Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Quantitation of the physicochemical properties of myelin using Nile Red fluorescence spectroscopy.

Myelin is a vital structure that is key to rapid saltatory conduction in the central and peripheral nervous systems. Much work has been done over the decades examining the biochemical composition and morphology of myelin at the light and electron microscopic levels. Here we report a method to study myelin based on the fluorescent probe Nile Red. This lipophilic dye readily partitions into live and chemicallyfixed myelin producing bright, well-resolved images of the sheath. Using spectral confocal microscopy, a complete emission spectrum of Nile Red fluorescence can be acquired for each pixel in an image. The solvatochromic properties of Nile Red cause its emission spectrum to change depending on the polarity of its local environment. Therefore, measuring spectral shifts can report subtle changes in the physicochemical properties of myelin. We show differences in myelin polarity in central versus peripheral nervous system and in different regions of central nervous system white matter of the mouse brain, together with developmental and sex variations. This technique is also well suited for measuring subtle changes in myelin properties in live ex vivo white matter specimens. We also demonstrate how light deprivation induces a myelin polarity change in adult mouse optic nerve underscoring a continuing myelin plasticity in response to axonal activity well into adulthood. The Nile Red spectroscopic method allows measurement of subtle physicochemical changes in myelin that can importantly influence its electrical properties and by extension, conduction velocities in axons.

Ageing and Polypharmacy in Mesenchymal Stromal Cells: Metabolic Impact Assessed by Hyperspectral Imaging of Autofluorescence.

The impact of age on mesenchymal stromal cell (MSC) characteristics has been well researched. However, increased age is concomitant with increased prevalence of polypharmacy. This adjustable factor may have further implications for the functionality of MSCs and the effectiveness of autologous MSC procedures. We applied hyperspectral microscopy of cell autofluorescence-a non-invasive imaging technique used to characterise cytometabolic heterogeneity-to identify changes in the autofluorescence signals of MSCs from (1) young mice, (2) old mice, (3) young mice randomised to receive polypharmacy (9-10 weeks of oral therapeutic doses of simvastatin, metoprolol, oxycodone, oxybutynin and citalopram), and (4) old mice randomised to receive polypharmacy. Principal Component Analysis and Logistic Regression Analysis were used to assess alterations in spectral and associated metabolic characteristics. Modelling demonstrated that cells from young mice receiving polypharmacy had less NAD(P)H and increased porphyrin relative to cells from old control mice, allowing for effective separation of the two groups (AUC of ROC curve > 0.94). Similarly, cells from old polypharmacy mice were accurately separated from those from young controls due to lower levels of NAD(P)H (p < 0.001) and higher porphyrin (p < 0.001), allowing for an extremely accurate logistic regression (AUC of ROC curve = 0.99). This polypharmacy regimen may have a more profound impact on MSCs than ageing, and can simultaneously reduce optical redox ratio (ORR) and increase porphyrin levels. This has implications for the use of autologous MSCs for older patients with chronic disease.

Early detection of prion protein aggregation with a fluorescent pentameric oligothiophene probe using spectral confocal microscopy.

Misfolding of the prion protein (PrP) and templating of its pathological conformation onto cognate proteins causes a number of lethal disorders of central nervous system in humans and animals, such as Creutzfeldt-Jacob disease, chronic wasting disease and bovine spongiform encephalopathy. Structural rearrangement of PrPC into PrPSc promotes aggregation of misfolded proteins into ß-sheet-rich fibrils, which can be visualized by conformationally sensitive fluorescent probes. Early detection of prion misfolding and deposition might provide useful insights into its pathophysiology. Pentameric formyl thiophene acetic acid (pFTAA) is a novel amyloid probe that was shown to sensitively detect various misfolded proteins, including PrP. Here, we compared sensitivity of pFTAA staining and spectral microscopy with conventional methods of prion detection in mouse brains infected with mouse-adapted 22L prions. pFTAA bound to prion deposits in mouse brain sections exhibited a red-shifted fluorescence emission spectrum, which quantitatively increased with disease progression. Small prion deposits were detected as early as 50 days post-inoculation, well before appearance of clinical signs. Moreover, we detected significant spectral shifts in the greater brain parenchyma as early as 25 days post-inoculation, rivaling the most sensitive conventional method (real-time quaking-induced conversion). These results showcase the potential of pFTAA staining combined with spectral imaging for screening of prion-infected tissue. Not only does this method have comparable sensitivity to established techniques, it is faster and technically simpler. Finally, this readout provides valuable information about the spatial distribution of prion aggregates across tissue in the earliest stages of infection, potentially providing valuable pathophysiological insight into prion transmission.

Quantitative detection of grey and white matter amyloid pathology using a combination of K114 and CRANAD-3 fluorescence.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that exacts a huge toll on the patient, the healthcare system and society in general. Abundance and morphology of protein aggregates such as amyloid ß plaques and tau tangles, along with cortical atrophy and gliosis are used as measures to assess the changes in the brain induced by the disease. Not all of these parameters have a direct correlation with cognitive decline. Studies have shown that only particular protein conformers can be the main drivers of disease progression, and conventional approaches are unable to distinguish different conformations of disease-relevant proteins. METHODS AND RESULTS: Using the fluorescent amyloid probes K114 and CRANAD-3 and spectral confocal microscopy, we examined formalin-fixed paraffin-embedded brain samples from different control and AD cases. Based on the emission spectra of the probes used in this study, we found that certain spectral signatures can be correlated with different aggregates formed by different proteins. The combination of spectral imaging and advanced image analysis tools allowed us to detect variability of protein deposits across the samples. CONCLUSION: Our proposed method offers a quicker and easier neuropathological assessment of tissue samples, as well as introducing an additional parameter by which protein aggregates can be discriminated.

A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

Amyloid dye pairs as spectral sensors for enhanced detection and differentiation of misfolded proteins.

Protein misfolding with subsequent formation of cross-ß-sheet-rich fibrils is a well-known pathological hallmark of various neurodegenerative conditions, including Alzheimer's disease (AD). Recent evidence suggests that specific protein conformations may be the primary drivers of disease progression, differentiation of which remains a challenge with conventional methods. We have previously described a unique phenomenon of light-induced fluorescence enhancement and spectral changes of the amyloid dyes K114 and BSB, and demonstrated its utility in characterizing different amyloid fibrils. In this study, we further characterize and explore the potential of photoconversion, coupled with dual-probe staining, for improved detection of heterogeneity of amyloids using silk fibers and 5xFAD mouse brain sections. BSB and K114 were paired with either Nile Red or MCAAD-3, aiming to increase the sensitivity and specificity of staining and misfolded protein detection via complementary binding and FRET. Principal component analysis of spectral data revealed significant differences between various amyloids, and was able to detect subtle amyloid pathology in the 5xFAD mouse background brain parenchyma.

Scytonemin redox status in a filamentous cyanobacterium visualized by an excitation-laser-line-scanning spontaneous Raman scattering spectral microscope.

Some cyanobacteria produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although scytonemin-containing dark sheaths are recognizable through optical microscopes and its redox changes have been known for decades, there has been no report to obtain images of both oxidized and reduced scytonemins at subcellular resolution. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of both the oxidized and reduced scytonemins in a filamentous cyanobacterium. The redox changes of scytonemin were supported by comparison in the Raman spectra between the cyanobacterial cells, solid-state scytonemin and quantum chemical normal mode analysis. Distributions of carotenoids, phycobilins, and the two redox forms of scytonemin were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The redox differentiation of scytonemin will advance our understanding of the redox homeostasis and secretion mechanisms of individual cyanobacteria as well as microscopic chemical environments in various microbial communities. The line-scanning Raman microscopy based on the 1064 nm excitation is thus a promising tool for exploring hitherto unreported Raman spectral features and distribution of nonfluorescent molecules embedded below nontransparent layers for visible light, while avoiding interference by autofluorescence.

Dual-probe fluorescence spectroscopy for sensitive quantitation of Alzheimer's amyloid pathology.

Protein misfolding is a prominent pathological hallmark of neurodegenerative disorders, including Alzheimer's disease (AD). Studies have shown that the diversity of ß sheet-rich protein deposits (such as amyloid ß plaques and neurofibrillary tangles), present across different brain regions, might underlie different disease phenotypes and only certain types of aggregates might be associated with cognitive decline. Conformationally sensitive fluorescent amyloid probes have the ability to report different structures of protein aggregates by virtue of their shifting emission spectra. Here we defined the binding affinity of the fluorescent amyloid probes BSB and MCAAD to disease-relevant protein aggregates, and combined the two probes to examine formalin-fixed paraffin-embedded mouse and human brain samples. Coupled with quantitative spectral phasor analysis, the dual-probe staining approach revealed remarkable heterogeneity of protein aggregates across the samples. Distinct emission spectra were consistent with certain types of deposits present in the mouse and human brain sections. The sensitivity of this staining, imaging and analysis approach outperformed conventional immunohistochemistry with the detected spectral differences between the greater parenchyma of cognitively normal and AD cases indicating a subtle yet widespread proteopathy associated with disease. Our method offers more sensitive, objective, and quantitative examination of protein misfolding pathology using conventional tissue sections.

A Spectral Principal Component Analysis-Based Framework for Composite Hard/Soft Tissue Fluorescence Image Investigation.

Traditional thin sectioning microscopy of large bone and dental tissue samples using demineralization may disrupt structure morphologies and even damage soft tissues, thus compromising the histopathological investigation. Here, we developed a synergistic and original framework on thick sections based on wide-field multi-fluorescence imaging and spectral Principal Component Analysis (sPCA) as an alternative, fast, versatile, and reliable solution, suitable for highly mineralized tissue structure sustain and visualization. Periodontal 2-mm thick sections were stained with a solution containing five fluorescent dyes chosen for their ability to discriminate close tissues, and acquisitions were performed with a multi-zoom macroscope for blue, green, red, and NIR (near-infrared) emissions. Eigen-images derived from both standard scaler (Std) and Contrast Limited Adaptive Histogram Equalization (Clahe) pre-preprocessing significantly enhanced tissue contrasts, highly suitable for histopathological investigation with an in-depth detail for sub-tissue structure discrimination. Using this method, it is possible to preserve and delineate accurately the different anatomical/morphological features of the periodontium, a complex tooth-supporting multi-tissue. Indeed, we achieve characterization of gingiva, alveolar bone, cementum, and periodontal ligament tissues. The ease and adaptability of this approach make it an effective method for providing high-contrast features that are not usually available in standard staining histology. Beyond periodontal investigations, this first proof of concept of an sPCA solution for optical microscopy of complex structures, especially including mineralized tissues opens new perspectives to deal with other chronic diseases involving complex tissue and organ defects. Overall, such an imaging framework appears to be a novel and convenient strategy for optical microscopy investigation.

What can biophotonics tell us about the 3D microstructure of articular cartilage?

Connective tissues such as articular cartilage have been the subject of study using novel optical techniques almost since the invention of polarized light microscopy (PLM). Early studies of polarized light micrographs were the main evidential basis for the establishment of quantitative models of articular cartilage collagen structure by Benninghoff and others. Even now, state of the art optical techniques including quantitative polarized light microscopy (qPLM), optical coherence tomography (OCT), polarization-sensitive optical coherence tomography (PS-OCT), second harmonic generation (SHG) microscopy, Fourier-transform infrared (FTIR) microscopy, Raman and optical hyperspectral reflectance and fluorescence imaging are providing new insights into articular cartilage structure from the nanoscale through to the mesoscale. New insights are promised by emerging modalities such as optical elastography. This short review highlights some key recent results from modern optical techniques.