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At least 127 records · Page 7Linked to original sources
PubMed1996

Differences in bull spermiograms using eosin-nigrosin stain, feulgen stain, and phase contrast microscopy methods.

The Society for Theriogenology recently adopted a minimum standard of 70% normal spermatozoa for the bull breeding soundness examination (BSE). We conducted this study to determine if spermiograms derived by brightfield microscopy of eosin-nigrosin stained semen smears (Society method) overestimated the proportion of normal spermatozoa. Comparison of the above method was made with that of phase contrast microscopy (Phase method). We then evaluated our ability to discern head abnormalities by comparing brightfield microscopy of Feulgen-stained sperm DNA (Feulgen method) with those of the Society and Phase methods. Spermiograms were determined for each of the 181 beef bulls using all 3 methods. Only bulls that were being routinely tested prior to the 1993 breeding season were included. The mean percentage of normal spermatozoa surpassed the minimum standard with the Society (72.8%) but not the Phase (52.5%) method, which identified more distal cytoplasmic droplets that adhered to cells without distal midpiece reflexes. The Phase method also identified more total primary and fewer primary head abnormalities. We conclude that the Phase method is not a suitable substitute for the Society method when applying the minimum standard during a routine BSE. The Feulgen method identified more head abnormalities, especially in the pattern of DNA, than the other methods, however, when compared to the minimum standard that improvement was not clinically important. Both the Phase and Feulgen methods are better than the Society method for monitoring changes in abnormal spermiograms over time.

Journal Article↗
PubMed2005

Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.

Cell Survival↗
PubMed2002

Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM.

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.

Animals↗