditions comparable with those found in actual experimental situations. The performance of the method was also examined in experiments on artificial liposomes and fibroblast membranes labeled with fluorescent lipid and/or protein components. Our results indicate that: 1) the method was capable of extracting one- and two-component D values over a large range of conditions; 2) the D of a one-component recovery can be measured to within 10% with a small signal (100 prebleach photon counts per channel); 3) a two-component recovery requires more than 100-fold greater signal level than a one-component recovery for the same error; and 4) for two-component fits, multiple recovery curves may be needed to provide adequate signal to achieve the desired level of confidence in the fitted parameters and in the differentiation of one- and two-component diffusion.

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca2+; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca2+ in biological samples, we demonstrate thatintensity-based measurements of Ca2+ with single-wavelength Ca2+ probes such as Calcium Crimson may falsely report the actual Ca2+ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.

Cellular determinants of the lateral mobility of neural cell adhesion molecules

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using fluorescence recovery after photobleaching (FRAP). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode or in the size of their cytoplasmic domain, were expressed in NIH 3T3 cells and C2C12 muscle cells. When the various isoforms were compared in 3T3 cells, FRAP studies showed both GPI-anchored and transmembrane isoforms diffused rapidly and only small differences in either the diffusion coefficients (D) or the mobile fractions (mf) were measured, suggesting the importance of the ectodomain in regulating lateral diffusion. However, the mobility of all NCAM isoforms was greatly reduced in regions of cell-cell contact, presumably due to homophilic trans interactions between NCAMs on adjacent cells. NCAM isoforms transfected into C2C12 cells which express NCAM naturally usually displayed a significantly lower D compared to the same isoforms transfected into 3T3 cells. Thus, NCAM lateral mobility is modulated in regions where cells interact and by the structure of the host cell membrane.

Measurement of Fluorescence Resonance Energy Transfer in the Optical Microscope

Fluorescence resonance energy transfer (FRET) can be used as a spectroscopic ruler to study and quantify the interactions of cellular components with each other, as well as the conformational changes within individual molecules at the molecular level (Herman, 1998). FRET is a process by which a fluorophore (donor) in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor) nonradiatively through dipole—dipole interactions. This energy transfer manifests itself as both quenching of donor fluorescence intensity and lifetime (in the presence of acceptor) as well as an increase in the emission of acceptor fluorescence (sensitized emission). Because FRET decreases in proportion to the inverse sixth power of the distance between the donor and acceptor, this phenomenon is effective at measuring separation of the donor- and acceptor-labeled molecules when they are within 10–100 A of each other.

High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 A). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca 2+ ) i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

Fluorescence lifetime imaging in cell biology

The measurement of fluorescence lifetimes offers the advantages of being independent of local intensity and concentration of the fluorophore, and can provide information regarding the molecular environment in a single living cell. Historically, measurements of fluorescence lifetimes have employed photomultipliers as detectors, providing high sensitivity but sacrificing spatial information. Fluorescence Lifetime Imaging Microscopy (FLIM) provides a 2- or 3D spatial map of the distribution of fluorescent lifetime(s) in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to fluorophore containing living cells on the stage of a fluorescent microscope, and images of the fluorescence emission at various times during the decay of the fluorescence lifetime are collected using a high speed nanosecond-gated multichannel plate image intensifier. FLIM promises to substantially enhance the information obtainable from living cells and tissues, and will allow observations of the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques.