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

Serend-ip

1) AFM

Service

for adherent

cells

Diagnostic-Assays

DERMATACT

VERUM INDEX SUI ET FALSI

TBA

(Metastasis)

2) Computer Vision

for AFM (patented)

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

Main FOCUS:

Reading the TEXTure of Cells

= NANOScaleTopography for diagnostIC purpose

adhesive tape

corneocytes

stratum spinosum

stratum granulosum

stratum corneum

SKIN: Tape strips are non-invasive biopsiesDERMATACT

VERUM INDEX SUI ET FALSI

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

Electron Microscopy:

healthy control (smooth)

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)

SE

M

20 µm

AF

M

80 µm 5 µm

Method comparison

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

Measuring principles

Intensity Height !

SEM AFM

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

AD Non-lesional Sites

DTI < 50: OK DTI > 100: at risk

Clinical study on 24

children < 2y

seeking advice

because of AD

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)

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)

Corneocyes are mini-biopsies

comprising

The DTI indicates skin (barrier) integrity

Genetics (FLG)

Biochemistry (NMF)

Physiology (TEWL)

Clinics (SCORAD)

6 weeks treatment with glucocorticoids

Monitoring the EFFECT of THERAPY

Corticosteroids ameliorate SC nano-structure:

DERMATACT can monitor healing effects

Impact of irritantswith Dr. Lars Rüther, Dermatest GmbH, Münster

1d SDS

(detergent)

6d sampling

40 µm40 µm

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

Sun damage(unpublished)

CBA

D

A DCB

untreated:

Actinic Keratosis patients,

before and after therapy

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

✚ 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

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.