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!