histoquest and tissuequest, the microscopy based...
TRANSCRIPT
HistoQuest and TissueQuest, the
microscopy based FACS-like equivalents
for cells in tissue and smears
Heinrich Bürgers
TissueGnostics GmbH
Collagen Typ-3
From Image … to Analysis
How many of the nuclei
are also stained in red?
Improve your measurements!
How many of the nuclei
are also stained in red?
Expert‘s estimations:
0,5% - 18% (n=20)
Observer independent
measurement:18,57%
Make use of your controls!
Expert‘s estimations:
0,5% - 18% (n=20)
Observer independent measurement:
18,57%
Control staining sample staining
The concept:
1. Define your region of interest
2. Count all cells.
3. Evaluate each individual marker for all detected cells.
4. Generate scattergrams or histograms
to quantify and compare positive and negative cells.
How many of the nuclei
are also stained in red?
How many of the nuclei
are also stained in red?
Forward connection: from image to scattergram
The „Forward Connection“ feature allows to double click on
individual cells in any image in order to show its position in
the scattergrams, i.e.to show a cell‘s properties.
Backward connection: from scattergram to image
Basal Epithelial Cells
Secre
tory
Epith
elia
l C
ells
nuclear
cytoplasmic
perinuclear
Application:Intranuclear Translocation of AHR
TissueGnostics offers software to quantify
immunofluorescence
and IHC samples
Original image color separation
SEGMENTED NUCLEI
HistoQUEST: the analysis SW for immunohistochemistry
segmentation
Sample linking
Our method is made to be:
simple and efficient
fast
reliable
review safe
controllable at all steps
no expert knowledge necessary just controls
Discover new perceptions!
Size matters 1
Size matters 2
Compare sets
blue: Estrogen receptor
red: Progesteron receptor
Feulgen Staining of Colon Carcinoma – Analysis
blue: normal tissue
red: tumor area
Week 13 Week 22*Week 17M
ark
er
1M
ark
er
2
11.41%
0.27%
69.36%
1.30%
20.48%
0.72%
0.07%
Ma
rker
3 0.15% 0.0%
Courtesy Dr. R. Sgonc, Inst. for Anatomy, Med. Univ. Innsbruck, Austria
*) Sample was smaller.
Reason: Estimation versus Measurement
Relative Staining Intensity = gray value(GV)
GV = 0: BLACK
GV = 255: WHITE
What is the difference in
GV between A and B?
1. ∆ = 0
2. ∆ ≤ 20
3. ∆ ≤ 50
4. ∆ ≤ 100
5. ∆ > 100
Reason: Estimation versus Measurement
∆ = 0 !!!!
Reason 2: Estimation versus Measurement
Relative Staining Intensity = gray value(GV)
Assume A and B are 2
samples stained for:
• Tumor marker
• Cytokine expression
• Apoptosis
• Proliferation
• …..
The import Wizard enables to use:JPEGPNGBMPTIFNDP (Nanozoomer)
…
for extreme high flexibility
Tissue FAXS – eases working with TMA
Even empty
positions are
recognized
Our Products:
TissueQuest
image analyzing of fluorescence samples
HistoQuest
image analyzing of bright field samples
TissueFAXS and HistoFAXS
microscopic platforms to capture automatic Images
Steps to the results
1. Staining, FISH, IHC,
2. Capture ImageGrayscale image from each channel per Field of view by fluorescence or using TG TissueFAXS
3. set parameter / load profile
4. Print out the results
Our requests in general:
overall staining, nuclear staining
e.g.: DAPI, Hemalaun
Our request in bright field:
High-contrast marker combination
e.g.: blue vs. red,
green vs. brown
1. Controls
Application: Counting of Bacteria (Spirochaeta)
Courtesy Dr. M.J. Flaig, Dep. of Dermatology, LMU Munich, Germany
Aim:
Automatically count
bacteria per mm² in
infected skin.
Density: 2198 bac./mm²
Application:Find rare events?
Courtesy Prof. M. Maurer, Inst. f. Pathophysiologie, Univ. of Heidelberg, Germany
Aim:
Measure the moleular expression levels of certain markers in a rat model.
1,87%
Red: nuclei (Propidium Iodide)
Green: young oligodendrocyte marker
Application:Viability Testing in Cell Cultures
Courtesy Dr. Schenkel, Central Mouse Facility, DKFZ Heidelberg, Germany
Aim:
Quality control of sperm cells after
thawing. By applying a specific stain
that labels exclusively nuclei of live
cells and a second stain that
exclusively labels nuclei of dead cells
an automated viability test can be
established by using TissueFAXS™.
Tissue FAXS – NEW „DotFinder“ Algorithm
Identifies and
quantifies
dots
optionally in
the nucleus
or in the
cytoplasm
For FISH & globular staining pattern
• Ecker A, Simma O, Hoelbl A, Kenner L, Beug H, Moriggl R*, Sexl V*.
The dark and the bright site of Stat3: proto-oncogene and tumor-suppressor. * equal
correspondence, (Frontiers in Bioscience, in press)
• Harir N, Boudot C, Friedbichler K, Sonneck K, Kondo R, Kenner L, Kerenyi M, Gouilleux-Gruart V,
Gondry J, Lassoued K, Valent P, Gouilleux F, Moriggl R.
Oncogenic Kit controls neoplastic mast cell development through a Stat5/PI 3-kinase signaling
cascade. * equal contribution and correspondence (Blood, in press)
• Eferl R, Hasselblatt P, Rath M, Popper H, Zenz R, Komnenovic V, Idarraga MH, Kenner L and Wagner
E. Development of Pulmonary Fibrosis through a Pathway Involving the Transcription Factor
Fra-2/AP-1. (Proc. Natl. Acad. Sci., in press)
• Maurer M, Feldmann R, Bürgers H and Kuschinsky W (2008).
Glycogen Synthase Kinase 3β (GSK3β) Regulates Differentiation and Proliferation in Neural Stem
Cells from the Rat Subventricular Zone, BMC Neuroscience 9:7
• Bürgers H, Schelshorn D, Wagner W, Kuschinsky W, Maurer M. (2008)
Acute anoxia stimulates proliferation in adult neural stem cells from the rat brain (Experimental
Brain Research 188, 33-43
• Maurer M, Brömme J, Feldmann R Jr, Järve A, Sabouri F, Bürgers H, Schelshorn D, Krüger C,
Schneider A, Kuschinsky W. (2007).
Glycogen synthase kinase 3beta (GSK3beta) regulates differentiation and proliferation in neural
stem cells from the rat subventricular zone. J. Proteome Res. 6(3):1198-208.
The TissueGnostics Group
Headquarter
TissueGnostics GmbH
Taborstrasse 10/2/8
1020 Vienna
Austria
Tel: +43 1 2161190
Fax: +43 1 2161190 90
Email: [email protected]
Web: www.tissuegnostics.com
International Offices
TissueGnostics USA Inc.
Los Angeles
TissueGnostics Romania SRL
Iasi, Romania
Distributors: Japan, Taiwan, Singapore, Israel, Lithuania, Latvia,
Feulgen Staining of Colon Carcinoma – Overview
At 40x magnification a total of 1464 field of view was scanned.
Slide Overview at 2.5x Magnification
Application:Leukocyte Cytokine Expression in situ
Chang-Rodriguez et al.2004, Journal of Leukocyte Biology 76(3):657-66
Aim:
Quantify the expression of Interleukin-10
of dermal leukocytes in a knock-out
mouse model. Different mice (adult,
newborn and knock-out) were used to
determine the IL-10 expression level in
situ and thereby learn to understand
developmental changes throughout the
ontogeny of the mouse immune system.
It could be demonstrated that autocrine
IL-10 partially prevents differentiation of
neonatal dendritic epidermal leukocytes
into Langerhans cells
From Image …to Analysis
Positive cells Positive cells
Intensity of DNA staining
Inte
nsity o
f antigen e
xpre
ssio
n
Each cell is indicated as one dot
The reactivity of two channels is plotted on the x- and y-axes
Negative Control Normal Skin UV-Irradiated Skin
Courtesy Prof. Dr. L. Kemeny, Dept. of Dermatology, University of Szeged, Hungary
Aim:
Quantify the effect of UV-radiation (or any other treatment) on human skin on the
molecular basis.
The expression level of target molecules (in green) is determined in skin biopsies of
normal and UV-treated samples in correlation to isotype-matched negative control.
Application:Effect of Irradiation / Therapy
Blue: DAPI (nuclei)
Green: Specific marker
(upregulated upon
UV-irradiation)
Virtual
sample
composed of
84 FOV Detail view
Courtesy Dr. R. Sgonc, Inst. for Anatomy, Med. Univ. Innsbruck, Austria
The TissueFAXS™ product family
TissueFAXS-plus
TissueFAXS
HistoFAXS
TissueFAXS-i
+ paraffin sections
+ cryocut sections
+ biopsy material
+ tissue microarrays
TissueFAXS Applications
+ cells in suspension
+ cultured cells
+ cell smears
+ cytospins
…with TissueQuestTM algorithms
original
The newly developed
and patented
method for single cell
identification.
TissueQuest algorithms dynamically search for nuclei which are
• isolated or in close proximity
• intensly or weakly stained
• large or small
• round or elongated
• compact or ring shaped
• in focus or somewhat blured (beause they are in a different focus plain)
First step
detection of cell nuclei and cells
Master channel
Second step
Definition of cell body
Antigen measure mask
TissueQuest analysis in human skin
Distinguish between Nucleus and Cytoplasm!
3 channel overlay
DAPI
Anti-Tubulin
Anti-beta-COP
Not only analyze single cells – even analyze subcellular compartments on a single cell basis!
Courtesy Dr. R. Pepperkok, Advanced Light Microscopy Facility, EMBL Heidelberg
HistoFAXS: the system for immunohistochemistry
Original image
Scattergrams are used to present data of individual cells
in two or more color channels
Dot plot display for immunohistochemistryA
nti-C
D3 r
eactivity
Hemalaun reactivity
Feulgen Staining of Colon Carcinoma – Analysis
blue: control tissue
red: tumor area
Week 13 Week 22*Week 17M
ark
er
1M
ark
er
2
11.41%
0.27%
69.36%
1.30%
20.48%
0.72%
0.07%
Ma
rker
3 0.15% 0.0%
Courtesy Dr. R. Sgonc, Inst. for Anatomy, Med. Univ. Innsbruck, Austria
*) Sample was smaller.
TissueGnostics GmbH Austria
Dr. Rupert Ecker (CEO/CTO)
Georg Steiner (CEO/CSO)
Taborstrasse 10/2/8
A – 1020 Vienna
Tel: +43-1-2161190
Fax: +43-1-3161190 90
Mobile: ++43-1-664 2058588 & ++43-1-664 8338041
Email: [email protected]
Web: www.tissuegnostics.com
Contact Information
TissueGnostics Romania SRL
(K2RG Research Group SRL)
DI Daniel Haisan (CEO)
Nicolae Iorga Boulevard nr. 51C
RO – 700213 Iasi
Tel: +40-332-405866
Fax: +40-332-405867
Email: [email protected]
TissueGnostics USA Inc.
Georg Steiner (President)
Hamid Barang (Vice President)
420 N. Larchmont Boulevard
Los Angeles
CA 90004
USA
Tel: +1-323-466-0499
Mobile: +1-818-9843182
Email: [email protected]
TissueFAXSThe Microscopic Equivalent to Flow Cytometry
Application Notes
Application Note 1:Measurement of Neuronal Markers
Courtesy Prof. M. Maurer, Inst. f. Pathophysiologie, Univ. of Heidelberg, Germany
Aim:
Measure the moleular expression levels of certain markers in neuronal cells in a rat model.
1,87%
Red: nuclei (Propidium Iodide)
Green: neuronal marker
Application Note 2:Viability Testing in Cell Cultures
Courtesy Dr. Schenkel, Central Mouse Facility, DKFZ Heidelberg, Germany
Aim:
Quality control of sperm cells after
thawing. By applying a specific stain
that labels exclusively nuclei of live
cells and a second stain that
exclusively labels nuclei of dead cells
an automated viability test can be
established by using TissueFAXS™.
Please note: The single event in the upper right
quadrant is a doublet of a living and a dead cell
tightly sticking together and can be easily gated out.
Why?
Why do we need biomedical image analysis?
• The human brain is a potent interpolater that can hardly be
replaced by software!
• An experienced observer is much faster than a computer!
• What could a computer „see“ in an image that a human
observer cannot recognize?
• What does it help me to know how many tumor cells are
there? All I need to know is whether there are any tumor cells!
Steiner et al. 2000, Journal of Immunological Methods 237 (1-2);39-50
Cytotoxic
T-cells
T-Helper
cells
Application Note 3:Phenotype of Tissue Infiltrating Leukocytes
Aim:
Determine the immune status in samples of different renal cell carcinoma patients
by phenotypic characterization of tissue infiltrating leukocytes in situ.
CD45 was used to identify all leukocytes.
Dot plots show the reactivity of anti-CD3
(x-axis) versus anti-CD8 (y-axis).
Blue: CD45 – all leukocytes
Red: CD3 – T-lymphocytes
Green: CD8 – zytotoxic T-lymphocytes
Application Note 4:In-situ Quantification of Apoptosis
Aim:
Determine the apoptotic rate of dendritic cells in mouse skin-sheets after treatment
with certain drugs
Normal ControlStrong Apoptotic
InducerWeak Apoptotic
Inducer
Hoetzenecker et al. 2004, Journal of Investigative Dermatology 122(3):673-84
Hoetzenecker et al. 2005, Journal of Allergy and Clinical Immunology, 115(6):1276-83
Green: Apoptotic cells
Red: Langerhans cells
Epidermal sheets are stained after Elidel treatment and the number of apoptotic cells was analyzed.
TUNEL+ : 0.64%
Application Note 5:Leukocyte Cytokine Expression in situ
Chang-Rodriguez et al.2004, Journal of Leukocyte Biology 76(3):657-66
Aim:
Quantify the expression of Interleukin-10
of dermal leukocytes in a knock-out
mouse model. Different mice (adult,
newborn and knock-out) were used to
determine the IL-10 expression level in
situ and thereby learn to understand
developmental changes throughout the
ontogeny of the mouse immune system.
It could be demonstrated that autocrine
IL-10 partially prevents differentiation of
neonatal dendritic epidermal leukocytes
into Langerhans cells
Negative Control Normal Skin UV-Irradiated Skin
Courtesy Prof. Dr. L. Kemeny, Dept. of Dermatology, University of Szeged, Hungary
Aim:
Quantify the effect of UV-radiation (or any other treatment) on human skin on the
molecular basis.
The expression level of target molecules (in green) is determined in skin biopsies of
normal and UV-treated samples in correlation to isotype-matched negative control.
Application Note 6:Effect of Irradiation / Therapy
Blue: DAPI (nuclei)
Green: Specific marker
(upregulated upon
UV-irradiation)
Application Note 7: Cellular Changes during Tumor Formation
Courtesy Prof. H. Klocker, Dep. of Urology, Med. Univ. of Innsbruck, Austria
Aim:
Provide an automated
and observer
independent data basis
for clinical diagnosis of
prostate cancer based
on a specific tumor
marker and changes in
the composition of
prostatic glands
characterized by
different types of
cytokeratin expressed
by epithelial cells.
Application Note 8: Counting of Bacteria (Spirochaeta)
Courtesy Dr. M.J. Flaig, Dep. of Dermatology, LMU Munich, Germany
Aim:
Automatically count
bacteria per mm² in
infected skin.
Density: 2198 bac./mm²
Compare your samples online
Before and after treatment
Controls and samples
