Photobleaching Problems Research Articles

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues

FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.

Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

Controlled bimolecular collisions allow sub-diffraction limited microscopy of lipid vesicles

The concentration and vesicle size-controlled collisions of single molecules with target biological assemblies allow sub-diffraction limited optical images to be obtained that are not subject to the usual photobleaching problems with single molecule experiments. For example, single molecules of the probe Nile Red in aqueous solution emit a burst of fluorescence when they collide with a 50 nm hydrophobic vesicle situated on the surface in the laser focus. The bimolecular kinetics of the bursts is defined by their on- and off-time distribution functions which depend on the concentration and diffusion of the probe and the vesicle size. The mean burst frequency changes much more sharply than does the fluorescence intensity when a vesicle is raster scanned through the laser focus. This sharpness allows the spatial resolution of two objects to be improved and separations less than the diffraction limited resolution of the conventional optical microscope to be measured. The principle of this method of trajectory time distribution optical microscopy (TTDOM) could be used in a far field optical microscopic system with a resolution of several nanometers.

Improving the signal sensitivity and photostability of DNA hybridizations on microarrays by using dye-doped core-shell silica nanoparticles.

The development of new highly sensitive and selective methods for microarray-based analysis is a great challenge because, for many bioassays, the amount of genetic material available for analysis is extremely limited. Currently, imaging and detection of DNA microarrays are based primarily on the use of organic dyes. To overcome the problems of photobleaching and low signal intensities of organic dyes, we developed a new class of silica core-shell nanoparticles that encapsulated with cyanine dyes and applied the dye-doped nanoparticles as labeling in the DNA microarray-based bioanalysis. The developed nanoparticles have core-shell structure containing 15-nm Au colloidal cores with 95 dye-alkanethiol (dT)20 oligomers chemisorbed on the each Au particle surface and 10-15-nm silica coatings bearing thiol functional groups. To be utilized for microarray detection, the dye-doped nanoparticles were conjugated with DNA signaling probes by using heterobifunctional cross-linker. The prepared nanoparticle conjugates are stable in both aqueous electrolytes and organic solvents. Two-color DNA microarray-based detection was demonstrated in this work by using Cy3- and Cy5-doped nanoparticles in sandwich hybridization. The use of the fluorophore-doped nanoparticles in high-throughput microarray detection reveals higher sensitivity with a detection limit of 1 pM for target DNA in sandwich hybridization and greater photostable signals than the direct use of organic fluorophore as labeling. A wide dynamic range of approximately 4 orders of magnitude was also found when the dye-doped nanoparticles were applied in microarray-based DNA bioanalysis. In addition, the use of these dye-doped nanoparticles as the labeling in hybridization also improved the differentiation of single-nucleotide polymorphisms. This work offers promising prospects for applying dye-doped nanoparticles as labeling for gene profiling based on DNA microarray technology.

12] Functional imaging of mitochondrial redox state

The use of intrinsic fluorophores allows evaluation of the function of mitochondrial oxidative phosphorylation without having the phototoxic side effects of the various fluorescent mitochondrial dyes. To detect the changes of the mitochondrial redox state in response to alterations of oxidative phosphorylation (OxPhos) at the single cell level, the fluorescence changes of the α-lipoamide dehydrogenase flavin moiety by confocal microscopy is investigated. Functional imaging of the mitochondrial redox state can be performed by microscopic detection of the flavoprotein fluorescence of the mitochondrially localized α-lipoamide dehydrogenase, which is in tight redox equilibrium with the mitochondrial NAD system. Direct detection of the NAD(P)H fluorescence signal for mitochondrial NAD redox state determinations can be performed in digitonin-permeabilized cells or saponin-permeabilized muscle fibers at considerably higher sensitivity. The procedure is applicable for cultured cells having low mitochondrial content. For the determination of putative heterogeneities of the mitochondrial redox state in permeabilized muscle fibers, the application of Fp/NAD(P)H ratio imaging is advantageous, because it offers an increased sensitivity with respect to metabolic alterations of the fluorescence signals. In sum, the monitoring of intrinsic fluorophores allows one to obtain insights into the mitochondrial function of single living cells, avoiding the problems of phototoxicity and photobleaching.

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts.

Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acid N-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.