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Live-Cell Imaging:

1. Epifluorescent and Confocal Microscopy


Epifluorescent and confocal microscopy are routinely used for in our laboratory for biochemical characterization of cells.

    As illustrative examples, below are shown the characterization of two types of cells in the two research areas of primary interest to us - Vision and Cancer.

 Example 1: Characterization of Retinal Ganglion Cell Model - Differentiated RGC-5 Cells


    In-vitro retinal ganglion cell model is being developed for establishing quantitative assays of their pathological response to elevated pressure in glaucoma patients or a retinal prosthetic device. We have differentiated RGC-5 with staurosporine and characterized these using immunocytochemistry. The differentiated RGC-5 cells exhibit neuronal characteristics, including postmitotic state without apoptosis; neuronal morphology such as soma, neurites, and growth cones; and other neuronal markers. The differentiated RGC-5 cells can thus provide a robust platform for establishing quantitative assays from which refinements can be evolved to establish assays of acute and chronic impact of elevated pressure.

Characterization of RGC-5 Cell Line (Undifferentiated and Differentiated)


 

    Retinal ganglion cell line RGC-5 was differentiated with non-specific protein kinase staurosporine. The differentiated RGC-5 showed the neuronal morphology, whereas the RGC-5 cells untreated with staurosporine were in fibroblast-like morphologies. Staurosporine promotes the formation of dendritic neurites in RGC-5 cells. Formation of growth cone-like structure,focal adhesion of the growth cone, typical fibroblast-like cell structure of F-actin and focal adhesion distribution were observed on differentiated RGC-5 via F-actin/Viniculin labeling. The differentiated RGC-5 cells showed the expression synaptophysin, a presynaptic vesicle protein was expressed in differentiated RGC-5 cells, suggesting the presence of synaptic vesicle availability to form synaptic connection between ganglion cells.

Example 2: Autofluorescent Spectrum of Cancerous Cells.

    In any disease detection scheme which utilizes fluorescent labeling (E.g. Dye molecules, Quantum Dots) of specific biomarkers, the autofluorescence from cells limits the ultimate detection sensitivity (the minimum detectable concentration of labeled biomarkers). For illustration purpose, an autofluorescent spectrum from breast cancer cell SKBR3 is shown in the figure below. The excitation is with 488nm Ar+ laser. Fluorescence spectra in the 500-1000nm and 1000-1500nm regime are obtained, respectively, with CCD and InGaAs PD array detectors and are glued together. Obviously, the autofluorescence in the near infrared (900-1500nm) is significantly lower than that in the visible regime. Hence, for high sensitive detection, near infrared is a more advantageous wavelength for fluorescent labels. One of our research interests is indeed to develop InAs and PbS based near infrared nanocrystal quantum dot fluorescent probes for live cell imaging.

 

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