Optical coherence microscopy with a split-spectrum image reconstruction method for temporal-dynamics contrast-based imaging of intracellular motility.

Biomedical researchers use optical coherence microscopy (OCM) for its high resolution in real-time label-free tomographic imaging. However, OCM lacks bioactivity-related functional contrast. We developed an OCM system that can measure changes in intracellular motility (indicating cellular process states) via pixel-wise calculations of intensity fluctuations from metabolic activity of intracellular components. To reduce image noise, the source spectrum is split into five using Gaussian windows with 50% of the full bandwidth. The technique verified that F-actin fiber inhibition by Y-27632 reduces intracellular motility. This finding could be used to search for other intracellular-motility-associated therapeutic strategies for cardiovascular diseases.

Three-dimensional imaging in reflection phase microscopy with minimal axial scanning.

Reflection phase microscopy is a valuable tool for acquiring three-dimensional (3D) images of objects due to its capability of optical sectioning. The conventional method of constructing a 3D map is capturing 2D images at each depth with a mechanical scanning finer than the optical sectioning. This not only compromises sample stability but also slows down the acquisition process, imposing limitations on its practical applications. In this study, we utilized a reflection phase microscope to acquire 2D images at depth locations significantly spaced apart, far beyond the range of optical sectioning. By employing a numerical propagation, we successfully filled the information gap between the acquisition layers, and then constructed complete 3D maps of objects with substantially reduced number of axial scans. Our experimental results also demonstrated the effectiveness of this approach in enhancing imaging speed while maintaining the accuracy of the reconstructed 3D structures. This technique has the potential to improve the applicability of reflection phase microscopy in diverse fields such as bioimaging and material science.

Efficacy Evaluation of SDF-1α-Based Polypeptides in an Acute Myocardial Infarction Model Using Structure-Based Drug Design.

Stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) mediates the migration of circulating cells to desired sites for tissue development, homeostasis, and regeneration and can be used to promote cardiac regeneration by recruiting stem cells. However, the use of SDF-1α in the injured heart necessitates not only higher binding affinity to its receptor, CXCR4+, but also better robustness against enzymatic degradation than other SDF-1 isoforms. Here, we conduct a screening of SDF-1α analog peptides that were designed by structure-based drug design (SBDD), a type of computer-aided drug design (CADD). We have developed in vitro and in vivo methods that enable us to estimate the effect of peptides on the migration of human mesenchymal stem cells (hMSCs) and cardiac regeneration in acute myocardial infarction (AMI)-induced animals, respectively. We demonstrate that one type of SDF-1α analog peptide, SDP-4, among the four analog peptides preselected by SBDD, is more potent than native SDF-1α for cardiac regeneration in myocardial infarction. It is interesting to note that the migratory effects of SDP-4 determined by a wound healing assay, a Transwell assay, and a 2D migration assay are comparable to those of SDF-1α. These results suggest that in vivo, as well as in vitro, screening of peptides developed by SBDD is a quintessential process to the development of a novel therapeutic compound for cardiac regeneration. Our finding also has an implication that the SDP-4 peptide is an excellent candidate for use in the regeneration of an AMI heart.

Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique.

Optical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.

Development of a 3-D Physical Dynamics Monitoring System Using OCM with DVC for Quantification of Sprouting Endothelial Cells Interacting with a Collagen Matrix.

The extracellular matrix (ECM) plays a key role during cell migration, proliferation, and differentiation by providing adhesion sites and serving as a physical scaffold. Elucidating the interaction between the cell and ECM can reveal the underlying mechanisms of cellular behavior that are currently unclear. Analysis of the deformation of the ECM due to cell-matrix interactions requires microscopic, three-dimensional (3-D) imaging methods, such as confocal microscopy and second-harmonic generation microscopy, which are currently limited by phototoxicity and bleaching as a result of the point-scanning approach. In this study, we suggest the use of optical coherence microscopy (OCM) as a live-cell, volumetric, fast imaging tool for analyzing the deformation of fibrous ECM. We optimized such OCM parameters as the sampling rate to obtain images of the best quality that meet the requirements for robust digital volume correlation (DVC) analysis. Visualization and analysis of the mechanical interaction between collagen ECM and human umbilical vein endothelial cells (HUVECs) show that cellular adhesion during protrusion can be analyzed and quantified. The advantages of OCM, such as fine isotropic spatial resolution, fast time resolution, and low phototoxicity, make it the ideal optic tool for 3-D traction force microscopy.

Macrophages-Triggered Sequential Remodeling of Endothelium-Interstitial Matrix to Form Pre-Metastatic Niche in Microfluidic Tumor Microenvironment.

The primed microenvironment of future metastatic sites, called the pre-metastatic niche, is a prerequisite for overt metastasis. However, a mechanistic understanding of the contributions of recruited cells to the niche is hindered by complex in vivo systems. Herein, a microfluidic platform that incorporates endothelial cells and extracellular matrix (ECM) scaffolds is developed, and the distinct role of recruited monocytes and macrophages in establishing pre-metastatic niches is delineated. It is observed that monocyte-derived matrix metalloproteinase 9 facilitates cancer cell extravasation through destruction of endothelial tight junctions. Furthermore, subsequent cancer cell invasiveness is significantly enhanced. Close examination of ECM structures reveals that cancer cells move within characteristic "microtracks" generated by macrophages, suggesting that macrophages could serve as a compensatory mechanism for the reduced migratory capacity of cancer cells. Thus, the first evidence of monocyte/macrophage-induced remodeling is shown, and these findings will open up new horizons for improving characterization of the pre-metastatic niche and corresponding immunotherapies.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis.

Single-shot digital holographic microscopy for quantifying a spatially-resolved Jones matrix of biological specimens.

Field-based polarization measurements are essential for the completeness of information when exploiting the complex nature of optical responses of target objects. Here, we demonstrate digital holographic microscopy for quantifying a polarization-sensitive map of an object with a single-shot measurement. Using the image-splitting device generating four different copies of an object image and a separate reference beam of an off-axis configuration enables single-shot and multi-imaging capability. With the use of two polarization filters, four complex field images containing an object's polarization response are obtained simultaneously. With this method, we can construct a complete set of 2-by-2 Jones matrix at every single point of the object's images, and thus clearly visualize the anisotropic structures of biological tissues with low level of birefringence. This method will facilitate the high-precision measurements for fast dynamics of the polarization properties of biological specimens.

Hemodynamic responses in rat brain during transcranial direct current stimulation: a functional near-infrared spectroscopy study.

In the present study, we monitored hemodynamic responses in rat brains during transcranial direct current stimulation (tDCS) using functional near-infrared spectroscopy (fNIRS). Seven rats received transcranial anodal stimulation with 200 µA direct current (DC) on their right barrel cortex for 10 min. The concentration changes of oxygenated hemoglobin (oxy-Hb) were continuously monitored during stimulation (10 min) and after stimulation (20 min). The trend of hemodynamic response changes was modeled using linear regression, and the relationship between incremental and decremental rates of oxy-Hb was investigated by correlation analysis. Our results showed that the oxy-Hb concentration was almost linearly increased and decreased during and after stimulation, respectively. In addition, a significant negative correlation (p < 0.05) was found between the rate of increase of oxy-Hb during stimulation and the rate of decrease of oxy-Hb after stimulation, indicating that the recovery time after tDCS may not depend on the total amount of hemodynamic changes in the stimulated brain area. Our results also demonstrated considerable individual variability in the rate of change of hemodynamic responses even with the same direct current dose to identical brain regions. This suggests that individual differences in tDCS after-effects may originate from intrinsic differences in the speed of DC stimulation "uptake" rather than differences in the total capacity of DC uptake, and thus the stimulation parameters may need to be customized for each individual in order to maximize tDCS after-effects.