Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution

Spectral properties of single gold nanoparticles in close proximity to biological fluorophores excited by 2-photon excitation

Metallic nanoparticles (NPs) are able to modify the excitation and emission rates (plasmonic enhancement) of fluorescent molecules in their close proximity. In this work, we measured the emission spectra of 20 nm Gold Nanoparticles (AuNPs) fixed on a glass surface submerged in a solution of different fluorophores using a spectral camera and 2-photon excitation. While on the glass surface, we observed the presence in the emission at least 3 components: i) second harmonic signal (SHG), ii) a broad emission from AuNPS and iii) fluorescence arising from fluorophores nearby. When on the glass surface, we found that the 3 spectral components have different relative intensities when the incident direction of linear polarization was changed indicating different physical origins for these components. Then we measured by fluctuation correlation spectroscopy (FCS) the scattering and fluorescence signal of the particles alone and in a solution of 100 nM EGFP using the spectral camera or measuring the scattering and fluorescence from the particles. We observed occasional fluorescence bursts when in the suspension we added fluorescent proteins. The spectrum of these burst was devoid of the SHG and of the broad emission in contrast to the signal collected from the gold nanoparticles on the glass surface. Instead we found that the spectrum during the burst corresponded closely to the spectrum of the fluorescent protein. An additional control was obtained by measuring the cross-correlation between the reflection from the particles and the fluorescence arising from EGFP both excited at 488 nm. We found a very weak cross-correlation between the AuNPs and the fluorescence confirming that the burst originate from a few particles with a fluorescence signal.Fil: Anzalone, Andrea. University of California at Irvine; Estados UnidosFil: Gabriel, Manuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina. University of California at Irvine; Estados UnidosFil: Estrada, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina. University of California at Irvine; Estados UnidosFil: Gratton, Enrico. University of California at Irvine; Estados Unidos. University of New England; Australi

Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging

Both multi-cell biochemical assays and single cell fluorescence measurements have revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Employing high-speed 3D fluorescence nanoimaging we resolve here at the single cell level multiple, distinct regions of mRNA synthesis within a labeled transgene array. By employing phasor analysis, a fluorescence fluctuation spectroscopy technique, we demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm, and we extract the local PolII elongation rate. While we detect a range of 10-100 bp/s for PolII elongation from cell to cell, we are now also able to measure up to a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell. Furthermore, we are able to visualize changes of PolII elongation as a function of time. We observe a correlation between the average elongation rate measured in a locus and its local mobility. Finally, by cross-correlating the transcriptional activity with the nm-sized movements of the active loci, we provide evidence of an active molecular mechanism determining displacements of the transcription sites concomitant to increases in transcriptional activity. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.Comment: 56 pages, 5 main figures and 10 supplementary figure

Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.

The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone FRET in live cells co-expressing H2B-eGFP and H2B-mCherry. The data was acquired in the frequency domain [5] and processed by the phasor approach to lifetime analysis [6]. The data can be valuable to researchers interested in using the histone FRET assay since it highlights the impact of cellular autofluorescence and acceptor-donor ratio on quantifying chromatin compaction. The data is related to the research article "Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response" [4]

Quantitative image mean squared displacement (iMSD) analysis of the dynamics of profilin 1 at the membrane of live cells.

Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories

In Vivo Imaging of Single-Molecule Translocation Through Nuclear Pore Complexes by Pair Correlation Functions

BACKGROUND: Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu. METHODOLOGY: The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which single particle tracking (SPT) has given results that cannot be confirmed by traditional single-point FCS measurements because of the lack of spatial resolution. CONCLUSIONS: We show that obstacles to molecular flow can be detected and that the pCF algorithm can recognize the heterogeneity of protein intra-compartment diffusion as well as the presence of barriers to transport across NE

Two-photon imaging of cancer cell extravasation in live mice

Abstract MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-GFP channel. New approaches will allow the tracking of the tip of the protrusion to determine if focal adhesions are forming there as the cells extravasate. This is important in establishing the mechanism of cell extravasation and migration in tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2011-141