Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of
Förster Resonance Energy Transfer (FRET) for single molecular complexes recently lead to the development of a novel
method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al.,
Nature Photon., 3, 2009). An appealing feature of this method is its ability to provide such important information while
being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98, 2010). We
will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the
cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the
membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data
for determination of the quaternary structure of some proteins (or the protein complex structure).
Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule
in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the
cell under study is scanned a number of times in order to accumulate enough spectral information to accurately
determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may
change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average
over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of
spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level
spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in
oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single
spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This
presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to
the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor,
Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.
Resonant Energy Transfer (RET) from an optically excited molecule to a non-excited molecule residing nearby has been
used to detect molecular interactions in living cells. Information such as the number of proteins forming a molecular
complex has been obtained so far for a handful of proteins, but only after exposing the samples sequentially to at least
two different excitation wavelengths. Changes in the molecular makeup of a cellular region occurring during this lengthy
process of measurement has limited the applicability of RET to determination of cellular averages. We developed a
method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a
spectrally-resolved two-photon microscope. The use of diffractive optics in a non-descanned configuration allows
acquisition of a full set of spectrally-resolved images after only one complete scan of the excitation beam. This
presentation will briefly describe our basic experimental setup and a simple theory of RET in oligomeric complexes, and
it will review our recent results on determination of the geometry and size of oligomeric complexes of several proteins in
yeast as well as in mammalian cells. This method basically transforms RET into a method for performing veritable
structural determinations of protein complexes in vivo.
KEYWORDS: Molecules, Fluorescence resonance energy transfer, Microscopes, Luminescence, Proteins, Sapphire lasers, Spectral resolution, Green fluorescent protein, Yeast, Molecular energy transfer
Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a
broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative
Förster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states,
require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In
addition, for studies of dynamics in biological systems the time required for tuning the excitation should be
commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked
Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse
shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup,
represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us
to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may
be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo.
We have developed a fast, sensitive multiphoton microscope employing a tunable femtosecond laser and an electron-multiplying
CCD camera (EMCCD) in a non-descanned detection scheme. In one configuration, the microscope
provides "standard" multiphoton fluorescence images with a relatively high acquisition rate (limited only by the speed of
the scanners) over a broad band of emission wavelengths. In a second configuration, the use of a diffractive optic
element allows acquisition of one full set of spectrally-resolved images after only one complete scan. Increased speed is
achieved in this configuration by setting the integration time of the camera such that an entire line is scanned at once,
with the spectrum at each point being perpendicular to the scanned line. In both configurations, a suitable optical
arrangement permits acquisition of high resolution images for both multiphoton excitation of fluorescence and widefield
transmission microscopy. To illustrate the performances of our microscope we present spectrally-resolved images
of individual fluorescent molecules spread over a surface as well as fluorescence images of yeast cells expressing a
membrane receptor tagged with a variant of the green fluorescent protein.
KEYWORDS: Fluorescence resonance energy transfer, Molecules, Luminescence, Proteins, Green fluorescent protein, In vivo imaging, Molecular energy transfer, Molecular interactions, Energy efficiency, Energy transfer
Fluorescence Resonance Energy Transfer (FRET) - a process of nonradiative energy transfer from an optically excited molecule (donor, D) to an unexcited nearby molecule (acceptor, A) - is a powerful tool in studies of protein-protein interactions in living cells. FRET can be quantified, e.g., by measuring an increase in the donor fluorescence after inactivating the acceptor through photobleaching. In spite of its sheer simplicity, this method also introduces donor bleaching, which often complicates the interpretation of data. Correction methods are complicated by the fact that D photobleaching depends on whether a D molecule is free or coupled to an A molecule. In this communication we show that, instead of being a nuisance, donor bleaching actually can be harnessed to provide invaluable information about a population of interacting proteins in vivo. We present data on proteins tagged with fluorescent molecules, which indicate that donor and acceptor bleaching kinetics reveal quantitative information on the stoichiometry of proteinprotein
interaction. Under appropriately chosen conditions, we are able to model the bleaching kinetics of D and A (both interacting and noninteracting) and determine the stoichiometry of the protein interaction. This provides a method for imaging protein complexes in living cells, which opens a way for testing the law of mass action in vivo.
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