robert dunstan, luke jandreski and the comparative pathology laboratory
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Robert Dunstan, Luke Jandreski and the Comparative Pathology Laboratory. Fluorescence for fluorophobes--Virtual fluorescence using chromagens and histochemical stains. Lecture outline. 1.Introduction 2.Using “non-fluorophores” for fluorescence Histochemical stains - PowerPoint PPT PresentationTRANSCRIPT
Robert Dunstan, Luke Jandreski and the Robert Dunstan, Luke Jandreski and the Comparative Pathology LaboratoryComparative Pathology Laboratory
Robert Dunstan, Luke Jandreski and the Robert Dunstan, Luke Jandreski and the Comparative Pathology LaboratoryComparative Pathology Laboratory
Fluorescence for fluorophobes--Virtual fluorescence using chromagens and histochemical stains
Fluorescence for fluorophobes--Virtual fluorescence using chromagens and histochemical stains
2 November 13, 2003
Lecture outlineLecture outline
1. Introduction
2. Using “non-fluorophores” for fluorescence
• Histochemical stains
• Use of red IHC chromagens for fluorescence
3. Use of registration/co-registration
4. Conclusions
3 November 13, 2003
Vision for virtual imaging and analysisVision for virtual imaging and analysis
“Increase signal, decrease noise with consistency”
4 November 13, 2003
Brightfield
Pro--Morphologic assess- ment
Con--Lower signal:noise--Non-linear expression of chromagens--Colocalization difficult
Fluorescence
Pro--High signal to noise--Linear expression of fluorophores--Colocalization easy
Con--Morphologic assessment difficult
Vision for virtual imaging and analysisVision for virtual imaging and analysis
5 November 13, 2003
Brightfield
Pro--Morphologic assess- ment
Con--Lower signal:noise--Non-linear expression of chromagens--Colocalization difficult
Fluorescence
Pro--High signal to noise--Linear expression of chromagens--Colocalization
Con--Morphologic assessment difficult
--Virtual microscopy
--“Non-fluoro-phores” for fluorescence
--Co-registration of images
Vision for virtual imaging and analysisVision for virtual imaging and analysis
6 November 13, 2003
Using non-fluorophores for fluorescence--Using non-fluorophores for fluorescence--histochemical stainshistochemical stains
• Thioflavin S and T and Congo Red
• Toluidine Blue O
• Eosin > hematoxylin
• Gentian Violet
• Neutral Red
Example of histochemical stains that fluoresce
7 November 13, 2003
Using non-fluorophores for fluorescence--Using non-fluorophores for fluorescence--histochemical stainshistochemical stains
What determines which stains fluoresce?
“Of the dyes with conjugated bond numbers (CBNs) of 29 or less, 90% showed fluorescence; 70% of the dyes whose CBNs exceeded 29 did not . . . “
The number of conjugated bonds
Juarranz et al, Histochem ’86
8 November 13, 2003
Histochemical Stains
Eosin fluoresces!
Using non-fluorophores for fluorescence--Using non-fluorophores for fluorescence--histochemical stainshistochemical stains
9 November 13, 2003
Histochemical Stains--Thioflavin S
Brain (TG-2576 mouse with amyloid plaques)
Using non-fluorophores for fluorescence--Using non-fluorophores for fluorescence--histochemical stainshistochemical stains
10 November 13, 2003
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Not perfect but very good
Bad
• Not as specific as standard antibody-bound fluorophores
• Some red chromagens will smear
Good
• Can assess morphology and fluorescence
• Will colocalize: structures > cells > regions within cells
• Higher signal:noise with fluorescence than bright field
•Improved morphometry
11 November 13, 2003
Epitope
1o Ab
2ndry Ab
Avidin-biotin complex with alkaline
phosphatase
Vector Fast Red
Biotin
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Comparing fluorescence from a chromagen with fluorescence from a fluorophore
12 November 13, 2003
CD-138 Red chromagen CD-138 Alexofluor 488
Human Lymph Node
Comparing fluorescence from a chromagen with fluorescence from a fluorophore
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
13 November 13, 2003
CD31 (endothelial cell)
Smooth muscle actin (smooth muscle)
CD31 (endothelial cell)
Human to mouse xenograft
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
14 November 13, 2003
CD31 (endothelial cell)
Human to mouse xenograft
Is anything gained?
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Fluorescent Deconvolution of brightfield image
15 November 13, 2003
CD138 (plasma Cell)
VS38C (plasma Cell)
CD138 (Plasma cell)
Human lymph node
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
16 November 13, 2003
Human lymph node
CD138 (Plasma cell)
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Fluorescent Deconvolution of brightfield image
Is anything gained?
17 November 13, 2003
Mouse spleen
B220 (B cell)
F480 (macrophage)
B220 (B Cell)
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
18 November 13, 2003
Mouse spleen
B220 (B Cell)
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Fluorescent Deconvolution of brightfield image
Is anything gained?
19 November 13, 2003
Mouse liver
B220 (B Cell)
F480 (macrophage)
B220 (B Cell)
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
20 November 13, 2003
Mouse liver
B220 (B Cell)
Using non-fluorophores for fluorescence—Red Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence IHC chromagens for fluorescence
Fluorescent Deconvolution of brightfield image
Is anything gained?
21 November 13, 2003
Registration of the same imageRegistration of the same image
Bright field
Fluo-rescent
Merged
Image registration--the process of transforming 2 or more related images into one coordinate system
22 November 13, 2003
Registration of the same imageRegistration of the same image
B220 (B cell)
F480 (macrophage)
B220 (B Cell)
Mouse spleen
23 November 13, 2003
Registration of the same imageRegistration of the same image
Cleaved caspase 3/B220
CD31/Smooth muscle actin
B220/F480
24 November 13, 2003
Registration of virtual imagesRegistration of virtual images
Using AE1 and AE3 (pancytokeratin) as a
mask on TMAs Ki-67 +’ve cell in
tumor cluster
Ki-67 +’ve cell Fluorescent Brightfield Registration
25 November 13, 2003
Registration of different imagesRegistration of different images
Pseudo color
Merged
3um step
sections Chromagen 1 Chromagen 2
Pseudo color
26 November 13, 2003
Registration of different imagesRegistration of different images
CD31
3um apart
CD31
27 November 13, 2003
Registration of different imagesRegistration of different images
CD31 pseudocolored
3um apart
CD31 pseudocolored
28 November 13, 2003
Registration of different imagesRegistration of different images
CD31 pseudocolored
CD31 pseudocolored+ Registered
29 November 13, 2003
Registration of different imagesRegistration of different images
CD31
3um apart
Smooth muscle actin (SMA)
30 November 13, 2003
Registration of different imagesRegistration of different images
3um apart
SMA pseudocolored
CD31 pseudocolored
31 November 13, 2003
Registration of different imagesRegistration of different images
CD31 pseudocolored
SMA pseudocolored+ Registered
32 November 13, 2003
Registration of virtual imagesRegistration of virtual images
Automatic section alignment Final result
Michael Grunkin, Visopharm
33 November 13, 2003
Registration of virtual imagesRegistration of virtual images
Michael Grunkin, Visopharm
34 November 13, 2003
Registration of different imagesRegistration of different images
Bright field
Fluo-rescent)
Merged
3um step
sections Chromagen 1 Fluorophore
35 November 13, 2003
Registration of different imagesRegistration of different images
Paul Kowalski, Bruker Daltonics
Imaging Mass Spectrometry
36 November 13, 2003
ConclusionsConclusions
• Virtual microscopy is just beginning to meet its potential as a tool to analyze morphologic changes
• Advances in virtual fluorescence, fluorophores, image registration and evolving image analysis programs will make image analysis easier and more accurate than ever
• Increase signal, decrease noise in a consistent manner