A high-resolution multimode digital microscope system.
This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components-the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae.
Department of Biology, University of North Carolina, Chapel Hill, North Carolina, USA.
Edward D
ED
Salmon
Sidney L
SL
Shaw
Jennifer C
JC
Waters
Clare M
CM
Waterman-Storer
Paul S
PS
Maddox
Elaine
E
Yeh
Kerry
K
Bloom
D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D064246:Mad2 Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D014981:Xenopus
C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins
Charge-coupled device; Green fluorescent protein; Hamamatsu; MetaMorph digital imaging system; Nikon FXA microscope