Confocal cell design

Confocal Raman microspectroscopy is now yielding spectra at micrometer spatial (lateral) resolution, and can be carried out in situ (Figure 6). Diffusion profiles can also be mapped. The potential for FTIR microspectroscopy is also apparent, and results have been obtained that are superior to those from OTTLE cells, with simpler cell design, thanks to the use of microelectrodes. But the discrimination against solvent absorption is poor for in situ work it is hoped that higher intensity sources, such as synchrotron sources, in conjunction with PM-IR-RAS, can overcome this problem. 

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

The test equipment of crystal type of gas hydrates consists of a laser Raman spectrometer, gas supply system, jacketed cooling type high-pressure visual cell, temperature control system, data acquisition and other parts. The experiment using a laser Raman spectrometer for the JY Co. in French produced Lab RAM HR-800 type visible confocal Raman microscope spectrometer. Laboratory independently designed a cooled jacket visible in situ high-pressure reactor, reactor with sapphire window to ensure full transparency of laser, and high pressure performance, visual reactor effective volume 3 ml, compression 20 MPa effective volume, to achieve characteristics of gas hydrate non-destructive and accurate measurement. The schematic representation of equipment is shown in Eigure 1. 

Fig. 20 Copper bis(thiosemicarbazonato) complex designed by Pascu et al. imaged in HeLa cells using confocal microscopy, where a) is the fluorescent channel, b) the DIC and c) the overlay of each channel (Ref 127).

Another point deals with the constraints on the physical dimension of the optical setup. In confocal mode, the membrane system must be very close to the microscope objective since its working distance is usually of the order of a few millimeters. Therefore, the design of a cell depends on these considerations. For membrane transport studies, the system must contain the fluid channels, the membrane and an optical window in a strictly limited thickness. Figure 7.7 depicts such a cell with two channel and a membrane. Fluids can be gases [40] or liquids [38, 41], From a practical point of view the use of an immersion objective can enhance the solid angle collecting the emitted light. 

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