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