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Zeiss Axioscan‐Slidescanner

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Rob Campbell edited this page Dec 17, 2024 · 9 revisions

System Specs

  • Fully automated for high throughput
  • Z-drive with 3mm usable focus range
  • "Magazine" for 100 slides (size 26x76mm)
  • Precision scanning stage 300x100mm
  • Light source for bright field: Vis-LED
  • Light source for fluorescence: HXP 120 V (D) for fixed samples

Important Notices

  • All old scan profiles are in: C:\OLD_SCAN_PROFILES
  • You can populate your own scan profile directory by copying things into: C:\Scan Profiles
  • Shut down the microscope after use.
  • Power off the slide scanner itself using the button
  • Shut down the PC
  • The power to the camera is not controlled by the button on the AxioScan, so you need to turn off the power at the wall.

When loading slides in a holder please make sure that:

  • The cover-slip is hanging over the edge of the slide.
  • The corners of your microscope slide are intact
  • The slide scanner uses air objectives: make sure slides are clean and not covered in oil
  • Do not cover (e.g. with tape) the small round hole in the slide holder.
  • During loading if the microscope lights flashes red (indicating an error), DO NOT shut down the machine since the internal robotic arm might crash into a slide.

Usage Hints

  • It is possible to defining slide names and even profiles associated with slides by importing data from an Excel or .csv file.
  • Profiles that lead to very long acquisition times are probably doing so because of focus map parameters that are excessively thorough.
  • Saving the data using Zen's lossy compression (jpeg xr) at 85% has been shown to produce results almost indistinguishable from lossless compression.
  • Avoid auto-exposure with fluorescence imaging, it tends to produce odd results and makes quantification impossible.

General Analysis Options

In addition to the usual suspects, such as Fiji and Icy, you should be aware of:

Options for registering slices to the atlas

There are many options, some of which are summarised here.

Cameras

The system contains two high-end imaging cameras and one low-quality overview camera for taking images of the whole slide.

Bright-Field RGB Camera

  • Model: 3CCD Hitachi HV-F202SCL for Axio Scan.Z1 (O)
  • Effective number of pixels: 1600 x 1200
  • Pixel size: 4.4 μm x 4.4 μm
  • Effective Area: 7.04 mm x 5.28 mm
  • Max frame rate: 30 FPS RGB
  • Dynamic range: 12 bit
  • Sensitivity: 2000 Lux/F5.6

Monochrome Fluorescence Camera

  • Model: Orca Flash 4.0 V2 for Axio Scan.Z1 (O)
  • Effective number of pixels: 2048 x 2048
  • Pixel size: 6.5 μm x 6.5 μm
  • Effective Area: 13.31 mm x 13.31 mm
  • Spectral range: appr. 250 - 1000 nm
  • Full frame rate: 100 FPS
  • Dynamic range: 16 bit
  • Quantum efficiency (at 600 nm): > 70%
  • Noise at min EM gain: 1.6 electrons rms

Objectives

Name Mag. / NA Rayleigh resolution at 550 nm (µm) Microns/pixel FOV (mm) Medium Working distance
Fluar 5x / 0.25 1.34 1.3 1 mm air 12.5 mm
Plan-Apochromat 10x / 0.45 0.61 0.75 0.5 mm air 2.1 mm
Plan-Apochromat 20x / 0.8 0.42 0.325 0.25 mm air 0.55 mm

Note the system is operating below Nyquist. i.e. the number of microns per pixel is not less than half the Rayleigh resolution value. This is unlikely to present a problem, given the typical use cases of the system.

Filtersets

Fluorochrome Availability Excitation Dichroic Emission Filter set
DAPI Yes 387/11 409 447/60 49 DAPI shift free (E)
eGFP Yes 470/40 495 520 / 50 38 HE GFP shift free (E)
mRFP Yes 572/25 590 629/62 63 HE mRFP shift free (E)
Cy5 Yes 640/30 660 690/50 50 Cy5 shift free (E)

So if you want quick overview scans of the "where is my fluorescence" sort you should use the 5x with 4x4 binning. That will give 3.5 microns per pixel. For counting somata the 10x with 4x4 binning will work. The 20x will provide brighter images at the cost of longer acquisition times.

Other

  • Serial number: 4631000402
  • Bulbs: HXP R 120W/45C VIS

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