Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning...
Transcript of Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning...
![Page 1: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/1.jpg)
Molecular Imaging and 3D Visualisation
Gergely KOVÁCH Ph.D.
Product Developer
2017
![Page 2: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/2.jpg)
Pannoramic Confocal Whole Slide Scanner
3 in One Solution:
• Brightfield
• Widefield Fluorescent
• Confocal Fluorescent
![Page 3: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/3.jpg)
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
![Page 4: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/4.jpg)
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)
![Page 5: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/5.jpg)
Light source
LED Lightsource: • Brightrer than Metal Halid • Up to 3 W green light power • 20 000 hours lifetime • > 10 μs switch time
![Page 6: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/6.jpg)
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
![Page 7: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/7.jpg)
Colocaized BF image
3 chip equivalent Brightfield illumination:
Color separation with dichroics
• Precise color reproduction • Colocalized imaging
![Page 8: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/8.jpg)
Aperture pattern
Illumination pattern
Aperture
Aperture correlation technique
Lens
Lens
![Page 9: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/9.jpg)
Aperture pattern
Illumination pattern
Aperture correlation technique
Aperture
Lens
Lens
![Page 10: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/10.jpg)
Aperture pattern
Illumination pattern
Aperture correlation technique
Aperture
Lens
Lens
![Page 11: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/11.jpg)
CF mode: structured illumination
Image1 =
Image2 =
+
+
![Page 12: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/12.jpg)
Confocal image
Image1 = Image2 = Confocal image =
Image1 – Image2
+ +
![Page 13: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/13.jpg)
Optical path
![Page 14: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/14.jpg)
Structured Illumination Disc
• Important,
• this is not Nipkow Disk!!!
![Page 15: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/15.jpg)
Hi-speed filterwheel mechanism
Switching time: ~60ms for neighboring, ~100ms for opposite filtercube
![Page 16: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/16.jpg)
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
![Page 17: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/17.jpg)
Conventional vs Confocal imaging
Pannoramic Confocal Zeiss AxioImager
Same part of the tissue stained with Dapi, Fitc and Tritc
![Page 18: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/18.jpg)
Application: Neurobiology
Rat brain in FL mode Rat brain in CF mode
![Page 19: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/19.jpg)
![Page 20: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/20.jpg)
„Offline” investigation of slides
![Page 21: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/21.jpg)
Application: Cancer research
Colon tissue with 3 labelling: Green (SFRP1), Red (β-actin), White(mRNS)
![Page 22: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/22.jpg)
Whole Slide Confocal IHC
![Page 23: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/23.jpg)
Application: Whole Cell FISH
Conventional FL Confocal FL
![Page 24: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/24.jpg)
Visualisation of FL labelled spots
FL image
CF image
3D image
![Page 25: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/25.jpg)
Application: Flourescent Resonant Energy Transfer
animation by - Dr Othon Gervasio
![Page 26: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/26.jpg)
FRET image by Pannoramic Confocal
FRET: 536nm donor, 593nm akceptor
![Page 27: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/27.jpg)
3D reconstruction and animation
![Page 28: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/28.jpg)
3D reconstruction
![Page 29: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/29.jpg)
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
![Page 30: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/30.jpg)
Thank you for your attention!
http://www.3dhistech.com
![Page 31: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/31.jpg)
Part 2 – Choosing correct filtersets
• Single vs multiband filtersets
• 3DHISTECH`s dualband filtersets
• Relation with channel peak
• Optimization to camera characteristic
![Page 32: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/32.jpg)
Multi- vs Single band
![Page 33: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/33.jpg)
Lumencor light source
![Page 34: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/34.jpg)
Optimized TRITC
![Page 35: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/35.jpg)
Optimized FITC
![Page 36: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/36.jpg)
Overlap effect
![Page 37: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/37.jpg)
Solution: non-neighbor dual filters
![Page 38: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/38.jpg)
P.C.O. characteristic
![Page 39: Molecular Imaging and 3D Visualisation€¦ · • Widefield Fluorescent ... Laser scanning Confocal Spinning disc Pannoramic Confocal Scanning Speed Slow, 2-3 sec / image Very fast](https://reader034.fdocuments.in/reader034/viewer/2022042912/5f454bb5432e657a96025b6e/html5/thumbnails/39.jpg)
Conclusion
Main filter design aspects:
- Fluorophore combination
- Lumencor spectral distribution
- Camera characteristic