atomic force microsopy (afm)...1 atomic force microsopy (afm) tactile method marker-free...
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1
Atomic Force Microsopy (AFM)
tactile method
marker-free
ultra-resolution (1 nm! in height)
Nanoscale phenotyping for skin health
Dr. Christoph Riethmüller
© Serend-ip GmbH, Münster
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Serend-ip
1) AFM
Service
for adherent
cells
Diagnostic-Assays
DERMATACT
VERUM INDEX SUI ET FALSI
TBA
(Metastasis)
2) Computer Vision
for AFM (patented)
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Serendip‘s USP: Quantitation of cellular nanomaps
• Vacuoles
• Microvilli (Ex.
2)
• Intracellular
Fibers (f-actin)
• Cell Borders (Ex.1)
• Extracellular Matrix
US-Pat 8,798,935
EP 2 435 829
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Main FOCUS:
Reading the TEXTure of Cells
= NANOScaleTopography for diagnostIC purpose
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adhesive tape
corneocytes
stratum spinosum
stratum granulosum
stratum corneum
SKIN: Tape strips are non-invasive biopsiesDERMATACT
VERUM INDEX SUI ET FALSI
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Skin inflammation, a vitious cycle:
reduced
Barrier function
Infiltration
(germs, chemical noxes)
Immune reaction
(Itching)
Mechanical stress
(Scratching)
Atopic Dermatitis (AD): Non-curable, chronic disease, 3 Mio. patients in D
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Electron Microscopy:
healthy control (smooth)
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With higher magnifications (x2.700-4.000) villous projections could be seen in the cell membrane, as well as the irregular cell outline and some Figure 5).
Scanning electron microscopy - detail of the lower corneocytes showing irregular cell outline, intercellular clefts and villous projections (x2.700- 4.000).
Diseased skin exhibits
nano-protrusions
(known since 1979)
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SE
M
20 µm
AF
M
80 µm 5 µm
Method comparison
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raw data processed single image
statistics
object
classification
circularity
height (nm)
area (µm^2)
LDVolume (al)
n = 187
A
B C
n (
ob
ject
s)n
(ob
ject
s)n
(ob
ject
s)n
(o
bje
cts)
⇔⇔
D
500
550
650
Hei
gh
t (n
m)
600
1.0 1.5 2.0 2.5 x (µm)
DTI
de
rma
l te
xtu
re in
de
x
Text
com
pute
r vis
ion o
pera
ting p
rocedure
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Measuring principles
Intensity Height !
SEM AFM
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Severity of atopic dermatitis (IGA-Score)
Ob
jec
t c
ou
nt
Retrospective analysis on existing AFMdata (AFM-images recorded by Dr. Gorzelanny)
DTI
clinical window
subclinical
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AD Non-lesional Sites
DTI < 50: OK DTI > 100: at risk
Clinical study on 24
children < 2y
seeking advice
because of AD
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non-lesional DTI correlates to classical measures
Genetics (filaggrin) Biochemistry (NMF)
Physiology (Barrier function) Clinical Score (of lesional areas)
(Study on children < 2yrs, Riethmüller et al. JACI 2015)
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Age and Pigmentation do not matter
Outliers reveal a hereditary background,
(elevated count hints at individual risk)
Age (years)
outliers are healthy, but have a
hereditary background in skin disease
Skin Type
(Study on healthy individuals, Franz et al., SRT 2016)
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Corneocyes are mini-biopsies
comprising
The DTI indicates skin (barrier) integrity
Genetics (FLG)
Biochemistry (NMF)
Physiology (TEWL)
Clinics (SCORAD)
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6 weeks treatment with glucocorticoids
Monitoring the EFFECT of THERAPY
Corticosteroids ameliorate SC nano-structure:
DERMATACT can monitor healing effects
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Impact of irritantswith Dr. Lars Rüther, Dermatest GmbH, Münster
1d SDS
(detergent)
6d sampling
40 µm40 µm
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Kinetics of irritant impact(unpublished)
1day exposure to SDS (detergent), 6 days follow up
Moisturizing FactorBarrier function Nanomorphology
DTI
Max Effect on day 1-2 day 3 day 5
DTI
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Sun damage(unpublished)
CBA
D
A DCB
untreated:
Actinic Keratosis patients,
before and after therapy
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The skin barrier is subject to change by
Skin Barrier
external factors(chemicals, UV / sunlight,
mechanical stress, therapy)
internal factors(genetics, inflammation, lipids)
Change in DTI
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✚ painless
✚ easy
✚ precise
DERMATACT works at non-lesional skin areas
to:
➤ predict an individual risk for skin diseases (subclinically)
➤ abbreviate clinical studies (reduce study population)
➤ enable effective therapy monitoring (pro-actively)
Conclusion: DTI indicates skin stress level
DERMATACT
VERUM INDEX SUI ET FALSI
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Figure:Immunogold SEM of
corneodesmosin
Dr. M. Haftek, Uni
Lyon
FL
G +
/+F
LG
-/-
FL
G -
/+
References1: Soltanipoor M, Stilla T, Riethmüller C, Thyssen JP, Sluiter JK, Rustemeyer T, Fischer TW,
Kezic S, Angelova-Fischer I. Specific barrier response profiles after experimentally induced
skin irritation in vivo. Contact Dermatitis. 2018 Apr 2.
2: Engebretsen KA, Kezic S, Riethmüller C, Franz J, Jakasa I, Hedengran A, Linneberg A,
Johansen JD, Thyssen JP. Changes in filaggrin degradation products and corneocyte surface
texture by season. Br J Dermatol. 2018 Mar 7
3: Engebretsen KA, Bandier J, Kezic S, Riethmüller C, Heegaard NHH, Carlsen BC,
Linneberg A, Johansen JD, Thyssen JP. Concentration of filaggrin monomers, its metabolites
and corneocyte surface texture in individuals with a history of atopic dermatitis and controls. J
Eur Acad Dermatol Venereol. 2018 Jan 23
4: Koppes SA, Ljubojević Hadžavdić S, Jakasa I, Franceschi N, Riethmüller C, Jurakić Tončic
R, Marinovic B, Raj N, Rawlings AV, Voegeli R, Lane ME, Haftek M, Frings-Dresen MH,
Rustemeyer T, Kezic S. Effect of allergens and irritants on levels of natural moisturizing factor
and corneocyte morphology. Contact Dermatitis. 2017 May;76(5):287-295.
5: Bianco C, Visser MJ, Pluut OA, Svetličić V, Pletikapić G, Jakasa I, Riethmuller C, Adami G,
Larese Filon F, Schwegler-Berry D, Stefaniak AB, Kezic S. Characterization of silver particles
in the stratum corneum of healthy subjects and atopic dermatitis patients dermally exposed to
a silver-containing garment. Nanotoxicology. 2016 Dec;10(10):1480-1491
6: Franz J, Beutel M, Gevers K, Kramer A, Thyssen JP, Kezic S, Riethmüller C. Nanoscale
alterations of corneocytes indicate skin disease. Skin Res Technol. 2016 May;22(2):174-80
7: Riethmuller C, McAleer MA, Koppes SA, Abdayem R, Franz J, Haftek M, Campbell LE,
MacCallum SF, McLean WHI, Irvine AD, Kezic S. Filaggrin breakdown products determine
corneocyte conformation in patients with atopic dermatitis. J Allergy Clin Immunol. 2015
Dec;136(6):1573-1580.e2.