Digital imaging fluorescence microscopes

This substance penetrates into the cell where it is hydrolysed to fluorescein through the action of the enzyme s esterases. The quantity of the fluorescent quinoid form then depends on the local value of pH in the same way as in the case of phenolphthalein. The intensity of the fluorescein radiation is measured with a fluorescence microscope and then processed to a digital image which is the basis of a map of pH distribution in the cell (Fig. 1.13). 

The DNA in the sections is denatured by treatment with 70% formamide/2 x SCC for 5 min at 80°C. Ten microliters of the probe solution (hybridization buffer 7 pd, probe 1 pi, and distilled water 2 pi) is placed on the slide and coverslipped. The slide is placed in a microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of 15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear staining. The sections are promptly observed under a fluorescent microscope equipped with epifluorescence filters and a photometric CCD camera. The captured images are digitized and stored in an image analysis program. 

For the estimation of Chi formation in hairy roots, the root cells were examined on a laser scanning confocal image system attached to a microscope. The filter package with a laser (excitation 568 nm) and a 590-610 nm band pass barrier filter for red fluorescence was employed. Digital image analysis was performed by a computer-aided image processing system with software. 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

The techniques of FISH and mFISH used in conjunction with the resolving power and automated digital imaging capabilities of the fluorescence microscope offer a powerful combination of advantages that stand to benefit many areas of biology, from basic research to prenatal disease detection, cancer research, pathology, and cytogenetics. 

Shin, D., et al. A fiber-optic fluorescence microscope using a consumer-grade digital camera for in vivo cellular imaging. PloS One 5(6), el 1218 (2010)... 

Mixing was characterized by an optical microscope with an epifluorescence attachment [160], A 10 4 M fluorescein solution in sodium phosphate buffer (10 mM, pH 7, with trace of methanol) was applied. A filter cube was used for fitting the excitation and emission characteristics, allowing selective passages of the radiation. A CCD digital camera collected real-time fluorescence images. 

---