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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 TransplantationThomas E. Starzl Transplantation InstituteUniversity of Pittsburgh Medical Center
What is Quantum Dots (Qdots)?
Traditional Fluorescence Markers vs Qdots (1)
Adapted from 25 SEPTEMBER 1998 VOL 281 SCIENCE Adapted from Invitrogen.com
Rhodamine
Qdot
Qdot
Rhodamine
1H
3min
Traditional Fluorescence Markers vs Qdots (2)
• No photo-bleaching• Wide Stokes’ shift•Narrow emission spectra •Permanent•Multiple staining
• Expensive• Special filter
Qdots
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
• 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
• Avidin Block• Biotin Block• Non Serum Protein Block• 1st Primary Antibody• 1st Biotinylated Secondary Antibody• 1st Streptavidin Qdot
• Extra Blocking for each segment
• Amplify Signals by 2-step immunohistochemical staining
• 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
• 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)
CD3 Ab + Biotynilated anti-rabbit + streptavidin Qdot CD3 Ab + Anti-rabbit Qdot
Comparison of 2-Step and 3-Step IHC
2-Step 3-Step
Nuance420nm
720nm
unmix
420nm 720nm
Captured Image Pseudocolor Image
Unmixed Grayscale Individual Images
Subtract AF by program
Skeletal Muscle 2.36 Liver 1.84
Small Intestine 1.38Heart 1.51
Frozen Liver 1.21 Colon 1.03
Lung 1.0
Same AF pattern in different tissues
Autofluorescence (AF) in Different Tissue
+ =
+ =
LCA
CD68 CD3 CD68CD3
CD4 CD8 CD4CD8
LCACD68CD3CD4CD8
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
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 scannerZeiss/3D Histech scanner
What is Whole Slide Imaging (WSI)?
x40
Digital x20
Digital x10
Digital x100
12bit vs 8bit
•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
Digitally Preserving and Sharing the World’s Cultural Heritage
WSI : HLADRCK19CD31 (3D HISTECH/ CRi Pannoramic Viewer)
5 fields from liver, all portal tracts in the biopsy using three different antibodies + DAPI = 4 colorsTotal 19 cases------over 400 images
Data Obtained From WSI
CD31 in x4 CD31 in x4 CD31 in x20 CD31 in x20Nuance Mirax Nuance Mirax
0
5
10
15
20
25
30
%CD31 signal
Microscope x4 Microscope x20
WSI Digital x4 WIS Digital x20
CD31
Adapted from Zeiss “Microscopy From The Very Beginning”
Microscope vs WSI
Unfocused Areabecause of hardware
Shifting Problem Mechanical adjustment Layer adjustment in the software
Digitally subtract AF
Multiple focus points Dark field condenser
DAPI DAPI + Q705
Disadvantage of WSI
Limited Abs numbers because of AF and DAPI
http://www.farsight-toolkit.org/wiki/Main_Page
FarSight__ Developed by Dr. Badri Roysam
FarSight__Nucleus Editor HLADRIL10TGFbDAPI
HLADR expression and HLADR +TGFβ+/- cell numbers
Data Obtained From FarSight
N=3 N=8 N=8 N=4 N=10 N=10
X40 magnification, unknown field size x50 magnification, 230x350μm2
Vδ1+CD3+Vδ2+CD3+Vδ1+2+CD3+
N=8 N=10
Problem of FarSight or Human??
H&E after Qdot multiple staining Fluorescence signal
H&E Qdot multiple staining
Combitnation of H&E and MQDS
• 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
pH Recommendations >9 Not recommmended- Qdot® nanocrystals start to self-aggregate/clump.
(Qdot® nanocrystals are not degraded by basic pH. )
>6 to <9 Qdot® nanocrystals most optimal stability in this pH range.
>5 to <6 Marginal stability is shown in this range
<4 Not recommended- The polymer will dissociate; exposed core/shell will start to dissociate.
CD31CD34aSMA
MQDS H&EWSI
• 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
Microscope WSI
• 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
Thank you!Rensselaer Polytechnic InstituteRoysam Lab
Dr. Badri RoysamKedar Grama
Demetris Lab
Dr. DemetrisJohn Lunz IIISusan SpechtYoshiaki MizuguchiNatasha CorbittEnrico Pegolo
MIDI and system Consultant
Andrew Lesniak
RHS Lab
Lisa ChedwickLori PerezTrevor BenyackEleck WaltonTraci Ondik
ISH Lab
Kathy Cieply