TissueFAXS SL Fluorescent Slide Scanner

Specifications

  • TissueGnostics scanner with 120 slide loader and Zeiss upright microscope
  • Fast scanning of whole slides
  • Air objectives: 2.5x/0.09, 5x/0.16, 10x/0.45, 20x/0.5, 20x/0.8, 40x*/0.95
  • Oil objectives available upon request: 40x/1.3, 60x/1.4
  • Up to 7 fluorescent tags, including near IR (Cy7)
  • Sensitive back-thinned sCMOS monochrome camera (Hamamatsu ORCA-Fusion BT C15440)
  • Paired with StrataQuest workstation (Marlin) for whole-slide analysis

*Please use #1.5 coverslips with the the 40x/0.95 air lens

Sample Preparation

  • Scan area is limited to 50mm from clear edge.

  • DO NOT crowd  sections together. Spread them out for automated tissue detection.

  • DO NOT use antifade/mounting media with built-in DAPI*. Buy mountant without DAPI and do a separate DAPI stain and wash!!

  • DO NOT freeze your stained slides. Re-freezing slides will destroy your samples. Once stained, store them at 4°C.

  • DO NOT use the following flow-cytometry dyes. Detection (flow) and imaging (microscopy) have different requirements. Some flow dyes and reagents do not work for imaging – especially with multi-band filters. Choose alternatives to these broad-spectrum and other problematic dyes

    • ❌❌ PE (Phycoerythrin)
    • ❌❌ PerCP
    • ❌ APC (Allophycocyanin) - Cy5 or AF647 are preferred
    • ❌❌ Quantum Dots
    • ❌❌ Brilliant UV/Violet dyes other than BV421
    • ❌❌ Primary labeled antibodies in the green channel (FITC, AF488, etc)**

  • DO use one set of fluorescent dyes per slide. Camera/filter scanning settings apply per-slide.

  • DO check the filter list below for recommended fluorescent dyes

  • DO use a #1.5 coverslip over your sample

  • DO use an antifade mounting media (but one without built-in DAPI*)

  • Blank magazine layout templates are available in WORD and PDF formats

*Antifade with built-in DAPI is meant for high-volume clinics. Never buy it for research. The DAPI goes bad very quickly, diffuses into tissues poorly, and always leaves a background signal. Instead, do a separate DAPI stain and wash out the excess before mounting. Why spend hours, days, or months on an experiment and ruin it at the last minute with a poor reagent when a 5-15 minute DAPI stain will work much, much better!

**Tissue autofluorescence is strongest in the green channel. It can overwhelm weak primary-labeled antibody signals when imaging. Instead use a polyclonal fluorescent secondary antibody to amplify the signal. There is a similar but weaker autofluorescence in the orange channels (TRITC, AF548). Consider the no-labeled-primary rule for this color too. Remember, flow cytometry and microscopy are different beasts! What works for one doesn't always work for the other.

Two Scanning Software Configurations

AllBand (slow scanning) and QuadBand (fast scanning)

AllBand Fluorescent Filter Configuration

Slower scanning using partially separated filters, filters can be mixed-and-matched

Chroma single band DAPI, low bleed-through, better nuclear separation

Name # Std dyes Ex Em
DAPI 5 DAPI/Hoechst 390 460

Chroma wide-band, bright, up to 5 colors

Name #* Std dyes Cy series Alexa Ex Em
mDAPI 6 DAPI/Hoechst/BV421     390 435
mFITC 6 FITC Cy2 AF488 479 519
mTRITC** 6 TRITC Cy3 AF546 554 594
mTxRed** 7 RFP/TxRed Cy3.5 AF594/647 578 641
mCy5 6 APC Cy5/Cy5.5 AF647 637 695
mCy7 7 Cy7 Cy7 AF750 748 810

*# indicates multiband filter cube position.  Fluorophores on different cubes have less cross-talk
**These fluorophores are mutually exclusive on the Chroma filters. Use Spectra-Split filters below to image both in the same sample

Spectra-Split narrow-band, better separation, up to 7 colors

Name #* Std dyes Cy series Alexa Opal Atto Dylight Ex Em
ss380 1 DAPI/Hoechst/BV421           390 424
ss440 4 BV480     Opal p 480 Atto425   435 469
ss488 1 FITC Cy2 AF488 Opal 520 Atto488 Dylight488 491 525
ss546 2 TRITC Cy3 AF546 Opal 570 Atto542 Dylight549 546 569
ss594 3 TxRed   AF594 Opal 620 Atto590 Dylight594 594 624
ss647 4 APC Cy5/Cy5.5 AF647 Opal 690 Atto647 Dylight647 637 686
ss750 1   Cy7 AF750 Opal p.780 Atto740 Dylight750 748 799

*# indicates multiband filter cube position.  Fluorophores on different cubes will have less cross-talk

QuadBand Fluorescent Filter Configuration

Very fast scanning with Chroma multi-band filter, up to 4 colors without filter change delays

Name # Std dyes Cy series Alexa Ex Em
mDAPI 6 DAPI/Hoechst/BV421     390 435
mFITC 6 FITC Cy2 AF488 479 519
mTRITC 6 TRITC/TxRed Cy3 AF546/594 554 594
mCy5 6 APC Cy5/Cy5.5 AF647 637 695

Objectives and Extended Depth of Focus

Recommended Z-STEP sizes for extended depth-of-focus imaging

Objective OIL Z STEP Corr WD mm Z DOF µm
2.5x/0.085     EC Plan-Neofluar 8.80 106.71
5x/0.16     EC Plan-Neofluar 18.50 29.61
10x/0.45   4.0 µm Plan Apochromat 2.00 4.16
20x/0.5   2.5 µm EC Plan-Neofluar 2.00 2.85
20x/0.8   1.0 µm Plan Apochromat 0.55 1.27
40x/0.95*   0.8 µm Plan-Apochromat Korr 0.25 0.78
40x/1.3** OIL 0.6 µm EC Plan-Neofluar 0.21 0.68
63x/1.4** OIL 0.5 µm Plan Apochromat 0.19 0.54

*The 40x/0.95 air objective requires additional training
**Limited availability. See staff for OIL objective use

Data Files and Software

Acquired Data Files

  • The current acquisition version (7.xxx) uses a proprietary format, but with a free viewer for export to ome-tiff
  • The next acquisition version (8.xxx) will be store natively in ome-tiff
  • Acquired slide files include the small index file and ALL the subdirectories below them.
    • Jobs are indexed by "jobname.bfproj" and are a collection of 1 slide projects
    • Slides (1 slide projects) are indexed by "slidename.aqproj"
  • Keep a "golden master" of your data as it was acquired, not just the exported files.
    • Make sure to back up everything, not just the tiny .bfproj and .aqproj files.

Viewing/Export Software

  • TissueFAXS Viewer software is available for download here [StrataQuest link].
    • Make sure to download the version for "Systems with Slideloader".
    • It should have "SL" in the name
    • The viewer only runs in Microsoft Windows
    • The viewer can export the stitched image into ome-tiff format
    • The viewer also includes a command-line batch exporter