NIH P41 GM103540 Research Articles

Automatic Image Segmentation Based on Phasor Space Clustering

We present our latest developments in automatic image segmentation for measurements on spectral and lifetime fluorescence imaging. The algorithm operates on the basis of the phasor approach and makes use of a well-established machine learning method for clustering N-dimensional data points, in our case the pixel data points on the phasor space. The technique is based on Gaussian mixture models solved by the use of the expectation-maximization algorithm. We show the method is the best candidate out of the many machine learning clustering techniques available due to the nature of the phasor data and also due to the spreading of points in the phasor space for the cases of low pixel photon counts. In order to quantify the power of the method we performed simulations with synthetic phasor data and we tested the method by measuring its performance against a series of established clustering techniques. We also show results on lifetime and spectral data obtained from samples of live cells. The samples were prepared by staining with a series of commercially-available organelle dyes in order to target distinct structures in the cells. We show how by taking a single spectral and lifetime acquisition we resolve up to six populations in the phasor space corresponding to the six cellular staining probes. Furthermore, we use this method in combination with our other recent work, where we make use of higher harmonics of the phasor transform in order to solve for the fractions of known components and also for lifetimes of unknown components. This multi-harmonic multi-component method is highly sensitive to noise and heterogeneous distributions and here is where our clustering method simplifies the problem by preprocessing the data and fragmenting it into pixels containing distinct lifetime/spectral characteristics. (Supported by NIH P41-GM103540).

Primary Cilium Submicron Organization and Dynamics

The aim of this work is to contribute to the characterization of the biophysical properties of the ciliary microenvironment in order to understand central processes of the organelle's biology such as protein import, movement and crowding organization. First, we analyzed the movement of a small soluble protein (EGFP) to establish its pattern of diffusion in different regions of the ciliary compartment (body and distal tip). Using advanced fluorescence fluctuation correlation spectroscopy (Comprehensive correlation analysis) with the Zeiss Airyscan Detector, we characterized the organization and dynamic fingerprint of these subcompartments. Preliminary results showed that both ciliary body and distal tip have permeable microdomains explaining why EGFP presents a pattern of diffusion compatible with dynamic partition rather than free diffusion. However, at the ciliary body diffusion is more confined than at the ciliary tip. Measurements on cytoplasm and nucleus were made to compare with cilia crowding organization. Moreover, to study ciliary structural properties, we used environment-sensitive probes (ACDAN for the intra-ciliary compartment and LAURDAN for the ciliary membrane) combined with spectral phasor analysis to study molecular crowding and membrane's lipid order. Molecular crowding seems similar along cilia (base, body and distal tip). However, the ciliary base membrane was more ordered (less fluid) than the rest of the organelle. In conclusion, we put together a comprehensive protocol to study biophysical properties of primary cilium using state-of-the-art fluorescence techniques. This protocol will allow the evaluation of how different treatments or conditions affect the structure and dynamics of this organelle. (Acknowledgements: Work supported in part by NIH P41-GM103540 and Agencia Nacional de Investigación e Innovación Uruguay.)

Measuring the Spatial Distribution of Dipolar Relaxation in Live Zebrafish Eye Lenses during Development

This work presents image processing techniques we developed to analyze spectral images of in vivo zebrafish eye lenses using ACDAN (6-acetyl-2-dimethylaminonaphthalene) as a probe for intracellular dipolar relaxation (DR). Using these techniques we analyzed the development of regional DR in zebrafish lenses, as water homeostasis is crucial for normal development of structure and optical properties of the lens. We imaged lenses between two and nine days post-fertilization encompassing stages ranging from immature embryonic to functional emmetropic lenses at larval stages. ACDAN fluorescence was collected using a 32-channel spectral detector at anterior, equatorial and posterior planes of the ocular lens in anesthetized zebrafish incubated in ACDAN. We used the spectral phasor transformation to obtain a continuum of values within an arbitrarily-set interval in order to measure DR at each pixel of the images. From these images we interpolated radial DR profiles in all spatial directions from which a mean radial profile at each plane of the lens was calculated. We show that the mean radial profile presents a maximum in DR which shifts from the center of the lens towards the outer cortex during development. Moreover, the lens-averaged absolute value of DR decreases with age. Power-spectrum analysis of the radial profiles showed changes in cell size as a function of radial position which was also confirmed by watershed-based cell segmentation. Furthermore, complete high-resolution z-stacks allowed us to prove that the 3D spatial distribution of DR is spherically and not cylindrically symmetrical. The methods presented here allow us to quantify and map subcellular DR and analyze morphological properties of developing zebrafish lenses in vivo. (Supported by NIH P41-GM103540 and R01-EY5661).

Water Dynamics of a Model Protein Phase Separation via Fluorescence Lifetime and Spectral Analysis of ACDAN

Cells can organize many of their biochemical reactions in membrane-less compartments. These can be achieved by self-assembly of proteins leading to protein phase separations (PPS). The mechanism of de-mixing can be physiological (inner centromere) and/or pathological (stress granules). PPS organize cellular regions via confinement and exclusion of the molecules affecting their dynamics within and surrounding the compartment. PPS, also called coacervates, can express different types of phase, from liquid- to solid-like. To characterize that, investigations commonly starts with the definition of the phase separation diagram of the purified protein that drive the water/protein liquid demixing. In our opinion, to better understand these complex phases, the characterization of the water dynamics is fundamental. In this contest, we proposed the study of the water dipolar relaxation measuring the emission spectra and fluorescence lifetime at optical resolution of ACDAN (6-acetyl-2-dimethylaminonaphthalene, solvofluorochromic dye) that colocalize on a model PPS. Indeed, we implement the method characterizing the phase diagram of a model PPS obtained by the coacervation of bovine serum albumin (BSA) in several ratios with a crosslinker and a molecular crowder. Here, phasor analysis of the ACDAN optical properties (fluorescence lifetime and emission spectrum) allowed to highlight differences in terms of water dynamics of the BSA coacervates even if the shape and size of the PPS were the same. This method can improve the definition of the phase diagram in solution and in cellular environments. We believe this approach can be useful to track the inner dynamics of the PPS especially when they can evolve to severe pathological conditions. (Work Supported in part by Grant NIH P41-GM103540)

Airyscan Cca Provides Structural and Dynamics Fingerprinting of Subcellular Compartments in Living Cells

In this work we will present our current advances in the development of the CCA (Comprehensive Correlation Analysis) technique using the Zeiss Airyscan detector. This detector consists of 32 GaAsP PMT arranged in a hexagonal pattern, featuring a fast temporal sampling (down to 1.28 μs per frame). It can also be used in a super-resolution configuration as per the ISM [1] principle, while the arrangement of the detectors allows for the implementation of advanced state-of-the-art spatiotemporal correlation techniques. We will present a novel application of the spot-variation FCS technique [2] in super-resolution, as well as the implementation of the 2D-pCF [3], iMSD [4] and number and brightness [5] analysis using this fast detector array. This simultaneous analysis of the same dataset provides biophysical information regarding the fluorescent probe and the surrounding environment, such as diffusion coefficient, concentration, diffusion modality, environment organization, direction and anisotropy of molecular flows, diffusion connectivity and oligomerization state in a single analysis in a few seconds. Using CCA we obtained structural and dynamic fingerprints of several subcellular compartments of living NIH-3T3 cells, namely structured and unstructured cytoplasm, the nucleoli and heterochromatin- and euchromatin-rich regions in the nucleus. CCA provides a new methodology for the investigation of sub-resolution organelle structure and dynamics as well as insights on chromatin compaction states in living cells. The parallel acquisition of numerous biophysical parameters opens the doors to new multidimensional characterization and classification methods that can be applied to virtually any fluorescent or fluorescently-labeled probe diffusing in complex environments. [Work supported by NIH P41 GM103540 grant] [1] Sheppard et al Opt.Lett. (2013) [2] Wawrezinieck et al Biophys.J. (2005) [3] Hinde et al PNAS. (2010) [4] Di Rienzo et al Nat.Comm. (2014) [5] Digman et al Biophys.J. (2008)

Hyperspectral Imaging in Scattering Media using Two Filters

Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle and sample area. We have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes using epi detection, which in turn significantly increases imaging depth. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that two filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than displaying the entire spectrum. Using the properties of the spectral phasors we can resolve spectral components and perform the type of data analyses that are usually performed in hyperspectral image analysis. (Supported in part by the NIH grant NIH P41-GM103540).

Spectral Resolution in Fluorescence Microscopy in Strongly Scattering Media

Spectral phasors were introduced as alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation in membranes. The basic concept in the spectral phasor approach is that the entire spectrum is not needed for some analysis, but only some of the elements of the spectral distribution are sufficient. In the case in which the spectrum is dominated by few spectral bands, this approach is very efficient and provides fast and valid alternative to the analysis of the full spectrum. One example of a system that could benefit from the phasor analysis is in tissue spectroscopy in which the light to be collected has undergone multiple scattering so that it cannot be easily focused on the entrance slit of a dispersive element or transformed in a parallel beam for use with hyperspectral cameras. In a typical application of thick tissue spectroscopy, the light, after being transmitted through the tissue, is emitted over a very large cone angle. All the schemes designed to capture this transmitted (or fluorescence) light use a relatively narrow collection angle with severe reduction of the signal. In the microscopy field, we introduced in 2011 a microscope design (DIVER) that can capture a very large solid emission angle (close to 2pi) and we have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes using epi detection. A limitation of the DIVER design is that interference filters cannot be used in the emission path since the light is impinging on the surface of the filter in front of the detector with a very large angular distribution. Here we propose a simple device to overcome this problem (Supported by NIH P41-GM103540 and NIH P50-GM076516).

Water Activity Inside the Nucleus: Some Clues using ACDAN Fluorescence and its Implications in the Chromatin Supramolecular Organization

Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of ACDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used a heterochromatin marker based on the H2B-dsRed fluorescent protein. The nucleus at interphase was the most polar structure of the cell as reported by ACDAN. In the nucleus, we were able to discriminate at least two populations based on dipolar relaxation rates. Using the correlation between the ACDAN and H2B-dsRed, we associated the heterochromatin phase with regions of restricted ACDAN dipolar relaxations. The connectivity map shows that ACDAN inside the nucleus has areas with preferential/restricted diffusion. At cell division, there is a dramatic change in the dipolar relaxation profile. Changes in the dipolar relaxation can be explained as physical changes in the nucleus (phase transitions/molecular crowding) driven by the water activity. (Supported by NIH P41-GM103540 and NIH P50-GM076516).

Of Absolute Concentrations of NADH in Cells using the Phasor Flim Method

One of the properties of fluorescence lifetime is its independence on concentration. While this is considered to be an advantage, when analyzing images of cell and tissues we often need to also measure the absolute concentration of a fluorophore. The concentration value is commonly obtained by comparison of the intensity of the fluorophore in the tissue and in a solution of known concentration. The underlying hypothesis is that the quantum yield of the fluorophore is the same in the sample and in the solution used for calibration. In cells and tissue this assumption is not always valid especially for a fluorophore like NADH that changes the quantum yield by a large factor when free in solution with respect to when bound to enzymes. 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. Work supported in part by NIH grants NIH P41-GM103540 and NIH P50-GM076516.

Connectivity Map of the Cell Interior

We are developing an imaging platform that has the appropriate spatial and time resolution to investigate fundamental biological process in regard to signaling events in cells. The technology is based on fluctuation spectroscopy and the concept of spatial cross-correlation of fluctuations at different locations (the pair correlation function approach) originally developed in our lab. This technology is a radical departure from conventional fluctuation spectroscopy methods commonly done at single points in a cell or in small regions, yet providing the same information about diffusion and binding but with additional crucial advantages. The main advantage of the pair-cross-correlation approach is that it provides the full diffusion tensor at every point in the cell, which is crucial for the determination of the flow of molecules in the cell interior. The pair-cross-correlation function is zero if the two points are not in molecular communication while is different from zero if a molecule can go from one point to the other. We show how to derive a connectivity map which visualizes which compartments in a cell are in communication and how long a molecule takes to go from one point to the other. Supported by NIH P41-GM103540 and NIH P50-GM076516 grants.

Self-Assisted Optothermal Trapping of Gold Nanorods Under Two-Photon Excitation

We report self-assisted optothermal trapping and patterning of gold nanorods (GNRs) on glass surfaces with a femtosecond laser. We show that GNRs are not only the trapping targets, but also can induce convective flows that drive more particles toward the trap. The trapping phenomenon is the net result of thermophoresis and convective flow caused by localized heating, which is due to the conversion of absorbed photons into heat at GNR's longitudinal surface plasmon resonance wavelength. We investigated the optothermal trapping of GNRs at the glass surface which can be obtained with laser power as low as 0.5 mW at 840 nm. The attraction of particles toward the central hot spot can be larger than the typical field of view, e.g. attraction toward the trap was observed from a range of 210 µm × 210 µm. By moving the laser focus away from the glass surface, at certain distances, ring patterns of GNRs on the glass surface can be obtained visualizing the regions of flow. These patterns could be controlled by the laser power and the numerical aperture of the microscope objective. Moreover, we examined the spectral emission of GNRs under different trapping conditions using the spectral phasor approach to reveal the temperature and association status of GNRs. Our study will help understanding manipulation of flows in solution and in biological systems which could arise in future applications of GNRs induced heating and flows. Work done in part with funds from NIH P41-GM103540 and NIH P50-GM076516.

Real-Time Analysis of Endogenous Nuclear NADH in Differentiating Cells using the Spectral Phasor Approach

369-Pos Board B149 Real-Time Analysis of Endogenous Nuclear NADH in Differentiating Cells using the Spectral Phasor Approach Belinda K. Wright 1,2 , Mark R. Jones 1 , Michelle A. Digman 2 , Enrico Gratton 2 . School of Science and Health, University of Western Sydney, Richmond, Australia, 2 Biomedical Engineering, Laboratory of Fluorescence Dynamics (LFD), University of California, Irvine, CA, USA. NADH is an endogenous autofluorescent regulatory metabolite detected in the nuclear regions of live cells which when analysed for the bound and free form can aid in determining a cell’s metabolic status and molecular activity. Detect- ing the spectral differences of free and bound NADH in live cells is currently limited due to the very small differences in emission. The Spectral Phasor tech- nique enables not only examination of small shifts in spectral emissions but also provides the spatial location of spectrally different components in live cells without any prior knowledge of the species. The phasor representation enables direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells for global analysis. Here we describe the use of Spectral Phasors to spatially map NADH’s spectral emission in the nucleus of live cells under normal culture conditions and those stimulated into the early stages of differentiation. A comparison of undifferen- tiated cells and those stimulated to differentiate demonstrate differing spatial distributions of emission spectra associated with NADH. Undifferentiated cells displayed shorter emissions cantered in the nucleus, while longer wavelengths were localised around the perinuclear boarder. Cells stimulated into the early stages of differentiation displayed a redirection of the shorter emissions to regional clustering predominately in one area close to the nuclear/cytoplasmic boarder, while the longer wavelengths were localised throughout the remainder of the nucleus. Here we show the application of the Spectral Phasor technique to identify discrete wavelength shifts associated with endogenous NADH auto- fluorescence in the nucleus, and observed changes in their spatial distribution in live cells during the early stages of differentiation. Work in part supported by NIH P41-GM103540 and NIH P50-GM076516.

The Lifetime NAD(P)H Fingerprint of Salmonella Infection

The Lifetime NAD(P)H Fingerprint of Salmonella Infection

Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells

The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 microsecond, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intracellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluorescent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB-RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).

Fluorescence Fluctuation Microscopy Techniques to Study mRNA Synthesis and Dynamics

1627-Pos Board B578 Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells Carmine Di Rienzo 1,2 , Enrico Gratton 3 , Fabio Beltram 1,2 , Francesco Cardarelli 2 . NEST, Scuola Normale Superiore, Pisa, Italy, 2 Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering., University of California, Irvine, CA, USA. The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 micro- second, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intra- cellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluores- cent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB- RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).