Study Of Cellular Dynamics Research Articles

Application of Super-resolution SPEED Microscopy in the Study of Cellular Dynamics.

Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy. However, acquiring three-dimensional (3D) super-resolution information about structures and dynamic processes in live cells at high speed remains challenging. Recently, the development of high-speed single-point edge-excitation subdiffraction (SPEED) microscopy, along with its 2D-to-3D transformation algorithm, provides a practical and effective approach to achieving 3D subdiffraction-limit information in subcellular structures and organelles with rotational symmetry. One of the major benefits of SPEED microscopy is that it does not rely on complex optical components and can be implemented on a standard, inverted epifluorescence microscope, simplifying the process of sample preparation and the expertise requirement. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside submicrometer biological channels or cavities at high spatiotemporal resolution. The collected data are then subjected to postlocalization 2D-to-3D transformation to obtain 3D super-resolution structural and dynamic information. In recent years, SPEED microscopy has provided significant insights into nucleocytoplasmic transport across the nuclear pore complex (NPC) and cytoplasm-cilium trafficking through the ciliary transition zone. This Review focuses on the applications of SPEED microscopy in studying the structure and function of nuclear pores.

Cascade Forest-Based Model for Prediction of RNA Velocity.

In recent years, single-cell RNA sequencing technology (scRNA-seq) has developed rapidly and has been widely used in biological and medical research, such as in expression heterogeneity and transcriptome dynamics of single cells. The investigation of RNA velocity is a new topic in the study of cellular dynamics using single-cell RNA sequencing data. It can recover directional dynamic information from single-cell transcriptomics by linking measurements to the underlying dynamics of gene expression. Predicting the RNA velocity vector of each cell based on its gene expression data and formulating RNA velocity prediction as a classification problem is a new research direction. In this paper, we develop a cascade forest model to predict RNA velocity. Compared with other popular ensemble classifiers, such as XGBoost, RandomForest, LightGBM, NGBoost, and TabNet, it performs better in predicting RNA velocity. This paper provides guidance for researchers in selecting and applying appropriate classification tools in their analytical work and suggests some possible directions for future improvement of classification tools.

Cost-efficient boundary-free surface patterning achieves high effective-throughput of time-lapse microscopy experiments.

Time-lapse microscopy plays critical roles in the studies of cellular dynamics. However, setting up a time-lapse movie experiments is not only laborious but also with low output, mainly due to the cell-losing problem (i.e., cells moving out of limited field of view), especially in a long-time recording. To overcome this issue, we have designed a cost-efficient way that enables cell patterning on the imaging surfaces without any physical boundaries. Using mouse embryonic stem cells as an example system, we have demonstrated that our boundary-free patterned surface solves the cell-losing problem without disturbing their cellular phenotype. Statistically, the presented system increases the effective-throughput of time-lapse microscopy experiments by an order of magnitude.

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.

Quantitative phase microscopy with enhanced contrast and improved resolution through ultra-oblique illumination (UO-QPM).

Recent developments in phase contrast microscopy have enabled the label-free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra-oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field-of-view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria "spinning", which has not been reported before, further demonstrating the advantages of the proposed method.

Techniques for Studying Decoding of Single Cell Dynamics.

Cells must be able to interpret signals they encounter and reliably generate an appropriate response. It has long been known that the dynamics of transcription factor and kinase activation can play a crucial role in selecting an individual cell's response. The study of cellular dynamics has expanded dramatically in the last few years, with dynamics being discovered in novel pathways, new insights being revealed about the importance of dynamics, and technological improvements increasing the throughput and capabilities of single cell measurements. In this review, we highlight the important developments in this field, with a focus on the methods used to make new discoveries. We also include a discussion on improvements in methods for engineering and measuring single cell dynamics and responses. Finally, we will briefly highlight some of the many challenges and avenues of research that are still open.

Tracking Subcellular Dynamics with Multifocal Plane Microscopy

The study of cellular dynamics has often been hampered by the limited capabilities of standard microscopes. Classical microscopes are very well suited to the imaging of events that are constrained to one focal plane, but much less so for the investigation of fast dynamics that are not constrained to one focal plane. As cells are inherently three dimensional, this might account for the relatively limited amount of knowledge that is currently available regarding the specifics of subcellular trafficking.To address this problem we have introduced a microscopy modality into the study of cellular dynamics, multi-focal plane microscopy (MUM), which allows for the simultaneous imaging of several focal planes. We will review multifocal plane microscopy and discuss subsequent developments. Multifocal plane microscopy is an ideal tool for single molecule and single particle tracking in three dimensions. Single molecule trajectories by themselves, are, however, only of limited biological significance. It is the cellular environment that also needs to be imaged to provide the biological context for the observed dynamics. We will discuss our approaches to address this question.Antibodies form a central component of the human immune system. Over recent years, antibodies, especially IgG molecules, have also been developed as major new drugs. We will present data related to the trafficking of IgG molecules during endocytosis, exocytosis and sorting as imaged by multifocal plane microscopy.

Study of cellular dynamics on polarized CoCrMo alloy using time-lapse live-cell imaging

The physico-chemical processes and phenomena occurring at the interface of metallic biomedical implants and the body dictate their successful integration in vivo. Changes in the surface potential and the associated redox reactions at metallic implants can significantly influence several aspects of biomaterial/cell interactions such as cell adhesion and survival in vitro. Accordingly, there is a voltage viability range (voltages which do not compromise cellular viability of the cells cultured on the polarized metal) for metallic implants. We report on cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesion complexes in transiently transfected MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy surfaces polarized at the cathodic and anodic edges of its voltage viability range (−400 and +500mV (Ag/AgCl), respectively). Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) were also studied on the polarized metal at −1000, −400 and +500mV (Ag/AgCl). Our results show that at −400mV, where reduction reactions dominate, a gradual loss of adhesion occurs over 24h while cells shrink in size during this time. At +500mV, where oxidation reactions dominate (i.e. metal ions form, including Cr6+), cells become non-viable after 5h without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at −1000mV decreased sharply within 15min after polarization, which rendered the cells completely non-viable. No significant amount of ROS release by cells was detected on the polarized CoCrMo at any of these voltages.

Undesirable Charge-Enhancement of Isobaric Tagged Phosphopeptides Leads to Reduced Identification Efficiency

The study of cellular dynamics by proteomics using mass spectrometry requires a quantitation strategy that is robust, sensitive, and of sufficient resolution to deal with subtle changes in protein expression or post-translational modification. The major quantitation strategies are stable isotopic labeling of proteins and peptides for in vitro cell culture systems (stable isotope labeling using amino acids in cell culture, SILAC) or isobaric peptide labels such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) for both in vitro and in vivo systems. These quantitation strategies have also been successfully applied to phosphoproteomics studies for the investigation of signal transduction pathways. Here we describe major drawbacks associated with isobaric labeling for the identification and quantitation of phosphopeptides using electrospray tandem mass spectrometry. Phosphopeptide derivatization with isobaric tags results in significantly greater charging in electrospray ionization. This reduces phosphopeptide identification efficiency with multistage activation and HCD MS/MS by more than 50% and may contribute to the discrepancy observed between identifications observed for large cell- or tissue-based data sets from labeled and nonlabeled peptide mixtures. Ammonia vapor sprayed perpendicular to the electrospray needle during ionization resulted in an overall decrease in the average charge states and a concomitant increase in phosphopeptide identifications.

Studies of cellular dynamics by laser Doppler microscopy

AbstractThe sources and magnitudes of typical molecular, macromolecular and cytoplasmic motions in cells are reviewed and compared with the ability of the conventional light microscope to detect and quantify them. The principles of laser Doppler spectroscopy are introduced; the type and range of motions detectable are reviewed and found to be superior to conventional light microscopy in certain respects. A laser Doppler microscope system has been developed that allows accurate placement of the beam within the cell and combines visual monitoring of the cellular behaviour with the superior resolution of motions from spectroscopy. The system operates with a non‐destructive, low intensity, laser beam that permits continuous monitoring of diffusive and translational motions in living cells.