The Hadamard transform microscope image analysis was applied to analyze the DNA and protein in a single cell. By this method, the fluorescence emission spectrum, the fluorescence image and the fluorescence intensity of a single cell can be acquired which was tagged by a fluorescence probe. The potential application value of this system is shown for cytobiological aspects.Theory of the instrument. As shown in Fig. 1, the light signal captured by the objective can directly enter and be focussed on the focus-plane of the CCD camera when the mirror (M) is moved away from the optical path, then the image of analyzed sample can be Fresenius Fresenius' Journal of Fig. 1. Schematic diagram of the instrument of Hadamard transform microscope fluorescence image. P: dichroic beamsplitter, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 : lens, M: mirror
A novel system of Hadamard transform microscopic fluorescence imaging for single cells is presented, based on which the DNA ploidy of rat hepatocyte was quantitatively measured. The result shows that diploid rat hepatocyte has a stable DNA content, thus diploid rat hepatocyte was used to investigate the binding of five clinical anticancer agents, vincristine, cyclophosphamide, nitrogen mustard, cis-diamminedichloroplatinum(n) (CDDP) and mitomycin-C, with cellular DNA when acridine orange (AO) was used as the competitive fluorescence probe. Based on this model, some Schiff base complexes-cellular DNA interactions were investigated. The results indicate that all the twenty-two compounds, including Schiff base ligands of N-2-hydroxy-naphthaldehyde with D-glucoamine (NG) and the complexes of 3d-transitional metals ions with NO and with D-glucoamine (Glu) and the mixed complexes of NG and Glu series with alpha-glycine (GNG), have the ability to enter the cell membrane and interact with cellular DNA. Four of the compounds, CuGlu, Fe(II)NG, Fe(III)NG and CuGluG can intercalate with DNA like AO does and depress AO-DNA fluorescence to 70% or lower. An in intro UV-visible spectroscopic study on the compound-DNA spectra testified the above results and suggests that diverse interaction mechanisms coexist for all these complexes except intercalating mode. This study presents a new in vitro method for initial screening of anticancer compounds.
The technique of Hadamard transform microscopic fluorescence imaging is described. With an Acridine Orange (AO) staining cell, some factors that influence the fluorescence intensity of a cellular AO-DNA complex were studied; thereby the technique was applied to measure the nuclear DNA content (ploidy) in a breast tumor cell, and some parameters were employed to evaluate the tumor malignancy. A comparative study with conventional microfluorometry indicates that our system has some outstanding advantages. By using this system, the results of malignancy evaluation for 38 cases of breast tumor specimens were concordant with pathological diagnosis. This work demonstrates that this technique has a high potential value in medicine and cytobiology and may be applied as a new method for diagnostic and prognostic studies of tumor cases.
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