Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.
The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord
during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal
plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS)
microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides
chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti-
Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better
differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees
well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath
are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an
excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and
native 3D optical sectioning ability.
System CASA (computer-assisted semen analysis) is a medical applicable system which gets the sperm motility and its parameters using image processing method. But there is no any authoritative administration or academic organization gives a set of criterion for CASA now result in lowering the effective compare of work between the labs or researchers. The average path and parameters relative to it as average path velocity, amplitude of lateral head displacement and beat cross frequency are often unable to compare between systems because of different algorithm. The paper presents a new algorithm that could define the average path uniquely and compute those 3 parameters above quickly and handy from any real path.