Fluorescence Cell Imaging and Manipulation Using Conventional Halogen Lamp Microscopy

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.

[1]  P. Collas,et al.  Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation. , 1996, Theriogenology.

[2]  S. Kishigami,et al.  How to improve the success rate of mouse cloning technology. , 2010, The Journal of reproduction and development.

[3]  S. Kishigami,et al.  Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium. , 2007, The Journal of reproduction and development.

[4]  Maurizio Zuccotti,et al.  Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei , 1998, Nature.

[5]  S. Toyokuni,et al.  CD9 Is a Surface Marker on Mouse and Rat Male Germline Stem Cells1 , 2004, Biology of reproduction.

[6]  R Yanagimachi,et al.  Intracytoplasmic sperm injection in the mouse. , 1995, Biology of reproduction.

[7]  E. Sato,et al.  Effect of fluorescent mercury light irradiation on in vitro and in vivo development of mouse oocytes after parthenogenetic activation or sperm microinjection. , 2011, The Journal of reproduction and development.

[8]  Kazuo Yamagata,et al.  Long-term, six-dimensional live-cell imaging for the mouse preimplantation embryo that does not affect full-term development. , 2009, The Journal of reproduction and development.

[9]  A. Iritani,et al.  Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade , 2009, PloS one.

[10]  D. Keefe,et al.  The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. , 1999, Fertility and sterility.

[11]  H. Ohta,et al.  Establishment of mouse embryonic stem cell lines from somatic cell nuclei by nuclear transfer into aged, fertilization-failure mouse oocytes , 2007, Current Biology.

[12]  Rudolf Oldenbourg,et al.  A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes , 2000, Nature Biotechnology.

[13]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[14]  Hiroshi Kimura,et al.  Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling , 2011, Nucleic acids research.

[15]  P. Ross,et al.  Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope. , 2006, BioTechniques.