Fluorescence Cell Imaging and Manipulation Using Conventional Halogen Lamp Microscopy

Abstract

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

Early Development of Cloned Bovine Embryos Produced from Oocytes Enucleated by Fluorescence Metaphase II Imaging Using a Conventional Halogen-Lamp Microscope

“…It has been reported that phosphorylation of histone H3S10 occurs specifically in chromosomes of somatic cells and oocytes at the metaphase stage (Bui et al 2004;Swain et al 2007;Van Hooser et al 1998;Wei et al 1998). Recently, we demonstrated that the metaphase plate of the second meiotic division in mouse and cattle oocytes is clearly visible after injection of an antibody under a halogen-lamp microscope (Yamagata et al, 2012).…”

“…The microscope setup with a transmission fluorescence filter system used for manipulation and observation was described previously (Yamagata et al, 2012). Briefly, an inverted microscope (IX-70, Olympus, Tokyo, Japan) equipped with a 100 W halogen lamp was used for manipulation and observation.…”

“…Briefly, an inverted microscope (IX-70, Olympus, Tokyo, Japan) equipped with a 100 W halogen lamp was used for manipulation and observation. The filter adaptor previously made by us was placed on the top of the condenser [numerical aperture (NA) = 0.5] of the IX-70 (Yamagata et al, 2012). The optical axis of the condenser was adjusted before setting the adaptor to the condenser.…”

A Simplified Approach for Oocyte Enucleation in Mammalian Cloning

“…The alternatives to canonical (OH/UV) enucleation proposed so far are: (1) Polarized light microscopy, that locates the metaphase spindle (Liu et al, 2000); (2) the use of softer filter systems with traditional halogen lamps, associated with fluorescent antibody directed against the phosphorylated serine 10 of histone H3 (H3S10ph) binding to M-phase chromosomes (Yamagata et al, 2012); and (3) DAE (Yin et al, 2002).…”

“…The use of polarized light in large animal oocytes is less advisable due to the high lipid droplet content. The softer halogen lamp associated with fluorescent antibody anti-histone to locate the chromosomes reduces the phototoxic effects but adds the further step of antibody injection prior to enucleation (Yamagata et al, 2012), hence it is unpractical.…”