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Comparison of High Resolution Whole Slide Imaging (WSI) vs Conventional Fluorescence Microscopy for Viewing and Analyzing Multiplex Quantum Dot Immunostained.

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Presentation on theme: "Comparison of High Resolution Whole Slide Imaging (WSI) vs Conventional Fluorescence Microscopy for Viewing and Analyzing Multiplex Quantum Dot Immunostained."— Presentation transcript:

1 Comparison of High Resolution Whole Slide Imaging (WSI) vs Conventional Fluorescence Microscopy for Viewing and Analyzing Multiplex Quantum Dot Immunostained (MQDS) Microscopic Slides No more lights off in the lab! No more microscope! Kumiko Isse, M.D., Ph.D. Demetris Lab Department of Pathology, Division of Transplantation Thomas E. Starzl Transplantation Institute University of Pittsburgh Medical Center

2 What is Quantum Dots (Qdots)?

3 Traditional Fluorescence Markers vs Qdots (1)

4 Adapted from 25 SEPTEMBER 1998 VOL 281 SCIENCE Adapted from Invitrogen.com Rhodamine Qdot Rhodamine 1H 3min Traditional Fluorescence Markers vs Qdots (2)

5 No photo-bleaching Wide Stokes’ shift Narrow emission spectra Permanent Multiple staining Expensive Special filter Qdots

6 Enable pathologists to contribute to the molecular revolution in medicine by merging traditional morphologic examination with multiple markers to precisely characterize specific cell types and investigate intra-cellular signaling pathways Time consuming Complicated protocol Multiple Staining

7 Panoramic overview of tissue at low magnification Distribution, localization and cell-cell interactions visible Can unlock decades of human biology/pathology from paraffin blocks Connect to conventional morphology (H&E) Immediate sample collection and triage Complicated to analyze Artifacts (wrinkles, bubbles, dust, scratches, etc.) Tissue Staining, not Flow Cytometry

8 Avidin Block Biotin Block Non Serum Protein Block 1 st Primary Antibody 1 st Biotinylated Secondary Antibody 1 st Streptavidin Qdot Extra Blocking for each segment Amplify Signals by 2-step immunohistochemical staining Avidin Block Biotin Block Non Serum Protein Block 2 nd Primary Antibody 2 nd Biotinylated Secondary Antibody 2 nd Streptavidin Qdot Avidin Block Biotin Block Non Serum Protein Block 3 rd Primary Antibody 3 rd Biotinylated Secondary Antibody 3 rd Streptavidin Qdot Repeat for additional stainings Before starting the panel staining, titration of antibodies will be done by immunohistochemistry to decide the staining order Requires the best antigen retrieval for all antibodies that you are going to use Antigen retrieval Protocol for Multiplex Quantum Dot Immunostaining (MQDS)

9 CD3 Ab + Biotynilated anti-rabbit + streptavidin Qdot CD3 Ab + Anti-rabbit Qdot Comparison of 2-Step and 3-Step IHC 2-Step3-Step

10 Nuance 420nm 720nm unmix 420nm 720nm Captured Image Pseudocolor Image Unmixed Grayscale Individual Images Subtract AF by program

11 Skeletal Muscle 2.36 Liver 1.84 Small Intestine 1.38 Heart 1.51 Frozen Liver 1.21Colon 1.03 Lung 1.0 Same AF pattern in different tissues Autofluorescence (AF) in Different Tissue

12 + = + = LCA CD68 CD3 CD68CD3 CD4 CD8 CD4CD8 LCACD68CD3CD4CD8

13 Physical problems: Unpleasant usually isolated environment Limited availability = Data problems: Multiple layered image with lower opacity is unclear Individual colors need to be saved separately Mechanical problems: Repeated training often needed Precise adjustment of settings needed for good quality images Suboptimal at low magnification Disadvantage of Traditional Fluorescent Microscopes

14 Qdot Filter Total 12 slides are scanned in one time 10-20min by 20x lens, 20-40min by 40x lens for biopsy size tissue 2.2GB with 80% compression of JPG Using filters specific for Qdots Inside of the scanner Zeiss/3D Histech scanner What is Whole Slide Imaging (WSI)?

15 x40 Digital x20 Digital x10 Digital x100

16 12bit vs 8bit

17 Permanent data Share the same slide with many people at once Observe anytime, anywhere, portable. No need to reserve microscope Easy surveillance and analysis Preservation of context and detailed morphological information Saves space in your lab Large data Mechanical problems Requires lot of adjustment Cost Advantage of WSI

18

19 WSI : HLADRCK19CD31 (3D HISTECH/ CRi Pannoramic Viewer)

20 5 fields from liver, all portal tracts in the biopsy using three different antibodies + DAPI = 4 colors Total 19 cases over 400 images Data Obtained From WSI

21 Microscope x4Microscope x20 WSI Digital x4 WIS Digital x20 CD31 Adapted from Zeiss “Microscopy From The Very Beginning” Microscope vs WSI

22 Unfocused Area because of hardware Shifting Problem  Mechanical adjustment  Layer adjustment in the software  Digitally subtract AF  Multiple focus points  Dark field condenser DAPIDAPI + Q705 Disadvantage of WSI Limited Abs numbers because of AF and DAPI

23 FarSight __ Developed by Dr. Badri Roysam

24 FarSight__Nucleus Editor HLADRIL10TGFbDAPI

25 HLADR expression and HLADR +TGFβ+/- cell numbers Data Obtained From FarSight

26 N=3N=8 N=4N=10 X40 magnification, unknown field sizex50 magnification, 230x350μm 2 Vδ1+CD3+ Vδ2+CD3+ Vδ1+2+CD3+ N=8N=10 Problem of FarSight or Human??

27 H&E after Qdot multiple stainingFluorescence signal H&E  Qdot multiple staining Combitnation of H&E and MQDS

28 Eosin has wide spectrum ( Eosin) Eosin is strong Acid Hematoxylin is strong Base Eosin emission spectrum on the top of Qdots pH Ranges for Qdot® Nanocrystals pHRecommendations >9Not recommmended- Qdot® nanocrystals start to self-aggregate/clump. (Qdot® nanocrystals are not degraded by basic pH. ) >6 to <9Qdot® nanocrystals most optimal stability in this pH range. >5 to <6Marginal stability is shown in this range <4Not recommended- The polymer will dissociate; exposed core/shell will start to dissociate.

29 CD31CD34aSMA MQDS H&E WSI

30 Conventional Method Bottleneck because of limited resource (location and time) Microscope often difficult to use Not portable Limited triage to automated image analysis Looking at a tree and not the forest Easy to use, portable Stronger signal at lower magnification Combination of FL and Bright Field Direct connection to automated image analysis Unfocused areas Autofluorescence Adjustments needed MicroscopeWSI

31 Analyze whole slide image Automated whole slide image analysis using a selected region of interest (ROI) Better performance Hardware – computer, scanner, filter Software – imaging, analysis Future Plan

32 Rensselaer Polytechnic Institute Roysam Lab Dr. Badri Roysam Kedar Grama Demetris Lab Dr. Demetris John Lunz III Susan Specht Yoshiaki Mizuguchi Natasha Corbitt Enrico Pegolo MIDI and system Consultant Andrew Lesniak RHS Lab Lisa Chedwick Lori Perez Trevor Benyack Eleck Walton Traci Ondik ISH Lab Kathy Cieply


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