Fibrosis quantification using multiphoton excitation imaging of picrosirius red stained cardiac tissue.

Traditional methodologies for fibrosis quantification involve histological measurements, staining with Masson's trichrome and picrosirius red (PSR), and label-free imaging using second harmonic generation (SHG). The difficulty of label-free cardiac SHG imaging is that both collagen (i.e., collagen 1 fibrils) and myosin are harmonophores that generate SHG signals, and specific identification of either collagen or myosin is difficult to achieve. Here we present an alternate method of quantifying cardiac fibrosis by using PSR staining followed by multiphoton excitation fluorescence imaging. Our data from the deoxycorticosterone model of cardiac fibrosis shows that this imaging method and downstream analyses, including background correction, are robust and easy to perform. These advantages are due to the high signal-to-noise ratio provided by PSR in areas of collagen fibers. Furthermore, the hyperspectral and fluorescence lifetime information of PSR-stained area of fibrosis shows better quantification can eventually be obtained using more complex instrumentation.

Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases.

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Elucidating the Oligomerization and Cellular Interactions of a Trimer Derived from Aß through Fluorescence and Mass Spectrometric Studies.

Aß oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17-36 of Aß as model systems for studying Aß oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, indicating the presence of oligomers within cells. Collectively, these multiple complementary techniques help better understand the complex behavior of the 2AT-L model system.

In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging.

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.

Linear Combination Properties of the Phasor Space in Fluorescence Imaging.

The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling and storage. The reciprocity principle between the real and imaginary space-where the phasor and the pixel that the phasor originated from are linked and can be converted from one another-has helped the expansion of this method. The phasor coordinates calculated from a pixel, where multiple fluorescent species are present, depends on the phasor positions of those components. The relative positions are governed by the linear combination properties of the phasor space. According to this principle, the phasor position of a pixel with multiple components lies inside the polygon whose vertices are occupied by the phasor positions of these individual components and the distance between the image phasor to any of the vertices is inversely proportional to the fractional intensity contribution of that component to the total fluorescence from that image pixel. The higher the fractional intensity contribution of a vertex, the closer is the resultant phasor. The linear additivity in the phasor space can be exploited to obtain the fractional intensity contribution from multiple species and quantify their contribution. This review details the various mathematical models that can be used to obtain two/three/four components from phasor space with known phasor signatures and then how to obtain both the fractional intensities and phasor positions without any prior knowledge of either, assuming they are mono-exponential in nature. We note that other than for blind components, there are no restrictions on the type of the decay or their phasor positions for linear combinations to be valid-and they are applicable to complicated fluorescence lifetime decays from components with intensity decays described by multi-exponentials.

Method of transmission filters to measure emission spectra in strongly scattering media.

We describe a method based on a pair of transmission filters placed in the emission path of a microscope to resolve the emission wavelength of every point in an image. The method can be applied to any type of imaging device that provides the light in the wavelength transmission range of the filters. Unique characteristics of the filter approach are that the light does not need to be collimated and the wavelength response does not depend on the scattering of the sample or tissue. The pair of filters are used to produce the spectral phasor of the transmitted light, which is sufficient to perform spectral deconvolution over a broad wavelength range. The method is sensitive enough to distinguish free and protein-bound NADH and can be used in metabolic studies.

Phasor-based image segmentation: machine learning clustering techniques.

The phasor approach is a well-established method for data visualization and image analysis in spectral and lifetime fluorescence microscopy. Nevertheless, it is typically applied in a user-dependent manner by manually selecting regions of interest on the phasor space to find distinct regions in the fluorescence images. In this paper we present our work on using machine learning clustering techniques to establish an unsupervised and automatic method that can be used for identifying populations of fluorescent species in spectral and lifetime imaging. We demonstrate our method using both synthetic data, created by sampling photon arrival times and plotting the distributions on the phasor plot, and real live cells samples, by staining cellular organelles with a selection of commercial probes.

Blind Resolution of Lifetime Components in Individual Pixels of Fluorescence Lifetime Images Using the Phasor Approach.

The phasor approach is used in fluorescence lifetime imaging microscopy for several purposes, notably to calculate the metabolic index of single cells and tissues. An important feature of the phasor approach is that it is a fit-free method allowing immediate and easy to interpret analysis of images. In a recent paper, we showed that three or four intensity fractions of exponential components can be resolved in each pixel of an image by the phasor approach using simple algebra, provided the component phasors are known. This method only makes use of the rule of linear combination of phasors rather than fits. Without prior knowledge of the components and their single exponential decay times, resolution of components and fractions is much more challenging. Blind decomposition has been carried out only for cuvette experiments wherein the statistics in terms of the number of photons collected is very good. In this paper, we show that using the phasor approach and measurements of the decay at phasor harmonics 2 and 3, available using modern electronics, we could resolve the decay in each pixel of an image in live cells or mice liver tissues with two or more exponential components without prior knowledge of the values of the components. In this paper, blind decomposition is achieved using a graphical method for two components and a minimization method for three components. This specific use of the phasor approach to resolve multicomponents in a pixel enables applications where multiplexing species with different lifetimes and potentially different spectra can provide a different type of super-resolved image content.

Resolution of 4 components in the same pixel in FLIM images using the phasor approach.

In several cellular systems, the phasor FLIM approach has shown the existence of more than 2 components in the same pixel, a typical example being free and bound NADH. In order to properly quantify the concentrations and the spatial distributions of fluorescence components associated with different molecular species we developed a general method to resolve 3 and 4 components in the same pixel using the phasor approach. The method is based on the law of linear combination of components valid after transformation of the decay curves to phasors for each pixel in the image. In principle, the linear combination rule is valid for an arbitrary number of components. For 3 components we use only the phasor position for the first harmonic, which has a small error, while for 4 components we need the phasor location at higher harmonics that have intrinsically more noise. As a result of the noise in the higher harmonics, caused by limited photon statistics, we are able to use linear algebra to resolve 4 components given the position of the phasors of 4 independent components in mixtures of dyes and 3 components for dyes in cellular systems.

Barriers to Diffusion in Cells: Visualization of Membraneless Particles in the Nucleus.

Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is difficult to demonstrate experimentally. We describe an approach based on the 2-dimensional pair correlation function (2D-pCF) analysis of the spatial connectivity in a cell. The educational aim of the article is to present both a model suitable for explaining diffusion barrier measurements to a broad range of courses and examples of biological situations. If there are no barriers to diffusion, particles could diffuse equally in all directions. In this situation the pair correlation function introduced in this article is independent of the direction and is uniform in all directions. However, in the presence of obstacles, the shape of the 2D-pCF is distorted to reflect how the obstacle position and orientation change the flow of molecules. In the example shown in this article, measurements of diffusion of enhanced green fluorescent protein moving in live cells show the lack of connectivity at the nucleolus surface for shorter distances. We also observe a gradual increase in the connectivity for longer distances or times, presumably because of molecular trajectories around the nucleolus.

Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/ß1-Independent.

Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/ß1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-ß2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-ß2 might also collaborate in Gli2 nuclear entry. How does Imp-ß2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation.