Masilamani Elangovan

Nanosecond fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell

Visualizing and quantifying protein–protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity‐based FRET microscopy is limited by spectral bleed‐through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed‐through is not an issue in FRET–FLIM imaging. In this paper we describe the development of a nanosecond FRET–FLIM microscopy instrumentation to acquire the time‐resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one‐ and two‐component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.

Confocal FRET Microscopy to Measure Clustering of Ligand-Receptor Complexes in Endocytic Membranes

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

7 – FRET Data Analysis: The Algorithm

This chapter discusses the development of computer-based algorithms to measure and then remove the contaminating signals from the Forster resonance (radiationless) energy transfer (FRET) image based on single-labeled reference samples. The algorithm-based image acquisition and processing approach to correct spectral bleedthrough (SBT) represents an advancement over the other methods, particularly if the algorithm is used for the estimation of the rate of energy transfer efficiency (E%) or the distance between the donor and the acceptor molecules or for higher-level analysis. In the algorithm, the data processing is considered in various intensity ranges that would normalize the expression level variation in the cellular images. This algorithm allows the estimation of the SBT correction for linear and nonlinear intensity distributions. This method can be used for any intensity-based FRET imaging techniques, including wide-field, total internal reflection (TIRE), confocal, and multiphoton microscopy FRET data analysis.

Wide-Field, Confocal, Two-Photon, and Lifetime Resonance Energy Transfer Imaging Microscopy

The light microscope has been used for almost a century to produce images of cells, and this approach has contributed enormously to our understanding of cellular structure and function (Bright and Taylor, 1986; Herman, 1998; Inoue and Spring, 1997; Pawley, 1995; Periasamy and Herman, 1994). In turn, molecular biological studies over the past few decades have shown that cellular events, such as signal transduction and gene transcription, require the assembly of proteins into specific macromolecular complexes. What we require now are methods to visualize these protein—protein associations as they occur in the living cell. Recent advances in digital imaging coupled with the development of new fluorescent fluorophores now provide the tools to begin the study of protein-protein interactions in the intact cell. In this chapter we describe four different imaging techniques that apply the method of fluorescence resonance energy transfer (FRET) to surpass the optical limitations of the light microscope, allowing detection of the physical interactions of proteins in the living cell.

Bleed-through and photobleaching correction in multiphoton FRET microscopy

Fluorescence resonance energy transfer (FRET) microscopy provides a tool to visualize the protein with high spatial and temporal resolution. In multi-photon FRET microscopy one experiences considerably less photobleaching compared to one-photon excitation since the illumination is the diffraction limited spot and the excitation is infrared-pulsed laser light. Because of the spectral overlap involved in the selection of the fluorophore pair for FRET imaging, the spectral bleed-through signal in the FRET channel is unavoidable. We describe in this paper the development of dedicated software to correct the bleed-through signal due to donor and acceptor fluorophore molecules. We used living cells expressed with BFP-RFP (DsRed)-C/EBP(alpha) proteins in the nucleus. We acquired images of different combinations like donor alone, acceptor alone, and both acceptor and donor under similar conditions. We statistically evaluated the percentage of bleed-through signal from one channel to the other based on the overlap areas of the spectra. We then reconstructed the images after applying the correction. Characterization of multi-photon FRET imaging system taking into account the intensity, dwell time, concentration of fluorophore pairs, objective lens and the excitation wavelength are described in this paper.

Microscopy Core Facilities: Results of an International Survey

Introduction Core centers in the life sciences at academic institutions are well established [1], providing services for histology, sequencing, genomics, biostatistics, etc. Creating and running a microscopy core facility, however, presents unique challenges and opportunities [2, 3]. Cutting edge, sophisticated instrumentation provides researchers with many opportunities to advance their research goals, while perhaps requiring expert advice, training, or consultation from core center staff. The requirements of instrument maintenance, data storage, analysis, interpretation of results, and facility finances put a premium on the technical and management skills of core center directors and staff. The heterogeneous nature of microscopy cores, most likely based on historical developments, leads to multiple units in some academic institutions and sparks calls for consolidation, streamlining of systems for greater efficiency, and better resource utilization [3]. This article presents results of a survey constructed to analyze the functionality and management of biological microscopy core facilities. The range of questions included in the survey covered various aspects of facility structure and organization, instrumentation, financial support, productivity tools, and user profiles. Analysis of these data provides insights into how best to organize and manage a microscopy core facility. These survey data should provide institutions with some guidelines for operation of a core facility of this nature. Institutions newly adopting a shared resource system would benefit from input on how core facilities are run in different parts of the world. The responses allow new facilities to better understand the demands of a business-like environment combined with a technological service function when planning a new microscopy core center. Existing core facilities may find some interesting practices, which may help them to better manage their own organization, increase their numbers of users, work more efficiently, and possibly find new sources of financial support.

FRET-FLIM microscopy

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy coupled with the development of new fluorescent probes provide the tools to study protein interactions in living specimens. Spectral bleed-through or cross talk is a problem in one- and two-photon microscopy to recognize whether one is observing the sensitized emission or the bleed-through signals. In contrast, FLIM (fluorescence lifetime imaging microscopy) or lifetime measurements are independent of excitation intensity or fluorophore concentration. The combination of FLIM and FRET will provide high spatial (nanometer) and temporal (nanoseconds) resolution when compared to steady state FRET imaging. Importantly, spectral bleed-through is not an issue in FLIM imaging because only the donor fluorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the FRET efficiency and the distance between donor- and acceptor-labeled proteins. Moreover, the FRET-FLIM technique allows monitoring more than one pair of protein interactions in a single living cell.

Comparison of one- and two-photon fluorescence resonance energy transfer microscopy

The physics and chemistry of fluorescent resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years, but only with recent technical advances has it become feasible to apply FRET in biomedical research. FRET microscopy is a better method for studying the structure and localization of proteins under physiological conditions than are X-ray diffraction, nuclear magnetic resonance, or electron microscopy. In this study, we used four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides 2-D spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleed through and photo bleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. We will discuss the advantage of fluorescence lifetime methods to measure FRET signals at the moment of the protein-protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution.

Kinetics and comparison of δ-aminolevulinic-acid-induced endogenous protoporphyrin-IX in single cell by steady state and multiphoton fluorescence imaging

Photodynamic Therapy has emerged as a new modality in the treatment of various nonmalignant and malignant diseases. It involves the systemic administration of tumor specific photo-sensitizers with the subsequent application of visible light. This combination causes the generation of cytotoxic species, which damage sensitive targets, producing cell injury and tumor destruction. Although, photofrin is the only photosensitizer currently approved for PDT and tumor detection, its concomitant cutaneous photosensitization poses a significant problem. Hence, δ-aminoleuvulinic acid (δ-ALA) a precursor for the endogenous production of Protoporphyrin IX, through heme biosynthesis pathway, has gained significant importance in the Photodynamic Therapy. Though δ-ALA is present naturally in the cells, exogenous δ-ALA helps to synthesis more of PpIX in the tumor cells, as the fast growing tumor cells take up the administered δ-ALA more than the normal cells. Based on these facts, many invasive studies have been reported on the kinetics of δ-ALA at cellular level by chemical extraction of PpIX from the cells. In the present study we have studied the kinetics of δ-ALA induced PpIX fluorescence from Hela cells by perchloric/Methanol extraction method. However, the amount of PpIX synthesized in the cells at different point of incubation time by noninvasive methods has not been reported. Hence we have also used a noninvasive technique of measuring the kinetics δ-ALA induced PPIX fluorescence from Hela, an epithelial cell derived from human cervical cancer by both single photon (steady state) and multi photon excitation. From the studies it is observed that the δ-ALA induced PpIX is more at 2 hours incubation time for 2 mM of δ-ALA concentration. Further, it is observed that with steady state fluorescence imaging method, the excitation light itself cause the Photodynamic damage, due to the prolonged exposure of the cells than in multi photon excitation, leading to the rounding of cells. This may be due to the activation of PpIX in cells by the excitation light source.

Use of laser scanning confocal and two-photon FRET microscopy to image and quantify the co-localization of fluorophore-labeled ligands in MDCK epithelial cells

Here, we use fluorescence resonance energy transfer (FRET) imaging techniques to assay and study the organization and dynamics of endosomes in epithelial cells. We use polarized epithelial MDCK cells stably transfected with polymeric IgA-receptor (pIgA-R) to analyze the co-localization, within 10 -100A of pIgA-R-ligand complexes labeled with different fluorophores in the apical endosome. When internalized at 17 degree(s)C for four hours, these complexes co-localize in the apical endosome, which is located underneath the apical plasma membrane. While the transport pathways crossing are thought to be understood, the actual morphology of this endosome has not been completely characterized. Here, we compare the ability of laser scanning confocal and Two-Photon FRET microscopy to image the co-localization of differently labeled (donor: Alexa488, acceptor: Cy3) receptor-ligand complexes in the apical endosome. While the preliminary results are broadly similar, we have found that Two-Photon FRET microscopy possesses significant advantages over laser confocal microscopy FRET. In confocal microscopy FRET, the actual FRET signal in the acceptor channel, following donor excitation, is contaminated by the excitation of the acceptor molecule by the donor wavelength as well as by the cross-talk between donor and acceptor emissions in the acceptor channel. We are in the process of testing an algorithm that will correct this problem. In Two-Photon microscopy, we were able to prevent the excitation of the acceptor molecule by the donor wavelength. Furthermore, we have developed a method to manipulate the Two-Photon FRET data post-acquisition to remove the remaining contaminating signal, i.e. the cross-talk between donor and acceptor emissions in the acceptor channel. Since Two-Photon microscopy avoids out-of-focus bleaching which allows repeated scanning in multiple focal planes, a more detailed picture of intracellular events can be obtained. Therefore, we should be able to use this technology to assay the FRET signal along the vertical axis of polarized epithelial cells, such as MDCK cells. In summary, our results indicate that the Two-Photon FRET microscopy is a powerful tool to assay intracellular protein-protein co-localization/interaction events.