1. Robert Dunstan, Luke Jandreski and the Comparative Pathology Laboratory Fluorescence for fluorophobes-- Virtual fluorescence using chromagens and histochemical stains

2. 2 November 13, 2003 Lecture 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. 3 November 13, 2003 Vision for virtual imaging and analysis “Increase signal, decrease noise with consistency”

4. 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 analysis

5. 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 analysis

6. 6 November 13, 2003 Using non-fluorophores for fluorescence-- histochemical stains Thioflavin S and T and Congo Red Toluidine Blue O Eosin > hematoxylin Gentian Violet Neutral Red Example of histochemical stains that fluoresce

7. 7 November 13, 2003 Using non-fluorophores for fluorescence-- histochemical 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. 8 November 13, 2003 Histochemical Stains Eosin fluoresces! Using non-fluorophores for fluorescence-- histochemical stains

9. 9 November 13, 2003 Histochemical Stains--Thioflavin S Brain (TG-2576 mouse with amyloid plaques) Using non-fluorophores for fluorescence-- histochemical stains

10. 10 November 13, 2003 Using non-fluorophores for fluorescence—Red 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. 11 November 13, 2003 Epitope 1 o Ab 2ndry Ab Avidin-biotin complex with alkaline phosphatase Vector Fast Red Biotin Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Comparing fluorescence from a chromagen with fluorescence from a fluorophore

12. 12 November 13, 2003 CD-138 Red chromagenCD-138 Alexofluor 488 Human Lymph Node Comparing fluorescence from a chromagen with fluorescence from a fluorophore Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence

13. 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 IHC chromagens for fluorescence

14. 14 November 13, 2003 CD31 (endothelial cell) Human to mouse xenograft Is anything gained? Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence FluorescentDeconvolution of brightfield image

15. 15 November 13, 2003 CD138 (plasma Cell) VS38C (plasma Cell) CD138 (Plasma cell) Human lymph node Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence

16. 16 November 13, 2003 Human lymph node CD138 (Plasma cell) Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence FluorescentDeconvolution of brightfield image Is anything gained?

17. 17 November 13, 2003 Mouse spleen B220 (B cell) F480 (macrophage) B220 (B Cell) Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence

18. 18 November 13, 2003 Mouse spleen B220 (B Cell) Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence FluorescentDeconvolution of brightfield image Is anything gained?

19. 19 November 13, 2003 Mouse liver B220 (B Cell) F480 (macrophage) B220 (B Cell) Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence

20. 20 November 13, 2003 Mouse liver B220 (B Cell) Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence FluorescentDeconvolution of brightfield image Is anything gained?

21. 21 November 13, 2003 Registration 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. 22 November 13, 2003 Registration of the same image B220 (B cell) F480 (macrophage) B220 (B Cell) Mouse spleen

23. 23 November 13, 2003 Registration of the same image Cleaved caspase 3/B220 CD31/Smooth muscle actin B220/F480

24. 24 November 13, 2003 Registration 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. 25 November 13, 2003 Registration of different images Pseudo color Merged 3um step sections Chromagen 1 Chromagen 2 Pseudo color

26. 26 November 13, 2003 Registration of different images CD31 3um apart CD31

27. 27 November 13, 2003 Registration of different images CD31 pseudocolored 3um apart CD31 pseudocolored

28. 28 November 13, 2003 Registration of different images CD31 pseudocolored + Registered

29. 29 November 13, 2003 Registration of different images CD31 3um apart Smooth muscle actin (SMA)

30. 30 November 13, 2003 Registration of different images 3um apart SMA pseudocolored CD31 pseudocolored

31. 31 November 13, 2003 Registration of different images CD31 pseudocolored SMA pseudocolored + Registered

32. 32 November 13, 2003 Registration of virtual images Automatic section alignment Final result Michael Grunkin, Visopharm

33. 33 November 13, 2003 Registration of virtual images Michael Grunkin, Visopharm

34. 34 November 13, 2003 Registration of different images Bright field Fluo- rescent) Merged 3um step sections Chromagen 1 Fluorophore

35. 35 November 13, 2003 Registration of different images Paul Kowalski, Bruker Daltonics Imaging Mass Spectrometry

36. 36 November 13, 2003Conclusions 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