Confocal fluorescent labeling

Peachey L D, Ishikawa H and Murakami T 1996 Correlated confocal and intermediate voltage electron microscopy imaging of the same cells using sequential fluorescence labeling fixation and critical point dehydration Scanning Microsc. (SuppI) 10 237-47... 

In conventional chip experiments, fluorescence scanners are used for chip read-out. In the case of laser scanners, HeNe lasers are used as excitation sources and photomultiplier tubes as detectors, whereas CCD-based scanners use white light sources. The optical system can be confocal or non-confocal. Standard biochip experiments are performed using two fluorescent labels as... 

Fig. 12 CNTs act as a vector for drug delivery into living cells. After incubation of HeLa cells with AlexaFluor594-labeled SWNTs for 12 h at 37 °C, living cells were observed under confocal fluorescence microscope for a CNT uptake study, (a) Images show dual confocal detection of AlexaFluor594-SWNT (red) internalized into cells with the membrane stained by AlexaFluor488 (green), (b) Series of images of different z-focal scanning planes down through cells. (Adapted from [61])...

In case of fluorescent labelled secondary antibody mount the slide and visualise under fluorescent or Confocal microscope. For peroxide visualisation incubate with a DAB solution till brown color is seen followed by stopping the reaction under running tap water. 

When fluorescently labeled biological specimens are viewed with a conventional wide-field microscope, a haze of out-of-focus fluorescence is usually created hy the overlapping structures within the sample. As we focus through the specimen, our hrains have a remarkable ability to discern substantial structural detail. However, the resolution of the images we record on film is degraded hy the out-of-focus fluorescence. The confocal microscope can reject out-of-focus information and enhance the contrast of an image because the illumination and the detection are confined to an identical (small) region of the specimen. An overview of the basic principles of a confocal microscope is presented in Fig. 1 and outlined helow. 

Vesicular transport of bile acids has not been demonstrated under normal conditions, shown by using isolated rat hepatocyte couplets and fluorescently labelled bile acids. In these experiments confocal microscopy found no evidence of sequestering into clusters and colchicine disruption of microtubular function did not affect bile-acid transport. This makes it unlikely that vesicle transport plays a role and it is now believed that bile acids traverse the hepatocyte by diffusion through the cytosol while bound to soluble proteins. It is worth considering the caveat that fluorescently labelled bile acids, while very useful tools, do differ structurally from endogenous bile acids with increased hydro-phobicity leading to greater retention by cells. ... 

Confocal laser scanning microscopy can be used in conjunction with microwave heating for examining the three-dimensional structure and cellular interrelationships in sections of paraffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG molecular weight 300) for 90 sec in a microwave oven. The use of thick paraffin sections (15 (xm) and fluorescently labeled antibodies is preferred. 

Fluorescent labelling of protein/RNA or DNA combined with optical or confocal microscopy imaging is a powerful widely used imaging technique that can (like Raman spectroscopy) also be applied to live cells or tissues. This technique, however, is limited by the number of fluorescent molecular... 

The distribution of LCM in the brain parenchyma was further analyzed using fluorescently labeled LCM and confocal laser scanning microscopy. As in the case of unlabeled LCM, rats bearing 9L gliosarcoma tumors were injected intravenously (i.e., via tail vein) with diO-LCM and sacrificed 2 min later. The brains were processed as described elsewhere (ref. 531). In this case,... 

P 66] Equal streams of 1 mM solutions of fluorescein-labeled polymer (polyethylen-imine, molecular weight 500 000) in water-glycerol mixtures (0 and 80% glycerol) and a clear solution were injected into the channel [44], The flow was achieved by compressed air at constant pressure. Imaging was achieved by applying a confocal fluorescence microscope. 

Describe, with a labeled diagram, the principle of confocal fluorescent detection. (4 marks)... 

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