From Image to Analysis:Current State of Quantitative

Glenn Yang. TEL:8862-27855860 CELL:0953062485Cell-B

io 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

Tissomics

Life Science Research Scale

GenomicGenomic ProteomicProteomic CytomicCytomic

SuspensionSuspensionCell SampleCell Sample

AdherenceAdherenceCell SampleCell Sample

FunctionalFunctionalFluorescenceFluorescence

StainingStaining

Flow BasedCytometry

Image BasedCytometry

Quantitative Tool from Cell to Organic

Slide Based Cytometry

OrganismOrganism??

CellCytomic

TissueTissomics

Slide base cytometry (SBC)Tissomics is • Slide base cytometry• High throughput• High content• Easy to re-analysis• Fully automatic• Quantitative• Toward to 3D

Requirement of Tissomics• Digitalized slide image• Analyzed slide image• Quantified Slide image • Export visualized data

Quantitative Tissue Cytometry :Tissomics

Check your data Check your data even out of officeeven out of office

Recheck your data Recheck your data again, again again, again

and againand again

Preserve your data Preserve your data forever and freshforever and fresh

Share your data toShare your data toall over the world all over the world

Virtual Virtual SlideSlide

1.Digitalized your Slide

Quantitative Tissue Cytometry :Tissomics

2.Analyze your Slide

DAPI Alexa488 Cy3 APC

Segment cell in Immunoflu orescence slide

1.Using filter system and monochrome CCD to capture single dye image

2.Segment cell and organelle for each fluorescence channel

Quantitative Tissue Cytometry :Tissomics

Segment cell in Immuno chem istry slide

1.Separate color image into single grey value display(Software or Hardware base)

2.Segment cell and organelle for each grey channel

Visible SpectrumSoftware based color separationSoftware based color separation Hardware based color separationHardware based color separation

2.Analyze your Slide

Quantitative Tissue Cytometry :Tissomics

3.Quantity your Slide and export to visualize data

Quantitative Tissue Cytometry :Tissomics

Stitching, in this context, means assembling any number of small detail images to create one large image. The main purpose of TissueStitching is to create large overviews of tissue specimens without losing accuracy in detail.Comparedto tradi t ional microscopy, this technology blends a magnification factor of 1:400 for detailed resolution with a magnification factor of 1:25 or 1:50 for broad overview.

Background

Fluorescence SeparationFilter system to separate florescence channel.

We can offer 10 filter block in our system!

IHC Color SeparationIn order to ease analysis IHC sample HistoQuest® performs a color separation.

1. Pickup markers Color from original image

2. Color adjustment by manipulating the gain, the color space and blur.

3. Separate into gray scale Image

Blue shade of Nuclei

Brown shade of Nuclei

Brown shade of Cytopla

Nuclear SegmentationAutomatic nuclear segmentation is achieved via HistoQuest patented set of algorithm

Nuclear segmentation in HistoQuest®is completely automatic after the input of a few starting values:

•Average nuclear size

•Discrimination by area

(Exclusion of smaller nuclear sections)

•Discrimination by gray value

(Exclusion of weakly stained nuclei)

•Background threshold

(Default setting is automatic)

Single cell identificationHistoQuest® provides two algorithm sets for the identification of the cell cytoplasm .

•The first one creates a statistical ring mask which measures marker intensity between an interior and an exterior radius, measured from the border of identified nuclei.

•The second algorithm created on this mask identifies cell cytoplasm by effectuating simultaneous and incremental growth phases on marker intensity from the cell border.

Ring Mask around nuclear Growing Mask for cytoplasm Single cell cytoplasm mas k

List mode file for parameters of cellHistoQuest® provides List mode data and presentation just like FACS. (dot=cell)

3

1

1

0

58

P2

0.7121

6.822

4.005

3.204

2.1343

sizeP1ROI

8 5.221N

List mode data

100%43839.230Overall

59.13%25959.334Right

40.87%17910.140Left

PercentCountM1-MeanQuadrant

4.880

1.347

0.972

92.266

0.000

Cyclin A

0.00%00.000UL

3.90%4296124.815UR

100%110.281115.104Overall

95.88%105.604114.104LR

0.24%27142.249LL

PercentCountHamalinQuadrant

Data presentation and statisticHistoQuest® provides plot and statistic simultaneously just like FACS.

Histogram and Statistic Dot plot and Statistic

Hamaluan intensityC

yclin

A N

ucle

ar In

tens

ityCyclin A Intensity

Negative Cell

Dim positive Cell

Strong positive Cell

R1

G1=R1Gating with Strong positive Cell

Region, gating and overlay comparisonMeasurement data from different ROI’sand/or analysis selections

CyclinA Nuc.Int. CyclinA Cyt.Int. CyclinA Nuc.Int. VS cyt.int.

CyclinA Nuc.Int. CyclinA Cyt.Int. CyclinA Nuc.Int. VS cyt.int.

Dot plot Overlay

Histogram Overlay

Unt

reat

men

tT

reat

men

t

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 inthe scattergrams, i.e.to show a cell‘s properties.

Backward connection: from scattergram to image

Basal Epithelial Cells

Sec

reto

ryE

pith

elia

lCel

ls

Comparison of HistoQuest Analysis and Manual Counts

The Reinheckel Group at the Institute of Molecular Medicine and Cell Research of the Albert-Ludwigs University Freiburg, Germany, conducted validation experiments with HistoQuest®. The aim was to quantify Ki67 expression in pulmonal metastasis of 14 month-old MMTV-PyMT mice with HistoQuest and do manual counts in comparison.

TissueFAXSThe Microscopic Equivalent to

Flow CytometryApplication Notes

Application Note 1:Identification of Prostatic GlandsAim:

Automated distinction of secretory and basal epithelial cells in prostatic glands in order to allow cell type-specific measurements of further molecular markers.

Courtesy Prof. G. Kramer, Dep. of Urology, Med. Univ. of Vienna, Austria

Blue: Nuclei (DAPI)Green: Cytokeratin 18+ secretory epithelial cells.Red: Cytokeratin 18+/Cytokeratin 8.12+ basal epithelial cells

Application Note 2: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 3:Viability Testing in Cell Cultures

Courtesy Dr. Schenkel, Central Mouse Facility, DKFZ Heidelberg, Germany

AimAim ::

Quality control of sperm cells afterthawing. By applying a specific stainthat labels exclusively nuclei of live cells and a second stain thatexclusively labels nuclei of dead cellsan automated viability test can beestablished by using TissueFAXS™.

Please note: The single event in the upper right quadrant is a doublet of a living and a dead celltightly sticking together and can be easily gated out.

Steiner et al. 2000, Journal of Immunological Methods 237 (1-2);39-50

Cytotoxic T-cells

T-Helper cells

Application Note 4:Phenotype of Tissue Infiltrating Leukocytes

Aim:

Determine the immune status in samples of different renal cell carcinoma patientsby 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 leukocytesRed: CD3 – T-lymphocytesGreen: CD8 – zytotoxic T-lymphocytes

Application Note 5:In-situ Quantification of Apoptosis

Aim:

Determine the apoptotic rate of dendritic cells in mouse skin-sheets after treatmentwith 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 cellsRed: Langerhans cells

Epidermal sheets are stained after Elidel treatment and the number of apoptotic cells was analyzed.

TUNEL+ : 0.64%

Application Note 6: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-outmouse model. Different mice (adult, newborn and knock-out) were used to determine the IL-10 expression level in situ and thereby learn to understanddevelopmental changes throughout theontogeny of the mouse immune system.

It could be demonstrated that autocrineIL-10 partially prevents differentiation of neonatal dendritic epidermal leukocytesinto 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 themolecular 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 7:Effect of Irradiation / Therapy

Blue: DAPI (nuclei)Green: Specific marker (upregulated upon UV-irradiation)

Application Note 8: Cellular Changes during Tumor Formation

Courtesy Prof. H. Klocker, Dep. of Urology, Med. Univ. of Innsbruck, Austria

Aim:

Provide an automatedand observerindependent data basisfor clinical diagnosis of prostate cancer basedon a specific tumormarker and changes in the composition of prostatic glandscharacterized bydifferent types of cytokeratin expressedby epithelial cells.

Application Note 9: Counting of Bacteria (Spirochaeta)

Courtesy Dr. M.J. Flaig, Dep. of Dermatology, LMU Munich, Germany

Aim:

Automatically countbacteria per mm² in infected skin.

Density: 2198 bac./mm²