Cells were observed using a Nikon Eclipse E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF super high-pressure mercury lamp and a 1.4 numerical aperture, 100× Plan Apo oil immersion objective lens with appropriate fluorescence filter sets or with differential interference contrast microscopy. Images were acquired using a cooled charge-coupled device digital camera (C4742–95–12NR; Hamamatsu Photonics, Hamamatsu, Japan) using the AQUACOS-MOS software (Hamamatsu Photonics). To visualize GFP- or mRFP-tagged proteins, cells were grown under the indicated conditions, harvested, and resuspended in SDA- or SD-based medium. Cells were mounted onto microslides and immediately observed using a GFP band-pass or G-2A (for mRFP) filter set.
C4742 95 12nr
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C4742-95-12NR is a lab equipment product from Hamamatsu Photonics. It is a digital CCD camera designed for scientific and industrial applications. The core function of this device is to capture and record high-quality digital images.
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5 protocols using c4742 95 12nr
1
Fluorescence Microscopy of Tagged Proteins
2
Visualizing Actin Cytoskeleton Dynamics
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Cells were observed using a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF superhigh-pressure mercury lamp and a 1.4 numerical aperture 100× Plan Apo oil immersion objective lens (Nikon Instec) with appropriate fluorescence filter sets (Nikon Instec) or differential interference contrast optics. Images were acquired using a cooled digital charge-coupled device camera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics). GFP- or mRFP-tagged proteins were observed in living cells, which were grown from early to mid-logarithmic phase, harvested, and resuspended in SD medium. Cells were immediately observed using a GFP bandpass (for GFP) or G2-A (for mRFP) filter set. Treatment with LAT-A (Wako Pure Chemical Industries Ltd.) was performed at 100 μmol/L by addition of a suitable volume of 20 mmol/L stock in dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries Ltd.) to the medium, as described (Ayscough et al. 1997 (link)).
3
Microscopic Imaging of Fluorescent Proteins
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Cells were observed using a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF superhigh-pressure mercury lamp and a 1.4 numerical aperture 100 × Plan Apo oil immersion objective lens (Nikon Instec) with appropriate fluorescence filter sets (Nikon Instec) or differential interference contrast optics. Images were acquired using a cooled digital charge-coupled device camera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics). GFP-, mRFP1-, or mCherry-tagged proteins were observed in living cells, which were grown to early to mid-logarithmic phase, harvested, and resuspended in SD medium. Cells were immediately observed using a GFP bandpass (for GFP) or G2-A (for mRFP1 and mCherry) filter set. Observations were compiled from examining at least 200 cells. For statistical analysis, the observation of 200 cells was repeated five times for each strain.
4
Fluorescence Microscopy of GFP-Tagged Proteins
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Cells were observed using a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF super-high-pressure mercury lamp and a 1.4 numerical aperture 100× Plan Apo oil immersion objective lens (Nikon Instec) with appropriate fluorescence filter sets (Nikon Instec) or differential interference contrast optics. Images were acquired using a cooled digital charge-coupled devicecamera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics). GFP-tagged proteins were observed in living cells, which were grown to early to midlogarithmic phase, harvested, and resuspended in SD medium. For detection of GFP fluorescence, cells were immediately observed using a GFP bandpass filter set. Observations were compiled from the examination of at least 100 cells.
5
Microscopic Imaging of Fluorescent Proteins
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Cells expressing fluorescent proteins were observed using a Nikon ECLIPSE E800 microscope equipped with a 1.4 numerical aperture 100 × Plan Apo oil immersion objective lens with appropriate fluorescence filter sets or differential interference contrast (DIC) optics (Nikon Instec, Tokyo, Japan). Images were acquired using a cooled digital charge-coupled device camera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics) with 1 × 1 binning.
GFP-Snc1p , GFP-Lact-C2, and Ena1p-GFP were observed in living cells, which were grown as described in figure legends, harvested, and resuspended in SD medium. Cells were immediately observed using a GFP bandpass filter set. Colocalization of Cfs1p -EGFP with Drs2p -mRFP1, Neo1p -mRFP1, or Sec7p -mRFP1 was examined in fixed cells. Fixation was performed for 10 min at 25° by direct addition of 37% formaldehyde to a final concentration of 0.2% (Drs2p -mRFP1 and Neo1p -mRFP1) or 2% (Sec7p -mRFP1) in the culture medium. After fixation, cells were washed with phosphate-buffered saline and immediately observed using a GFP bandpass or a G2-A (for mRFP1) filter set.
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