Slides were imaged using a Photometrics Coolsnap HQ2 CCD camera and a Zeiss AxioImager A1 fluorescence microscope with a Plan Apochromat 100x 1.4NA objective, a Nikon Intensilight Mercury based light source (Nikon UK Ltd, Kingston-on-Thames, UK) and either Chroma #89014ET (3 colour) or #89000ET (4 colour) single excitation and emission filters (Chroma Technology Corp., Rockingham, VT) with the excitation and emission filters installed in Prior motorised filter wheels. A piezoelectrically driven objective mount (PIFOC model P-721, Physik Instrumente GmbH & Co, Karlsruhe) was used to control movement in the z dimension. Step size for z stacks was set at 0.2 μm. Hardware control, image capture and analysis were performed using Nikon Nis-Elements software (Nikon UK Ltd, Kingston-on-Thames, UK). Images were deconvolved using a calculated point spread function with the constrained iterative algorithm of Volocity (Perkinelmer Inc, Waltham, MA). Image analysis was carried out using the Quantitation module of Volocity (Perkinelmer Inc, Waltham, MA). For DNA FISH, only alleles with single probe signals were analysed to eliminate the possibility of measuring sister chromatids.
Plan apochromat 100x 1.4na objective
Manufactured by Nikon
Sourced in United Kingdom
The Plan Apochromat 100x 1.4NA objective is a high-quality optical lens designed for use in microscopy applications. It features a numerical aperture of 1.4 and a magnification of 100x, providing a high level of optical performance and resolution. The Plan Apochromat design helps to minimize chromatic and spherical aberrations, ensuring accurate and detailed image reproduction.
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2 protocols using plan apochromat 100x 1.4na objective
1
Fluorescence Microscopy for DNA FISH
2
Imaging of dCas9 and TALE Constructs in U2OS Cells
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U2OS cells growing on slides were fixed with 4 % paraformaledhyde, permeabilised with Triton X-100 and DAPI stained 24h following transfection with dCas9 or TALE constructs. Slides were imaged using a Photometrics Coolsnap HQ2 CCD camera and a Zeiss AxioImager A1 fluorescence microscope with a Plan Apochromat 100x 1.4NA objective, a Nikon Intensilight Mercury based light source (Nikon UK Ltd, Kingston-on-Thames, UK) and Chroma #89014ET (3 colour) single excitation and emission filters (Chroma Technology Corp., Rockingham, VT) with the excitation and emission filters installed in Prior motorised filter wheels. A piezoelectrically driven objective mount (PIFOC model P-721, Physik Instrumente GmbH & Co, Karlsruhe) was used to control movement in the z dimension. Step size for z stacks was set to 0.2 μm. Hardware control and image capture were performed the acquisition module or Nikon Nis-Elements software (Nikon UK Ltd, Kingston-on-Thames, UK).
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