Capturing cell morphology dynamics with high temporal resolution using single-shot quantitative phase gradient imaging.

Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75 frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining. Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images. Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability. Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3 lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( ∼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55 µ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings. Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.

Osteopontin stabilization and collagen containment slows amorphous calcium phosphate transformation during human aortic valve leaflet calcification.

Calcification of aortic valve leaflets is a growing mortality threat for the 18 million human lives claimed globally each year by heart disease. Extensive research has focused on the cellular and molecular pathophysiology associated with calcification, yet the detailed composition, structure, distribution and etiological history of mineral deposition remains unknown. Here transdisciplinary geology, biology and medicine (GeoBioMed) approaches prove that leaflet calcification is driven by amorphous calcium phosphate (ACP), ACP at the threshold of transformation toward hydroxyapatite (HAP) and cholesterol biomineralization. A paragenetic sequence of events is observed that includes: (1) original formation of unaltered leaflet tissues: (2) individual and coalescing 100's nm- to 1 µm-scale ACP spherules and cholesterol crystals biomineralizing collagen fibers and smooth muscle cell myofilaments; (3) osteopontin coatings that stabilize ACP and collagen containment of nodules preventing exposure to the solution chemistry and water content of pumping blood, which combine to slow transformation to HAP; (4) mm-scale nodule growth via ACP spherule coalescence, diagenetic incorporation of altered collagen and aggregation with other ACP nodules; and (5) leaflet diastole and systole flexure causing nodules to twist, fold their encasing collagen fibers and increase stiffness. These in vivo mechanisms combine to slow leaflet calcification and establish previously unexplored hypotheses for testing novel drug therapies and clinical interventions as viable alternatives to current reliance on surgical/percutaneous valve implants.

Direct intracellular detection of biomolecule specific bound-water with Raman spectroscopy.

Lipids, proteins, and nucleic acids have closely associated water molecules (Bound water), which exhibit considerably different physical properties compared to bulk water. Here we investigate the possibility of resolving Raman spectra of the specific hydration shell of these biomolecules in intracellular regions using Raman imaging. Lipids and proteins + nucleic acids Raman spectral components resolved in the analysis showed associated water spectral features, which are uniquely different from that of bulk water. These spectral profiles agree with water spectral profile observed in the case of corresponding hydrated pure biomolecules. The results show the prospects of Raman imaging in examining intracellular hydration in biomolecules and its functional relation.

Mycelial differentiation linked avermectin production in Streptomyces avermitilis studied with Raman imaging.

Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolite production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of secondary metabolites at various stages of morphological differentiation. Under solid culture, avermectin was detected in the mycelia formed at the later stages of morphological differentiation (e.g., spore-bearing mycelium and spiral spore chains), but not in the early-stage substrate mycelium. On the contrary, under liquid culture condition, avermectin was found concentrated in the mycelial pellet formed at the early MII stage of differentiation. Furthermore, the chemical profiles of the mycelia were substantially different depending on the culture condition. Raman spectra corresponding to proteins, lipids, and cytochrome were observed in the mycelia irrespective of the stage of morphological differentiation, however, carotenoid was observed under solid culture condition particularly in spore-bearing mycelium and spiral spore chains. KEY POINTS: • Avermectin production is regulated during mycelial differentiation • Liquid and solid culture conditions affects mycelial differentiation • Raman microspectroscopic analysis reveals localization profiles of avermectin.

Direct imaging of intracellular RNA, DNA, and liquid-liquid phase separated membraneless organelles with Raman microspectroscopy.

Methodologies for direct intracellular imaging of RNA and DNA are necessary for the advancement of bioimaging. Here we show direct label-free imaging of RNA and DNA in single cells by isolating their accurate Raman spectra. Raman images of DNA from interphase cells show intact nucleus, while those from mitotic cells reveal condensed chromosome. The condensed chromosome images are accurate enough to assign the stage of mitotic cell division (e.g., metaphase). Raman spectral features indicate B-DNA double helical conformational form in all the cell lines investigated here. The Raman images of RNAs, on the other hand, reveal liquid-liquid phase separated (LLPS) membraneless organelles in interphase cells, which disappears during mitosis. Further, the Raman spectrum of proteins from the intracellular LLPS organelles indicates slight enrichment of amyloid-like secondary structural features. Vibrational imaging of intracellular DNA and RNA simultaneously would open myriad of opportunities for examining functional biochemical aspects of cells and organelles.

Reproducible and sensitive micro-tissue RNA sequencing from formalin-fixed paraffin-embedded tissues for spatial gene expression analysis.

Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 µm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at two years after fixation and embedding and detected approximately 7000 genes in micro-punched tissue-spots (thickness: 10 µm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

Raman Microspectroscopy Imaging Analysis of Extracellular Vesicles Biogenesis by Filamentous Fungus Penicilium chrysogenum.

The mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest due to their diverse roles in biological systems and are far from being completely understood. Even though cellular cargo releases mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study, this aspect is investigated in detail using Raman microspectroscopic imaging. Considerable evidence is provided to suggest that the release of antibiotic penicillin by the filamentous fungus Penicillium chrysogenum involves EVs. Further, the study also reveals morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs. The results suggest a possible general role of EVs in the release of antibiotics from the producing organisms.

Stability and utility of flow cytometric platelet activation tests: A modality to bridge the gap between diagnostic demand and supply.

Light transmission aggregometry (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs). The requirement of customized aggregometer, large blood volume, normal platelet count and processing within 4 hours of venipuncture for LTA makes platelet function testing inaccessible to wider population. Flow cytometric platelet activation test (PACT) may overcome these limitations. This study compares the performance of PACT with LTA, characterizes diagnostic patterns of PFDs on PACT and assesses the stability of PACT beyond 4 hours of venipuncture in controls (n = 5) at different temperature conditions. LTA and PACT were performed in 121 healthy controls and 66 patients with suspected PFD. PACT had excellent agreement (kappa = 0.93) with LTA and 94.1% sensitivity, 98.5% specificity. PACT had distinct patterns in Bernard Soulier Syndrome (n = 10), Glanzmann Thrombasthenia (n = 24), δ-granule disorder (n = 7), and other PFDs (n = 12). PACT could assess platelet function in patients (14%) with thrombocytopenia/lipemia wherein LTA was inconclusive. PACT was stable up to 24 hours in samples stored/transported at 2-8◦C. The results of utility and stability are only valid for the specific markers, agonist concentrations, and conditions investigated in this paper. PACT is a useful modality for the diagnosis of PFD, especially in children, thrombocytopenia cases or in the setup where an aggregometer is not readily available.

Deconstruction of Obscure Features in SVD-Decomposed Raman Images from P. chrysogenum Reveals Complex Mixing of Spectra from Five Cellular Constituents.

Raman imaging has transcended in recent times from being an analytical tool to a molecular profiling technique. Biomedical applications of this technique often rely on singular-value decomposition (SVD), principal component analysis (PCA), etc. for data analysis. These methods, however, obliterate the molecular information contained in the original Raman data leading to speculative interpretations based on relative intensities. In the present study, SVD analysis of the Raman images from Penicillium chrysogenum resulted in 11 spectral components and corresponding images with highly distorted spectral features and complex image contrast, respectively. To interpret the SVD results in molecular terms, we have developed a combined multivariate approach. By applying this methodology, we have successfully extracted the contribution of five biomolecular constituents of the P. chrysogenum filamentous cell to the SVD vectors. Molecular interpretability will help SVD/PCA surpass the realm of variance-based classification to a more meaningful molecular domain.