Robert Dunstan, Luke Jandreski and the Comparative Pathology Laboratory

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 • Use of red IHC chromagens for fluorescence • Use of registration/co-registration • Conclusions

Vision for virtual imaging and analysis “Increase signal, decrease noise with consistency”

Vision for virtual imaging and analysis Brightfield Fluorescence Pro --Morphologic assessment Con --Lower signal:noise --Non-linear expression of chromagens --Colocalization difficult Pro --High signal to noise --Linear expression of fluorophores --Colocalization easy Con --Morphologic assessment difficult

Vision for virtual imaging and analysis Brightfield Fluorescence --Virtual microscopy --“Non-fluoro-phores” for fluorescence --Co-registration of images Pro --Morphologic assessment Con --Lower signal:noise --Non-linear expression of chromagens --Colocalization difficult Pro --High signal to noise --Linear expression of chromagens --Colocalization Con --Morphologic assessment difficult

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

Using non-fluorophores for fluorescence--histochemical stains What determines which stains fluoresce? The number of conjugated bonds “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 . . . “ Juarranz et al, Histochem ’86

Using non-fluorophores for fluorescence--histochemical stains Histochemical Stains Eosin fluoresces!

Using non-fluorophores for fluorescence--histochemical stains Histochemical Stains--Thioflavin S Brain (TG-2576 mouse with amyloid plaques)

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Not perfect but very good • Good • Can assess morphology and fluorescence • Will colocalize: structures > cells > regions within cells • Higher signal:noise with fluorescence than bright field • Improved morphometry • Bad • Not as specific as standard antibody-bound fluorophores • Some red chromagens will smear

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

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Comparing fluorescence from a chromagen with fluorescence from a fluorophore CD-138 Red chromagen CD-138 Alexofluor 488 Human Lymph Node

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence 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 Is anything gained? CD31 (endothelial cell) Fluorescent Deconvolution of brightfield image Human to mouse xenograft

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence CD138 (plasma Cell) VS38C (plasma Cell) CD138 (Plasma cell) Human lymph node

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Is anything gained? CD138 (Plasma cell) Fluorescent Deconvolution of brightfield image Human lymph node

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence B220 (B cell) F480 (macrophage) B220 (B Cell) Mouse spleen

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Is anything gained? B220 (B Cell) Fluorescent Deconvolution of brightfield image Mouse spleen

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence B220 (B Cell) F480 (macrophage) B220 (B Cell) Mouse liver

Using non-fluorophores for fluorescence—Red IHC chromagens for fluorescence Is anything gained? B220 (B Cell) Fluorescent Deconvolution of brightfield image Mouse liver

Registration of the same image Image registration--the process of transforming 2 or more related images into one coordinate system Fluo-rescent Bright field Merged

Registration of the same image B220 (B cell) F480 (macrophage) B220 (B Cell) Mouse spleen

Registration of the same image Cleaved caspase 3/B220 CD31/Smooth muscle actin B220/F480

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 Brightfield Fluorescent Registration

Registration of different images 3um step sections Chromagen 1 Chromagen 2 Pseudo color Pseudo color Merged

Registration of different images CD31 3um apart CD31

Registration of different images CD31pseudocolored 3um apart CD31pseudocolored

Registration of different images CD31 pseudocolored CD31 pseudocolored Registered +

Registration of different images CD31 3um apart Smooth muscle actin (SMA)

Registration of different images CD31pseudocolored 3umapart SMA pseudocolored

Registration of different images SMA pseudocolored CD31 pseudocolored Registered +

Registration of virtual images Final result Automatic section alignment Michael Grunkin, Visopharm

Registration of virtual images Michael Grunkin, Visopharm

Registration of different images 3um step sections Chromagen 1 Fluorophore Fluo-rescent) Bright field Merged

Registration of different images Imaging Mass Spectrometry Paul Kowalski, Bruker Daltonics

Conclusions • 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

Robert Dunstan, Luke Jandreski and the Comparative Pathology Laboratory