Fluorescence lifetime imaging for studying DNA compaction and gene activities.

Optical imaging is a most useful and widespread technique for the investigation of the structure and function of the cellular genomes. However, an analysis of immensely convoluted and irregularly compacted DNA polymer is highly challenging even by modern super-resolution microscopy approaches. Here we propose fluorescence lifetime imaging (FLIM) for the advancement of studies of genomic structure including DNA compaction, replication as well as monitoring of gene expression. The proposed FLIM assay employs two independent mechanisms for DNA compaction sensing. One mechanism relies on the inverse quadratic relation between the fluorescence lifetimes of fluorescence probes incorporated into DNA and their local refractive index, variable due to DNA compaction density. Another mechanism is based on the Förster resonance energy transfer (FRET) process between the donor and the acceptor fluorophores, both incorporated into DNA. Both these proposed mechanisms were validated in cultured cells. The obtained data unravel a significant difference in compaction of the gene-rich and gene-poor pools of genomic DNA. We show that the gene-rich DNA is loosely compacted compared to the dense DNA domains devoid of active genes.

Cellular transformations in near-infrared light-induced apoptosis in cancer cells revealed by label-free CARS imaging.

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti-Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro-apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm2 (~58%) and 3 J/cm2 (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis-associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm2 . Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm2 ) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non-irradiated and irradiated with 10 J/cm2 . Furthermore, irradiation of the cells with the 0.3 J/cm2 dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.

Cycles of protein condensation and discharge in nuclear organelles studied by fluorescence lifetime imaging.

Nuclear organelles are viscous droplets, created by concentration-dependent condensation and liquid-liquid phase separation of soluble proteins. Nuclear organelles have been actively investigated for their role in cellular regulation and disease. However, these studies are highly challenging to perform in live cells, and therefore, their physico-chemical properties are still poorly understood. In this study, we describe a fluorescence lifetime imaging approach for real-time monitoring of protein condensation in nuclear organelles of live cultured cells. This approach unravels surprisingly large cyclic changes in concentration of proteins in major nuclear organelles including nucleoli, nuclear speckles, Cajal bodies, as well as in the clusters of heterochromatin. Remarkably, protein concentration changes are synchronous for different organelles of the same cells. We propose a molecular mechanism responsible for synchronous accumulations of proteins in the nuclear organelles. This mechanism can serve for general regulation of cellular metabolism and contribute to coordination of gene expression.

The impact of cell fixation on coherent anti-stokes Raman scattering signal intensity in neuronal and glial cell lines.

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid-related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti-stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.

Near-Infrared Irradiation Affects Lipid Metabolism in Neuronal Cells, Inducing Lipid Droplets Formation.

It is known that lipids play an outstanding role in cellular regulation, and their dysfunction has been linked to many diseases. Thus, modulation of lipid metabolism may provide new pathways for disease treatment or prevention. In this work, near-infrared (NIR) light was applied to modulate lipid metabolism and increase intracellular lipid content in rat cortical neurons (RCN). Using label-free CARS microscopy, we have monitored the intracellular lipid content in RCN at a single-cell level. A major increase in average level of lipid per cell after treatment with laser diode at 808 nm was found, nonlinearly dependent on the irradiation dose. Moreover, a striking formation of lipid droplets (LDs) in the irradiated RCN was discovered. Further experiments and analysis reveal a strong correlation between NIR light induced generation of reactive oxygen species (ROS), lipids level, and LDs formation in RCN. Our findings can contribute to a development of therapeutic approaches for neurological disorders via NIR light control of lipid metabolism in neuronal cells.

Biomolecular Component Analysis of Phospholipids Composition in Live HeLa Cells.

The alteration of the phospholipid composition within the cell, in particular the ratio between saturated and unsaturated fatty acids, can serve as an important biomarker to prognosis of the disease progression (e.g., fatty-liver disease, prostate cancer, or neurodegenerative disorders). Major techniques for lipid analysis in biological samples require a lipid extraction procedure that is not compatible with live cell studies. To address this challenge, we apply microRaman-Biomolecular Component Analysis (BCA) for comparative analysis of phospholipid composition and sensing the saturation degree of fatty acid lipid chain in live HeLa cells and lipids extracted from HeLa cells. After processing raw Raman data, acquired in lipid droplets (LDs) free cytoplasmic area, LDs and extracted lipids with BCA, the lipid component was isolated. Despite the similarity in general profiles of processed Raman spectra acquired in live cells and extracted lipids, some clear differences that reflect diversity in their phospholipids composition were revealed. Furthermore, using the direct relation between the number of double bonds in the fatty acid chain and the intensity ratio of the corresponding Raman bands, the saturation degree of fatty acids was estimated.

Macromolecular Profiling of Organelles in Normal Diploid and Cancer Cells.

To advance an understanding of cellular regulation and function it is crucial to identify molecular contents in cellular organelles, which accommodate specific biochemical processes. Toward achievement of this goal, we applied micro-Raman-Biomolecular Component Analysis assay for molecular profiling of major organelles in live cells. We used this assay for comparative analysis of proteins 3D conformation and quantification of proteins, RNA, and lipids concentrations in nucleoli, endoplasmic reticulum, and mitochondria of WI 38 diploid lung fibroblasts and HeLa cancer cells. Obtained data show substantial differences in the concentrations and conformations of proteins in the studied organelles. Moreover, differences in the intraorganellar concentrations of RNA and lipids between these cell lines were found. We report the biological significance of obtained macromolecular profiles and advocate for micro-Raman BCA assay as a valuable proteomics tool.

Molecular profiling of single organelles for quantitative analysis of cellular heterogeneity.

Recent developments in Raman spectroscopy instrumentation and data processing algorithms have led to the emergence of Ramanomics - an independent discipline with unprecedented capabilities to map the distribution of distinct molecular groups in live cells. Here, we introduce a method for probing the absolute concentrations of proteins, RNA and lipids in single organelles of live cultured cells by biomolecular component analysis using microRaman data. We found significant cell-to-cell variations in the molecular profiles of organelles, thus providing a physiologically relevant set of markers of cellular heterogeneity. At the same cell the molecular profiles of different organelles can strongly correlate, reflecting tight coordination of their functions. This correlation was significant in WI-38 diploid fibroblasts and weak in HeLa cells, indicating profound differences in the regulation of biochemical processes in these cell lines.