Advanced cytological imaging system


Location: Adrian Building G1.
Contact: Dr Kees Straatman (krs5/7085, 2263).

Available objectives

This is an upright BX61 automated microscope which can run under Volocity (see end of page) or using Scanview, Applied Spectral Imaging's scanning platform for various fully automatic scanning applications, which can take 8 slides. The system is equipped with a 12 Bit camera, which functions in two separate modes - direct view (Normal image acquisition) and Fringe mode - which is the spectral mode. The camera has a VDS 2/3 inch CCD chip, is monochrome (B&W) and is not cooled. The pixel size is 6.7 micron (in calculation don't forget to multiple the magnification factor) and the resolution is 1280x1024 pixels.

Although this is a very advanced imaging system it is also excellent to take single images from a slide. It has a unique aquisition mode that allows to convert 3D stacks into 2D images using only in focus signal (see image C below).

overview_3D_cyto.jpg

Figure: a-Tubulin labeling in HeLa cells. A: 8 sections through a cell taken on the cytological imaging system. B: Maximum intensity projection of the images in A. C: accurate Z-stacking using the spectral imaging software to see all signals in focus.

Filters:

  • Narrow-band UV filter U-MNU2 (DAPI, Hoechst): exciter filter BP360-370, beam splitter DM400, barrier filter BA420.
  • Narrow-band Blue filter U-MNB2 (GFP, Alexa488, FITC, Cy2): exciter filter BP470-490, beam splitter DM500, barrier filter BA515 (long pass filter).
  • Narrow-band Green filter U-MNG2 (Alexa546, TRITC, Cy3): exciter filter BP530-550, beam splitter DM570, barrier filter BA590 (long pass filter).
  • Sky filterset from Applied Spectral Imaging.
  • AaronKees_0901.jpg

Figure: Analysis of number of nuclear bodies in HeLa cells using automatic spot counting. In screens the final results are numbers, not pictures

Software modules available:

A second computer is available to acquire images using Volocity and an Hamamatsu ORCA-R2 digital camera (12 bit, pixel size 6.45 x6.45 µm). On top of the microscope controller is a box called ‘MANHATTAN Data Transfer Switch’. This box has two settings:

  • A is for using the DELL computer together with the large spectral imager.
  • B is for using Volocity under Windows on the Mac with the Hamamatsu camera.

Please contact Dr Kees Straatman (krs5) when you encounter any problems.

References:
Novel Automated Three-Dimensional Genome Scanning Based on the Nuclear Architecture of Telomeres (2011) Klewes, L., Hösch, C., Katzir, N., Rourke, D., Garini, Y., and Mai S. Cytometry Part A, 79A: 159-166.

Three-dimensional nuclear telomere architecture is associated with differential time to progression and overall survival in Glioblastoma patients (2010) Gadji, M., Fortin, D., Tsanaclis, A-M., Garini, Y., Katzir, N., Wienburg, Y., Yan, J., Klewes, L., Klonisch, T., Drouin, R. and Mai, S. Neoplasia 12, 183–191.

Chromosomal location targets different MYC family gene members for oncogenic translocations (2009) Gostissa1, M., Ranganath1, S., Bianco, J.M. and Alt F.W. PNAS 106: 2265–2270.

Spectral karyotyping of human, mouse, rat and ape chromosomes – applications for genetic diagnostics and research (2006) Schrock, E., Zschieschang, P., O’Brien, P., Helmrich, A., Hardt, T., Matthaei, A and Stout-Weider, K. Cytogenet Genome Res 114:199–221.

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