2. HISTOINDEX – BRINGING DIAGNOSIS INTO BIOPSY
World’s first suite of fully-automated, 3-D, quantitative, laser-
based imaging system for tissue imaging
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3. WHAT ARE CUSTOMERS LOOKING FOR?
PRICE
SPEED
TARGET
CUSTOMER
ACCURACY
SPECIFIC
OBJECTIVE
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Tissue-Specific Image Analysis and Quantification
for Research and Clinical Applications
4. WHAT DO WE OFFER?
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• How did the
disease
progress/re
gress?
• Why did
that happen?
• When is
treatment
needed?
• What
treatment
to give?
CLOUD-BASED
GENESIS®
LAENNEC™
DISEASE-SPECIFIC ASSESSMENT
5. HOW IT WORKS
LASER
cells
ECM
Improvement in accuracy of tissue diagnosis from 65%
to more than 95%
Bedossa et al., Hepatology 20, 15-20 (1994); Gronbaek et al., Journal of Viral Hepatitis 9, 443-
449 (2002); Theodossi et al., Gastroenterology 79, 232-241 (1980); Theodossi et al., Gut 35,
961-968 (1994); Westin et al., Liver 19, 183-187 (1999)
He et al., Journal of Biomedical Optics, 15(5) 056007, 2010.
Bredfeldt et al., Journal of Pathology Informatics, 5(1), 28, 2014
Raja et al., Journal of Biomedical Optics 15(5) 056016, 2010.
Gailhouste et al., Journal of Hepatology 52, 398-406, 2010.
Asselah et al, Journal of Hepatology 61, 193-195, 2014.
Tai et al., Hepatology 50(4) 1093, 2009.
Tai et al., Journal of Biomedical Optics, 14(4) 044013, 2009.
Xu et al., Journal of Hepatology, 61, 260-269, 2014.
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7. REVOLUTIONARY TECHNOLOGY
Stage 1 Stage 2 Stage 3 Stage 4
HistoIndex Stain-Free
Stage 1.2 Stage 2.5 Stage 3.1 Stage 4.7
Current Method
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8. KEY BENEFITS
1 – 3 days
Sample Staining Imaging Staging Diagnosis
0.5 – 1 hr
Diagnosis
HistoIndex
Stain-free Technology
Sample
Patients (Hospitals and Clinics):
Same day results – greater accuracy & efficiency
Users (Researchers, Drug companies & CROs):
• Higher specificity & sensitivity
• A "translational" program to measure drug efficacy from
the start of preclinical studies to the end of the clinical
trial
• Assessment of incremental improvement in treatment
efficacy
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9. WHAT IS THIS NEW STANDARD?
Hardware Genesis®200
Multi-photon microscope
utilizing a high power laser
Customized optics
Fully automated
One-touch operation
Unstained tissues
Multi-organ capabilities
Computer-assisted
tissue diagnostic
system
HISTOINDEX
is setting a
NEW STANDARD!
Software FibroIndex™ +
LiverCloud™
Quantification for decision
support database
3D visualization
Cloud-based database & online
assessment
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10. GLOBAL PRESENCE
Physical units placement
Clinical/Research sites with samples sending to HistoIndex’s global
Imaging Service Centres
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11. TISSUE-SPECIFIC IMAGE ANALYSIS &
QUANTIFICATION
FIBROSIS
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Research Development Validation Commercial
Liver
Kidney
Lung
Bone Marrow
Eye – AMD, Glaucoma
Heart
Brain – AD
Skin
Hair
Launch 2Q2016
Launch 2Q2016
Published
Explorations
In
Partnerships
12. TISSUE-SPECIFIC IMAGE ANALYSIS &
QUANTIFICATION
ONCOLOGY
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Research Development Validation Commercial
Breast
Prostate
Bladder
Vs Genomic Markers
3Q2016 3Q2018
Vs Genomic Markers
3Q2016 3Q2018
Vs Genomic Markers
3Q2016 3Q2018
Target Intent of Use:
To provide Physicians (e.g.
Urologists) with adjunctive
information to subclassify tumor
stages
13. FIBROSIS IN LIVER VALIDATION PATHWAY
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14. HistoIndex Pte.Ltd. property. All Rights Reserved
IMAGE ANALYSIS & QUANTIFICATION TOOL
Quantification of
Cell Damage
Quantification of
Collagens
Hepatocytes’s NAD(P)H and
Flavins intrinsic florescence.
Areas containing nucleus, lipid droplets, and the vacuoles
formed by degenerated hepatocytes appeared dark in the
TPEF image( lack of the fluorescent molecules)
Aggregated and
distributed collagens
are revealed and then
quantified
16. LIVER FIBROSIS
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• A SHG may open new opportunities in the quantification of collagen content in different
organs, which is of main importance in providing diagnostic information and evaluation
of therapeutic efficiency
Morphological changes at different stages 1 to 4 of liver fibrosis recorded with (a) to (d)
conventional Masson Trichrome staining, as well as (e) to (h) SHG and TPAF microscopies.
In stage 1 liver fibrosis, there was presence of pericellular collagen without the septa formation in
(a) and (e).
In livers with stage 2 fibrosis, (b) collagen aggregations formed incomplete septa from the portal
tract to central
vein, and (f) the bile duct proliferation was seen as dim regions in the SHG image.
For stage 3 liver fibrosis, profuse bile duct proliferation
was observed all over the tissue sample, where complete but thin collagen septa interconnected
with each other in (c) and (g).
In stage 4 fibrosis, thick collagen septa were observed, forming complete cirrhosis in (d) and (h).
Red: TPAF
Green: SHG
17. RENAL FIBROSIS
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• Combined SHG and TPAF imaging can provide simultaneous three-dimensional visualization
of collagen synthesis and assembly sites in transgenic animal models expressing GFP
constructs
• SHG is used to image collagen fibers in human and mouse unstained kidneys. This
approach allows quantitative 3-D imaging of interstitial fibrosis and arterial remodelling with
high accuracy.
SHG TPAF Overlay
SHG imaging showing increased tubulointerstitial
fibrosis and disrupted extracellular matrix in tissue
inhibitor of metalloproteinase-3 (TIMP3)
SHG and TPAF imaging in unfixed and unstained kidneys
showing the fibrillar collagen network (magenta) and tubular
endogenous autofluorescence (green) in wild-type (WT) and
TIMP3−/− kidneys at 3 days after unilateral urethral
obstruction (UUO)
18. PULMONARY FIBROSIS
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• Using TPAF, we can characterize normal and fibrotic pulmonary tissue in the bleomycin
model, and show that SHG by fibrillar collagen reveals the micrometer-scale three-
dimensional spatial distribution of the fibrosis
• TPAF is able to differentiate neoplastic from nonneoplastic lung tissue and identify
tumour subtypes. In the future larger studies, we foresee real time intraoperative
applications of TPAF
TPAF and hematoxylin and eosin (H&E) images of
progression from atypical lesion to various
patterns of invasive adenocarcinoma of lung
a) and b) Images of atypical adenomatous hyperplasia shows a focus of pneumocyte
proliferation along the alveolar wall
c) and d) Images of adenocarcinoma of lung with lepidic-predominant pattern and a few
clusters of free-floating tumour cells
e) and f) Images of adenocarcinoma of lung with acinar-predominant pattern
g) and h) Images showing solid pattern (arrows) with suggestion of gland formation
i) and j) Images showing papillary pattern
k) And l) Images showing micropapillary pattern, with complete destruction of normal
lung parenchyma
19. MYELOFIBROSIS
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• TPAF or SHG is a promising novel technology to be applied in quantification of
myelofibrosis, with performance equivalent to a stereology-based approach.
Further studies with large sample size are needed to validate the utility of this
method.
Fibrosis score = 0 Fibrosis score = 2+ Fibrosis score = 3+
Myelofibrosis is the proliferation of an abnormal clone of Hematopoetic Stem Cell (HSC) which results in fibrosis.
0 represents the least damage, while 3+ represents the most damage on the myeloproliferative neoplasm.
Green: SHG
Red: TPAF
20. DERMAL FIBROSIS
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• SHG is used to measure the significant differences in thickness of epidermis
and stratum corneum, and modified degrees of fibrosis in dermis, are clearly
demonstrated in in vitro studies
Transversal optically sectioned ex-vivo skin sample of healthy dermis
(a) SHG, (b) TPAF, (c) merge between the two images
Transversal optically sectioned ex-vivo sample of normal scar (NS)
(d) SHG, (e) TPAF, (f) merge between the two images
Area with fibroblastic proliferation inside a transversal optically sectioned ex-vivo
skin sample of keloid
(g) SHG, (h) TPAF, (i) merge between the two images
Area with keloidal fibers inside a transversal optically sectioned ex-vivo skin
sample of keloid
(j) SHG, (k) TPAF, (l) merge between the two images
Blue: TPAF
Green: SHG
21. SUBEPITHELIAL FIBROSIS ON CORNEA
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• Using SHG, we detected the presence of subepithelial fibrosis at the anterior stroma
and disorganized collagen lamellae at the posterior stroma of the bullous keratopathy
cornea
• SHG imaging of the anterior segment of edematous corneas revealed a normal
appearance of interwoven collagen lamellae in the anterior stroma
SHG images of the anterior segment of the stroma in
normal (A) and edematous corneas (B).
Short, narrow bundles of linear structures in random
orientations are apparent in both corneas. (C, D)
Image of Subepithelial Fibrosis Associated
with Corneal Stromal Edema by SHG
22. RETINAL PIGMENT EPITHELIUM
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• Dysfunction Retinal Pigment Epithelium (RPE) will cause damages to the retina. Hence,
TPAF is used because it is an elegant and highly efficient tool to delineate the thick, fragile,
and opaque retina-choroid complex, and may provide clues to the trigger events of age-
related macular degeneration
TPAF signals from the 19-year-old macular RPE cells were acquired with two
spectral windows colour coded in (a) green and (b) red
(c) Merged TPAF images of the 19-year-old RPE cells
(d), (e) and (f) Corresponding TPAF images of the 64 year-old macular RPE
cells respectively
23. ARTERIAL FIBROSIS
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SHG and TPAF provides label-free molecular imaging of atherosclerotic lesions with 3-D
submicrometric resolution suggests its potential application to the diagnosis of
atherosclerotic plaques, determination of their rupture risk, and design of individualized
drug therapy based on plaque composition
D SHG E TPAF SHG/TPAFF
SHG TPAF SHG/TPAFCA B
Healthy Arteries
Atherosclerotic Iliac Arteries
a) SHG image of collagen
b) TPAF image of elastin in a healthy artery
c) Overlaid image of a and b
d) SHG image of collagen
e) TPAF image of elastin in an atherosclerotic artery
f) Overlaid image of d and e
Green: SHG
Red: TPAF
24. CARDIAC FIBROSIS
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• By using SHG to analyze the arrangement of collagen fibers, we can provide explicit information about the
relationship between myocardial fibrosis and Atrial Fibrillation (AF)
• A significant increase in the density of collagen fibres between hypertrophic and control tissues was evident
using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red
staining of collagen
Co-localized SHG (shown in white) and TPAF (shown in blue)
images visualize collagen fibril organization and cardiac muscle
cell nuclei, respectively, in the histological section of infarcted
myocardium of
(a) an untreated infarcted rat heart;
(b) an ASCs-treated infarcted rat heart;
(c) an image obtained from a histological section of a non-MI rat
heart
25. ALZHEIMER’S DISEASE (AD)
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• Using SHG and TPAF, the results suggest that tissues from AD transgenic
models contain distinct intrinsic emissions, which can provide valuable
information about the disease mechanisms
TPAF
a) TPAF
b) SHG
c) overlay of the hippocampus of a 17-month old mouse
d) Senile plaques emit TPAF that were morphologically distinct
from other sources of intrinsic emissions and were missing from
wild type
Images from acute hippocampal brain slice
of transgenic Alzheimer’s disease mouse
models
26. FIBROSIS IN DUCHENNE MUSCULAR
DYSTROPHY (DMD)
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• Second harmonic generation (SHG) imaging may soon be available in routine
clinical diagnostics, and in this work we provide valuable imaging tools to track
and quantify ultrastructural worsening in Duchenne Muscular Dystrophy (DMD),
and to judge the beneficial effects of possible drug or gene therapies
Co-imaging of TPAF and SHG shows that
the fascicle with abnormal sarcomere
angles sticking out of the main trunk is fully
covered by membrane
Red: TPAF
Green: SHG
27. CONTRACTION MODEL OF WOUND REPAIR
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• Since structural remodelling of collagen is important in fibrosis, SHG and Two
Photon Auto Fluorescence (TPAF) signals can be used in tandem to provide
spatially resolved 3-dimensional structural remodelling of a collagen matrix
A) SHG image of collagen matrix
B) TPAF image of contracted fibroblasts
C) Overlaid image of collagen matrix and contracted fibroblast
A) SHG image of collagen matrix
B) TPAF image of non-contracted fibroblasts
C) Overlaid image of collagen matrix and non-contracted fibroblasts
Gray: SHG signal strength
Green: Elongated Cell Peripheries
29. PROSTATE CANCER
POSITIVE SAMPLE NEGATIVE SAMPLE
Benign Region
Cancerous Region
Benign Region
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30. PROSTATE CANCER
Gleason score 3+3
Cancerous region
Benign region
Patches within region
are randomly chosen
POSITIVE SAMPLE
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31. RESULTS IN 3D FEATURE SPACE
• 3+3 cancerous
• 4+3 cancerous
• Negative sample
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34. BREAST CANCER
DCIS DCIS I I I I II II
T2N0M0 T2N0M0 T2N0M0 T2N1M0 T3N3M0 T3N0M0
II II III III III III III III
T1N0M0 T2N0M0 T2N0M0 T3N0M0 T3N1M0 T3N1M0 T2N0M0 T3N0M0
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41. BLADDER CANCER
(A) & (B) TPAF and H&E image of Bladder Cancer image
at low magnification showing demarcation between
normal urothelium (a) and lamina propria (b).
[Lamina propria is composed of collagen bundles (red)
and elastin fibers (green)]
(C) & (D) TPAF and H&E image at high magnification
showing multi-layered urothelium and superficial umbrella
cells.
Umbrella cells in each image are boxed and shown at
higher digital zoom in the inset.
(E) & (F) TPAF image and H&E image at low
magnification showing von Brunn nests, some with cystic
dilatation
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42. OESOPHAGEAL CANCER
The content and distribution of collagen, elastic fibers and cancer cells in normal and
cancerous submucosa layer can determine the early stages of early oesophageal
cancer by using multiphoton technology
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a) SHG and TPAF images of normal tissues
b) SHG and TPAF images of oesophageal cancer tissues
Green: Collagen Fibres (SHG)
Red: Cellular Structure (TPAF)
SHG TPAF SHG/TPAF
(a)
Normal
(b)
Cancer
43. RECTAL CANCER
Multiphoton Technology is used to image mucosa layer, goblet cells, intestinal
glands, and collagen fibers of human rectal mucosa and submucosa. Results
shows that it can monitor early rectal cancer
Multiphoton Technology has the potential to provide real-time, non-invasive
optical diagnosis for surgical margins in rectal cancer
a) SHG image of colorectal carcinoma
b) TPAF image of colorectal carcinoma
c) SHG/TPAF image of colorectal carcinoma
d) Hemotoxylin and eosin (H&E) staining of
colorectal carcinoma
Green: Collagen Fibres (SHG)
Red: Cellular Structure (TPAF)
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44. LUNG CANCER
By using Two Photon Auto Fluorescence (TPAF) and SHG, it can capture
3D structural organization of ECM as well as cellular morphologies in lung
or airway tissue with spectral specificity and sensitivity
Representative SHG image originating from fibrillar collagen in C
Overlaid with the TPAF image of healthy alveolar tissue is shown in D
C) SHG D) SHG + TPAF
a) SHG image shows normal collagen matrix
b) TPAF image shows normal alveolar tissue
c) Spirally wound form of collagen band around the elastic fibers
d) and e) Representative SHG image originating from the collagen matrix (D) overlaid with
the TPAF image of lung undergoing emphysematous destruction is shown E
f) The size of the ECM structures, particularly collagen, showed considerable variations as
evident from highly optically magnified image
A) 3D [SHG] B) 3D [SHG+TPAF] C) XY Plane [SHG + TPAF]
D) 3D [SHG] E) 3D [SHG+TPAF] F) XY Plane [SHG + TPAF]
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45. GASTRIC CANCER
TPAF has the ability to exhibit not only the mucosal and submucosal
microstructures of normal and cancerous gastric tissues but also the
distribution and content of abnormal cells in these 2 layers
Rows 1 and 2 represent microstructures of the mucosa epithelium and lamina
propria.
Rows 3 and 4 are from the submucosa.
Row 1 represents the abnormal epithelial cells
In row 2, pink circles indicate the boundary locations of representative
gastric glands; red circles indicate the nuclei of representative cells; blue
circles indicate the position of the gland cavity.
In row 3, blue arrows, pink arrows, red circles, and the blue circle
indicate collagen bundles, elastic fibers, abnormal cell nuclei, and the
lumen of the blood vessel, respectively.
In row 4, red circles indicate the nuclei of abnormal cells and blue circles
indicate the cavity of glandular tissue
TPAF Image SHG Image Overlay Image
Row 1
Row 2
Row 3
Row 4
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46. COLON CANCER
SHG microscopy has the potential in label-freely imaging the changes of
basement membranes for effectively distinguishing between normal,
precancerous, and cancerous colonic tissues
Image of SHG on colon cancer tissues
Green: Collagen Fibres (SHG)
Red: Cellular Structure (TPAF)
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47. KIDNEY CANCER
SHG and TPAF gave a detailed insight in cancer morphology and composition,
enabling to discern between normal kidney tissue, tumour and necrosis. Several
features can be seen clearly without the use of staining. It also greatly improves
speed and quality of the analyses.
(a) Image of the superficial kidney renal cortex shows dark renal
interstitium (RI), dark cellular nuclei (N) and bright intrinsic fluorescent
cytoplasm (CY) that form the epithelial cells in the renal tubules (RT), SHG
signal from the tough fibrous layer that forms the renal capsule (RC), and
the dark blood filled lumen (L) inside the renal tubules.
(b) Image of the inner colon wall shows bright intrinsic fluorescent signal
from entrocytes (E) surrounding dark circular crypts (C).
(c) Image of the rat liver showing ~20 µm diameter hapatocytes (coarse
dashed line) with dark nuclei (N, solid line) chained together to form hepatic
chords (HC), a dark bile duct (BD, fine dashed line) and bright intrinsically
fluorescent bile salts (BS), as well as SHG emission from the septa (S) a
fibrous tissue bands that separates hapatocyte nodules.
(d) Image of the rat liver without labels shown for clarity
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48. PANCREATIC CANCER
SHG and TPAF is used in a non-invasive manner and it provides real-time
histological evaluation of pancreatic cancer. Both of it also has ability to visualize
the morphology of fresh tissues associated with histology.
The pancreatic tumor xenografts harvested at different stages.
(A) 5 days after implantation
(B) 10 days after implantation
(C) 20 days after implantation
(D) 30 days after implantation
The SHG images (red color-coded), the TPAF images (green
color-coded), and the 3-D superimposed SHG/TPAF images are
shown in the first three columns respectively.
H&E images and the Masson's trichrome images are displayed in
the last two columns.
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49. GENESIS IMAGING SERVICES
(GLASGOW, UK)
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Services Capabilities
On Demand Tissue Imaging Services
Imaging System Shared access time
Tissue Imaging Solutions development
Grants Partnership:
o EU grants e.g. H2020
o UK national grants e.g. SBRI, MRC
grants, InnovateUK grants, etc
o University internal grants e.g. P2D
Breakthrough non-destructive tissue
imaging technologies including:
o Non-Linear Microscopy
Two Photon Excited Fluorescence
Second Harmonic Generation
o Image Processing and Analysis
capabilities
o Co-localization of stain and unstain
images