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 <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.
MQDS
WSI
CD31CD34aSMA
H&E
Microscope
•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
WSI
•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
Future Plan
•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
MIDI and system
Consultant
Demetris Lab
RHS Lab
Dr. Demetris
John Lunz III
Susan Specht
Yoshiaki Mizuguchi
Natasha Corbitt
Enrico Pegolo
Lisa Chedwick
Andrew Lesniak
Lori Perez
Trevor Benyack
Eleck Walton
Traci Ondik
Rensselaer
Polytechnic Institute
Roysam Lab
Dr. Badri Roysam
Kedar Grama
ISH Lab
Kathy Cieply