Diffusion and binding analyzed with combined point FRAP and FCS.

To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes.

Quantification of protein interaction in living cells by two-photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed-through.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.

EMCCD-based spectrally resolved fluorescence correlation spectroscopy.

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

Specific nucleic acid detection using photophysical properties of quantum dot probes.

In the present study, we report a novel separation-free method to detect and quantify avian influenza virus A (H5N1) nucleic acid without amplification, based on the alteration of photophysical parameters of quantum dot (QD) probes after hybridization with specific complementary target DNA. The target DNA was quantified in a custom-made portable device by simultaneously measuring lifetime and quenching of the QD probes. QD probes (25-mer) showed a 30% lifetime reduction and 40% fluorescence quenching when hybridized with complementary 25-mer target DNA. In comparison with a conventional QD-based assay, this assay provides a simple quantitation of nucleic acids with a single labeling step.

Two-photon spectral imaging with high temporal and spectral resolution.

We introduce a fast spectral imaging system using an electron-multiplying charge-coupled device (EM-CCD) as a detector. Our system is combined with a custom-built two-photon excitation laser scanning microscope and has 80 detection channels, which allow for high spectral resolution and fast frame acquisition without any loss of spectral information. To demonstrate the efficiency of our approach, we applied this technology to monitor fluorescent proteins and quantum dot-labeled G protein-coupled receptors in living cells as well as autofluorescence in tissue samples.

Quantification of scattering changes using polarization-sensitive optical coherence tomography.

We demonstrate that changes in the degree of polarization (DOP) depend on changes in the scattering coefficient, and they can be quantified by using a polarization-sensitive optical coherence tomography (PS-OCT) system. We test our hypothesis using liquid and solid phantoms made from Intralipid suspensions and gelatin, respectively. We also quantify the DOP changes with depth caused by changes in the concentration of scatterers in the liquid and solid phantoms. It is clearly shown that the DOP change has a linear relationship with the scattering change. In our previous study, we showed that the axial slope of the DOP is different between normal and pathologic cervical tissues. Our results demonstrate that the quantification of the axial DOP slope can be used for the systematic diagnosis of certain tissue pathology.

Optical diagnosis of cervical intraepithelial neoplasm (CIN) using polarization-sensitive optical coherence tomography.

We present a polarization-sensitive optical coherence tomography (PS-OCT) technique that can quantify the polarization changes (the degrees of circular polarization, DOCP) caused by the scattering changes induced by cervical intraepithelial neoplasia (CIN). The axial and lateral resolutions of our PS-OCT system are 13 microm and 15 microm, respectively. Uterine cervical conization tissue samples from 18 patients were examined, and 71 areas were imaged for in vitro studies; about 2-4 areas per sample were imaged and processed for diagnosis. The scanned areas had a size of 2 mm (axial) X 2 mm (lateral) X 4 mm (transversal). We quantified the slope of the axial decay of the DOCP signal near the cervical epithelium by a linear fitting procedure. The excised samples were then investigated by two pathologists, and their histological findings were later compared with the PS-OCT results. Our results show that the sensitivity and specificity are 94.7% and 71.2%, respectively.