{"id":25373,"date":"2017-01-20T09:45:04","date_gmt":"2017-01-20T08:45:04","guid":{"rendered":"https:\/\/www.micropticsl.com\/microscopy-part-3\/"},"modified":"2017-10-06T12:13:16","modified_gmt":"2017-10-06T10:13:16","slug":"microscopy-part-3","status":"publish","type":"post","link":"https:\/\/www.micropticsl.com\/es\/microscopy-part-3\/","title":{"rendered":"Microscopy – part 3"},"content":{"rendered":"

Importance of microscopy for cell biologists in general and spermatologists specifically<\/h2>\n

Part 2: Specialized microscopic techniques for sperm
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In the second blog on Microscopy<\/a>, I indicated that brightfield microscopy, positive and negative phase microscopy and fluorescence microscopy are now routinely used for CASA analysis. Before proceeding to a more detailed discussion of fluorescence microscopy I will first discuss a complimentary technique to phase contrast microscopy namely Nomarski Differential Interference Microscopy.<\/p>\n

1. Nomarski Differential Interference Microscopy (NDIC)<\/h4>\n

It is a contrast enhancing technique that allows visualization of live cells without staining like phase contrast. However, Wollaston prisms are utilized that assist to provide remarkable sharp and three dimensional like images. It is particularly useful to study human and animal sperm. Moreover, it has been very extensively used in the field of IVF for egg and sperm manipulation or similar optics, called Hoffman optics.\u00a0 NDIC has a lot of potential in CASA but unfortunately is very expensive for routine CASA.\u00a0 In Fig. 1 sperm of two ferret species could be distinguished on the basis of the structure of the posterior border of the acrosome.<\/p>\n

\"NDIC

Fig. 1: NDIC images of domestic ferret on the left and black footed ferret sperm on the right (van der Horst et al. 1991).<\/p><\/div>\n

2. Fluorescence microscopy:<\/h4>\n

Three essential components of epifluorescence is a high intensity light source, a special cube (Fig. 2) controlling specific incoming and very specific emitting fluorescent wavelengths and special objective quarts lenses. Nikon in their webpage states: \u201cThe technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, due to attributes that are not readily available with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can simultaneously identify several target molecules simultaneously\u201d. It is potentially possible to determine almost any structure of a sperm cell such as the acrosome which is lectin specific (Fig. 3) or inorganic chemical compounds such as Calcium or various functional proteins and even molecules and in several cases multiple probes can be used.<\/p>\n\n\t\t