Molecular Imaging and 3D Visualisation

Gergely KOVÁCH Ph.D.

Product Developer

2017

Pannoramic Confocal Whole Slide Scanner

3 in One Solution:

• Brightfield

• Widefield Fluorescent

• Confocal Fluorescent

Automated scanning technology

Automated 12 slide loader

Advanced tissue detection:

- Darkfield

- Fast Fluorescent

- High resolution Fluorescent

Tissue scanning:

- Automatic focus map

- Automatic exposure time

- High speed area scan

Low and High resolution scanning modes

Optical Resolutionx,y or d0 = 1.22λ / [NAObj + NACon]

XY resolution = 300 nm Scanning time = 3 min (15mm x 15mm area)

XY resolution = 200 nm Confocality = 1 um

Scanning time = 6 min (1 cm2 / layer / channel)

Light source

LED Lightsource: • Brightrer than Metal Halid • Up to 3 W green light power • 20 000 hours lifetime • > 10 μs switch time

Signal detection

5,5 Mp sCMOS camera 1 electron noise, peltier cooling 300000:1 dinamic range > 60% quantum efficiency 16 bit dept 6,5 um * 6,5 um pixel size 100 frame/sec

Colocaized BF image

3 chip equivalent Brightfield illumination:

Color separation with dichroics

• Precise color reproduction • Colocalized imaging

Aperture pattern

Illumination pattern

Aperture

Aperture correlation technique

Lens

Lens

Aperture pattern

Illumination pattern

Aperture correlation technique

Aperture

Lens

Lens

Aperture pattern

Illumination pattern

Aperture correlation technique

Aperture

Lens

Lens

CF mode: structured illumination

Image1 =

Image2 =

+

+

Confocal image

Image1 = Image2 = Confocal image =

Image1 – Image2

+ +

Optical path

Structured Illumination Disc

• Important,

• this is not Nipkow Disk!!!

Hi-speed filterwheel mechanism

Switching time: ~60ms for neighboring, ~100ms for opposite filtercube

Application Images Pros

Cancer research Fast and whole slide scanning

Immunhistology Thick samples, Extended focus function

Neuro Science Large structure tracking

FISH microscopy Better resolution and spot detection

FRET Fine intensity change can be tracked

Pathology Uncutted cells (e.g. 15 um) can be investigated

Developing Biology Zebrafish, Drosop

Applications of Pannoramic confocal

Conventional vs Confocal imaging

Pannoramic Confocal Zeiss AxioImager

Same part of the tissue stained with Dapi, Fitc and Tritc

Application: Neurobiology

Rat brain in FL mode Rat brain in CF mode

„Offline” investigation of slides

Application: Cancer research

Colon tissue with 3 labelling: Green (SFRP1), Red (β-actin), White(mRNS)

Whole Slide Confocal IHC

Application: Whole Cell FISH

Conventional FL Confocal FL

Visualisation of FL labelled spots

FL image

CF image

3D image

Application: Flourescent Resonant Energy Transfer

animation by - Dr Othon Gervasio

FRET image by Pannoramic Confocal

FRET: 536nm donor, 593nm akceptor

3D reconstruction and animation

3D reconstruction

Competitive technologies

Laser scanning Confocal Spinning disc Pannoramic Confocal

Scanning Speed Slow, 2-3 sec / image Very fast (1000 point paralell)

Optimized for best signal/speed ratio

Bleaching and Phototoxicity

High, 1 point illumination Low Phototoxicity Lowest Phototoxicity

Magnification 5x, 10x, 20x, 40x, 63x, 100x 5x, 10x, 20x, 40x, 63x, 100x Specified by display size, resolution equals to 63x

Light source Laser, expensive Laser, expensive

Solid state LED lightsource lifetime >20000 hours

Light efficiency 99%, incoming light, 2-4% emitted light

70 % incoming light 4-5 % emitted light

50%, incoming light 99% emitted light

Pinhole flexibility

Continously adjustable No, fix pinhole (50um) 3 different grids, for 20x, 40x and 63x objectives

Flexibility Every microscopic options are available

Similar to a microscope, Hi framerate for live imaging

Colocalized BF, FL and CF imaging

Thank you for your attention!

http://www.3dhistech.com

Part 2 – Choosing correct filtersets

• Single vs multiband filtersets

• 3DHISTECH`s dualband filtersets

• Relation with channel peak

• Optimization to camera characteristic

Multi- vs Single band

Lumencor light source

Optimized TRITC

Optimized FITC

Overlap effect

Solution: non-neighbor dual filters

P.C.O. characteristic

Conclusion

Main filter design aspects:

- Fluorophore combination

- Lumencor spectral distribution

- Camera characteristic