View PPT slides - Digital Pathology Association

January 31, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
Share Embed Donate


Short Description

Download View PPT slides - Digital Pathology Association...

Description

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

What is Quantum Dots (Qdots)?

Traditional Fluorescence Markers vs Qdots (1)

Traditional Fluorescence Markers vs Qdots (2)

Rhodamine

Rhodamine

3min

Qdot Qdot

1H

Adapted from 25 SEPTEMBER 1998 VOL 281 SCIENCE

Adapted from Invitrogen.com

Qdots

•No photo-bleaching •Wide Stokes’ shift •Narrow emission spectra •Permanent •Multiple staining

•Expensive •Special filter

Multiple Staining 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

Tissue Staining, not Flow Cytometry • 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.)

Protocol for Multiplex Quantum Dot Immunostaining (MQDS) •Antigen retrieval •Avidin Block •Biotin Block •Non Serum Protein Block •1st Primary Antibody •1st Biotinylated Secondary Antibody •1st Streptavidin Qdot

•Avidin Block •Biotin Block •Non Serum Protein Block •2nd Primary Antibody •2nd Biotinylated Secondary Antibody •2nd Streptavidin Qdot •Avidin Block •Biotin Block •Non Serum Protein Block •3rd Primary Antibody •3rd Biotinylated Secondary Antibody •3rd Streptavidin Qdot •Repeat for additional stainings



Requires the best antigen retrieval for all antibodies that you are going to use

• Before starting the panel staining, titration of antibodies will be done by immunohistochemistry to decide the staining order • Extra Blocking for each segment

• Amplify Signals by 2-step immunohistochemical staining

Comparison of 2-Step and 3-Step IHC 2-Step CD3 Ab + Anti-rabbit Qdot

3-Step CD3 Ab + Biotynilated anti-rabbit + streptavidin Qdot

420nm

Nuance Pseudocolor Image

Captured Image

Subtract AF by program

Unmixed Grayscale Individual Images

420nm

unmix

720nm

720nm

Autofluorescence (AF) in Different Tissue

Same AF pattern in different tissues

Skeletal Muscle 2.36

Liver 1.84

Heart 1.51

Small Intestine 1.38

Frozen Liver 1.21

Colon 1.03

Lung 1.0

LCACD68CD3CD4CD8

LCA

+ CD68

=

+ CD4

CD68CD3

CD3

= CD8

CD4CD8

Disadvantage of Traditional Microscopes

Fluorescent

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

What is Whole Slide Imaging (WSI)?

Zeiss/3D Histech scanner

Inside of the scanner • 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

Qdot Filter

x40

Digital x20

Digital x10

Digital x100

12bit vs 8bit

Advantage of WSI •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

Digitally Preserving and Sharing the World’s Cultural Heritage

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

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

Microscope vs WSI

%CD31 signal 35

30 25

Microscope x4

Microscope x20

20 15 10 5 0

WSI Digital x4 CD31

CD31 in x4

CD31 in x4

CD31 in x20

CD31 in x20

Nuance

Mirax

Nuance

Mirax

Adapted from Zeiss From The Very Beginning” WIS“Microscopy Digital x20

Disadvantage of WSI Unfocused Area because of hardware  Multiple focus points  Dark field condenser

Limited Abs numbers because of AF and DAPI  Digitally subtract AF

Shifting Problem  Mechanical adjustment  Layer adjustment in the software DAPI

DAPI + Q705

FarSight__ Developed by Dr. Badri Roysam

http://www.farsight-toolkit.org/wiki/Main_Page

FarSight__Nucleus Editor

HLADRIL10TGFbDAPI

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

Problem of FarSight or Human??

N=3

N=8

N=8

N=8

X40 magnification, unknown field size

N=4

N=10

N=10

N=10

x50 magnification, 230x350μm2

Vδ1+CD3+ Vδ2+CD3+ Vδ1+2+CD3+

Combitnation of H&E and MQDS

H&E  Qdot multiple staining H&E after Qdot multiple staining

Fluorescence signal

Eosin emission spectrum on the top of Qdots •Eosin has wide spectrum ( - - - - - - - Eosin) •Eosin is strong Acid •Hematoxylin is strong Base

pH Ranges for Qdot® Nanocrystals

pH

Recommendations

>9

Not recommmended- Qdot® nanocrystals start to self-aggregate/clump. (Qdot® nanocrystals are not degraded by basic pH. )

>6 to 5 to
View more...

Comments

Copyright � 2017 NANOPDF Inc.
SUPPORT NANOPDF