Fluorescence scanning and FISH Annamaria Csizmadia, 3DHISTECH Ltd.
Fluorescence scanning and FISH
Annamaria Csizmadia, 3DHISTECH Ltd.
Fluorescence scan Fluorescence scanwith Confocal
MIDI
Confocal scan Semi-Confocalscan
Camera type PCO edge 4.2MPPCO edge 5.5MP
Point Grey 5MP
PCO edge 5.5 MPPCO edge 5.5 MP PCO edge 5.5 MP
Objective type20x;40x; 20x;40x; 20x;40x; 20x;40x;
Numeric Aperture 20x NA = 0,840x NA = 0,95
20x NA = 0,840x NA = 1,2
20x NA = 0,840x NA = 1,2
20x NA = 0,840x NA = 1,2
Immersion type NO 40x water40x water 40x water
Resolution 20x = 0,3µm/pixel40x = 0,16 µm/pixel
20x = 0,3µm/pixel40x = 0,16 µm/pixel
20x = 0,3µm/pixel40x = 0,16 µm/pixel
20x = 0,3µm/pixel40x = 0,16 µm/pixel
Magnification 20x = 30,740x = 61,53
------------------20x = 54,2
40x = 108,69
20x = 30,740x = 61,53
20x = 30,740x = 61,53
20x = 30,740x = 61,53
Spinning disc NO Not used YES YES
Differences between the fluorescence scanning modes
40x Zoom
Pannoramic MIDI 40x vs. PannoramicConfocal 40x
BPAE MIDI 40x FL scan BPAE Confocal 40x FL scan
Pannoramic MIDI 40x vs. Pannoramic Confocal 40x
10x Zoom
Pannoramic Confocal Normal FL 40x vs. Pannoramic Confocal 40x
Fluorescence scan with MIDI II
Fluorescence scan with Confocal MIDI Confocal scan
Fluorochrome list for FISH
Lumencore lamp- Single band, Multi band filter set
Sola lamp- Single band filter set
Fluorochrome Excitation EmissionLumencor excitation
wavelength
DAPI 358 405 386/23
Spectrum Aqua 438/24-25 483/32-25 438/24
FITC 490; 494 520;525 475/25
Spectrum Green 497/30;509/31 538/44;524/56 475/25
Cy3 514 566 550/88
Spectrum Gold 537 556 550/88
Spectrum Orange 543/22-25 586/20 550/88
Thric-Rhodamine 550 620 550/88
Rhodamine 550 573 550/88
Spectrum Red 587;587/35 612;612/51 550/88
Texas Red 595 620 550/88
Cy5 649 667 650/12
Different background color intensities between thequad band and the single band filters
Quad band filter FITC
Single band filter:Spectrum Green
Different background color intensities between thequad band and the single band filters
Quad band filter TRITC
Single band filter:Spectrum Orange
New functions of scanner software 2.0
• Take preview from all slides
• Pseudo colored, and zoomed preview
• Quick focusing with mouse in LIVE microscope mode
• New filter table
• More profiles to one slide
• Profile history tree
• Scanning history
• Separate scanning for TMA
• Live image Tools
Pannoramic Scanner Software 2.0
Brightfield preview from FISH tissue
New functions of scanner software 2.0
Pseudo color, and zoomed preview
Low resolution prescan previewHigh resolution prescan preview
New functions of scanner software 2.0
Scanning time of the whole slide was 5 minutes
Low resolution prescan
New functions of scanner software 2.0 High resolution prescan
Scanning time of the tissue was 2 minutes
High resolution prescan
Scanning time of the tissue was 2 minutes
High resolution prescan
Zoomed high resolution preview
High resolution prescan
Zoomed high resolution preview
New functions of scanner software 2.0
Live image tools:
- Convolution
- Inverter
- Histogram normalisation
- Overexposer
New functions of scanner software 2.0
Convolution
New functions of scanner software 2.0 Color inversion
Why is the best digital image quality important?
Difficulties of the quantification on digitalslide
• High background colour intensity
• Weak biological signals
• Different bleach effect on each field of view
• Stitching problems due to the lack of object
information
• Different thickness of the tissue section
• Different pre-analytic phase for the sample
fixation
Image Analysis
FISHQuant
Autofluorescence compensation
Scanner software options:
Flat field correction
Custom extended focus
Case Viewer options:
Enhance color settings
Autofluorescence filtering
Intensity statistics – pixel level image correction using compensation image calculated from image data
Intensity reducing – pixel-level analysis of the acquired image during which the background of the image reduced based on the comparison of the image
recorded by the camera and the compensation image.
Intensity equalization – pixel-level analysis of the acquired image, a one-by-one type image compensation method is applied.
Fluorescent flat field correction algorithms
Intensity statistic flat field correction
40.0x - Mouse Kidney Section
Intensity reducing flat field correction
FISH Her2/Cep17 DC Breast tissue
Intensity equalization flat field correction
S-CF-100062_BPAE Cells_40xFL_Slide_1_BPAE
How can we increase the image quality?
Custom Extended Focus
Background intensities
Background intensities
Intensity peaks of 2 FISH spots
Intensity peaks of 1 FISH spots
Spot intensity attenuation
Maxima – the final image is made up from all of the layers created, areas (pixel groups) with the highest
intensity combined together.
AVG – an average intensity of the layers is used for the final image.
Max-AVG – the final image is made up from all of the layers created, areas (pixel groups) with higher
intensity than the average combined together.
How can we increase the image quality?
Custom Extended Focus
Maxima extended focus mode
40x, Brain tissue section
Average extended focus mode
40.0x - Human Hepar (Liver) Section
Maxima- Average extended focus mode
40.0x - Rat Brain Section
CaseViewer Autofluorescence filtering
Fluorescence scan and slide management
Hardware configuration
Set the best scan profile:
- Exposition time
- Scan mode
- Flat field correction type
- Extended focus type
- Z stack scanning
Fluorescence Digital Slide
Clinical case: FISH digital slide
- Case Manager
- FISH quantification
Research case:
- Multiplex imaging
- Imaris
Case Manager
• Pathology information system
• Case overview
• Special staining order
• Forward case
• Consultation
• Signed case
• Label to Tumor board
Case Viewer 2.2
Slide rotation into the same position
Annotation handling in Case Viewer 2.2
Annotation handling in Case Viewer 2.2
Integrated FISHQuant application
The FISHQuant software solutionfor clinical demands
The FISHQuant software solutionfor clinical demands
Automatic colour matching
Load function of the previously adjusted parameters
Display of the probe genes with the labeled colour
Automatic spot threshold setting
Min. spot contrast parameter setting
Special parameter to define the fusion distance
Morphometrical and intensity parameters for the automatic nuclei segmentation
The FISHQuant software solutionfor clinical demands
The FISHQuant software solutionfor clinical demands
The FISHQuant software solutionfor clinical demands
The FISHQuant software solution forclinical demands
The FISHQuant software solution forclinical demands
Improvements in 2018
- New Pannoramic Scanner Software
- Microscope mode
- Live image plugins
- Planner mode for easy clinical use
- Prescan
- New case management
- Integrated FISH quantification into Case Viewer
- The FISH Quantification module is IVD approved application.
Thank you for your attention!