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