SNOTRA LLC.

Scanning probe nanotomography – a new technology for analysis and

diagnostics of biomaterials and polymers

The Problem We Address

Nanoscale structure analysis of majority (~80%) of biological and soft polymer materials require cryosectioning and investigation at low temperatures.

Existing approaches (f.e. cryoTEM) are very expensive and suffer from a number of issues (low EM contrast, beam damage, etc), especially for 3D analysis.

Thus a number of problems concerning nanoscale analysis of biological, polymer and other soft materials still do not have an adequate solution

SNOTRA LLC.The Goal:

Development and commercialization of methodology and combined analytical system for nanotomography – non-

destructive three-dimensional analysis of native nanoscale structures in a wide range of soft materials.

The Solution is based on combination of

scanning probe microscopy (nanoscale analysis of surface features)

and

cryoultramicrotomy (ultrathin sectioning of soft materials at temperatures down to -190°С).

Scanning probe cryonanotomography

Scanning probe microscopy (nanoscale surface analysis)

Cryo ultramicrotomy (ultrathin sections - down to 10 nm – of frozen samples)

2D (XY)

1D (Z)

+

=3D(XYZ)

SNOTRA LLC.A novel technology of scanning probe nanotomography

Is applicable to: biomaterials, biopolymers, other soft or hydrated polymers & materials (e.g. resins), nanoemulsions

Is ~ 10 times cheaper than currently available competing methods/devices (f.e.TEM electron tomography, SEM/FIB)

Provides access to unique information about nanostructure – not available by other existing methods.

How advantage is secured by the company IP protection (Russian patent filed, EU patent applied, exclusive

license agreement for EU patent application is signed), High-ranked and well-known scientists in the field are involved

(f.e. Dr. Prof. Ferdinand Hofer, Head of FELMI/ZFE Graz, Austria), Working prototype (proof-of-the-concept) of the system is

successfully built and 2 prototype systems are installed. Results are published in scientific journals and disseminated on

scientific conferences.

SNOTRA LLC.The company

Is established by inventors personally, Federal Research Center for Transplantology and Artificial Organs (Moscow), and Center of Innovative Enterprise (Moscow State University)

Is cooperating with one of the world leading scientific centers in the field of the project – Austrian Centre for Electron Microscopy (FELMI/ZFE Graz, Austria)

Is a participant of Skolkovo Foundation

AFM TEM

AFM versus TEM

AFM provides the data on molecular structure based on the analysis of the morphology and mecha-nical properties of the surface

The block sur- face is mea-sured directly inside the cryogenic chamber – in the environ-ment of the sectioning procedure

Ultrathin sections

( 10..90 nm ) are

transferred into the vacuum

chamber of the TEM

Low contrast on biological and polymer samples

Projection image is the average of all the planes that constitute the thickness of the section

Electronbeam

AFM probe Interacting atom

UrAc

Chemical da-mage inside the material

The methods of nanostructure analysis(biology&polymers)

Product orTechnology

ResolutionCost

Preparation and damage to the sample structureXY, nm Z, nm

SPM + CryoUMT(SNOTRA)

5..10 10-20~250

k$

Intact native structures of soft polymers and biomaterials are measured

(cryoultratomography and immediate measurement)

Conventional SPM, Bruker, Asylum, ...

5..10Нет !измеряет

поверхность

~200 k$

Structures are damaged (measurements at room temperature)

CryoSPM, Omicron, JEOL, ... 5..10

>300 k$

Hard to exploit(vacuum, liquid He or N2 environment –

not suited for bio/polymers)

SEM Tomography (Focused-ion-beam sectioning), FEI, Zeiss

10 ~10-20>1 M$

Structures are damaged(electron and ion beams, vacuum, metal sputtering), no cryo 3D at the moment

CryoTEM (electronic tomography), FEI, Hitachi

5

5Sample

thickness

<100 nm

> 1M$

Structures are damaged(electron and ion beams, vacuum), projection imaging, low contrast at

biological and polymer samples

No!Measures only the surface

Tuning fork-based AFM probe

Cryo-AFM module is installed directly into the cryochamber

Leica EM UC6/FC6 cryoultramicrotome performs ultrathin sectioning of soft materials at temperatures from -15 to -190°C. Section thickness ranges from 10 nm to 1 um.

SNOTRA LLC.

Applications and examples of results

by scanning probe nanotomography

Nitrile butadiene-rubber latex

(a) A topographical AFM image of an epoxy embedded latex stripe that was mounted in the cryo-chamber of SNOTRA, cryo-sectioned and immediately scanned at −120°C.(b) Immediately afterwards, the same sample was warmed to room temperature and then examined using the same AFM (c) A TEM image of the last thin section of the NBR latex sample. (d) A schematic description of the topographical change of the sample block phase that took place during sectioning and the following warming processes. The scale bars in (a, b, c) are 200 nm, and the topographical variations in (a) was 27.2 nm, and in (b) was

35.5 nm.

A. E. Efimov, H. Gnaegi, R. Schaller, W. Grogger, F. Hofer and N. B. Matsko,, Soft Matter, 2012, 8, 9756, DOI:10.1039/c2sm26050f

3D reconstructions of the antennal sensillas Placodea and Coeloconica 12.5×13.0×0.7 µm3, 11 sections, section thickness 70 nm. .

3D model of chitin organization of wasp antenna sensillasАFМ

ТЕМ

3D reconstruction of PA6/SAN polymer nanocomposite e) АFМ- topography after cryosection at -80°C. f) АFМ- topography after section at room temperature . g, h) 3D reconstruction at -80°С: 7.9×6.2×0.75 µm и 2.0×2.0×0.75 µm, correspondingly, 125 nm sections.

3D reconstruction: porous polyoxibutirate biodegradable cell matrix for regenerative medicine use. 20 sections, 41.2×34.1×8.5 µm

V. N. Vasilets, I. V. Kazbanov, A. E. Efimov and V. I. Sevastianov, Vestnik transplantologii i iskusstvennyh organov, 2009, 9(2), 47 (in Russian).

3D reconstruction of ABS copolymer: 15 sections x100 nm at room temperature. 13.5x13x1.5 µm

. .

3D reconstruction of conductive nanocomposites

3D-reconstruction of the conductivity of the network of graphene flakes inside the PS/ graphene nanocomposite, 2.5x2.5x0.34 umA. Alekseev et al, Adv. Func. Mater., 22, 1311 (2012).

3D-reconstruction of the conductivity of the nanotube network inside the polysty-rene/CNT composite, 1.8x1.6x0.26 um. A. Alekseev, A. Efimov, K. Lu, J. Loos, Adv. Mater., 21, 4915 (2009)

AFM image and 3D-reconstruction of cholesteric liquid crystal structure with implanted fluorescent CdSe/ZnS quantum dots, 14 sections (/100 nm)

3D study of liquid crystal / quantum dots nanocomposite

A. Bobrovsky et al, Advanced Materials, 2012, DOI: 10.1002/adma.201202227;

K. Mochalov et al, Proc. SPIE 8475, Liquid Crystals XVI, 847514, 2012, doi:10.1117/12.929729

a

3D study of liquid crystal / quantum dots nanocomposite

3D-AFM reconstruction and 2D AFM image (4x4 um) of cholesteric LC structure with implanted fluorescent CdSe/ZnS quantum dots (arrows indicate the same QD on AFM image and 3D AFM reconstruction. We observe that implanted QD do not distort the planar LC structure

K. Mochalov, A. Efimov et al, Proc. SPIE 8767, Integrated Photonics: Materials, Devices, and Applications II, 876708 (2013); doi:10.1117/12.2017088

Scientific publications for our technology1) A. E. Efimov, H. Gnaegi, R. Schaller, W. Grogger, F. Hofer and N. B. Matsko, Analysis of native structure of soft materials by cryo scanning probe tomography, Soft Matter, 2012, 8, 9756, DOI:10.1039/c2sm26050f 2) K. E. Mochalov; A. Yu. Bobrovsky; V. A. Oleinikov; A. V. Sukhanova; A. E. Efimov; V. Shibaev; I. Nabiev, Novel cholesteric materials doped with CdSe/ZnS quantum dots with photo- and electrotunable circularly polarized emission, Proc. SPIE 8475, Liquid Crystals XVI, 847514 (October 11, 2012)3) Bobrovsky, A., Mochalov, K., Oleinikov, V., Sukhanova, A., Prudnikau, A., Artemyev, M., Shibaev, V., Nabiev, I. Optically and electrically controlled circularly polarized emission from cholesteric liquid crystal materials doped with semiconductor quantum dots. Advanced Materials, 2012, DOI: 10.1002/adma.2012022274) A. Alekseev, D. Chen, E. E. Tkalya, M. G. Ghislandi, Yu. Syurik, O. Ageev, J. Loos, and G. de With Local Organization of Graphene Network Inside Graphene/Polymer Composites Adv. Funct. Mater. 2012, 22, 1311–1318 5) V.Mittal and N.B.Matsko, Tomography of the Hydrated Materials, in Analytical Imaging Techniques for Soft Matter Characterization, Engineering Materials, Springer-Verlag Berlin Heidelberg, 2012, pp. 85-93 6) A. Efimov; H. Gnaegi; V. Sevastyanov; W. Grogger; F. Hofer; N. Matsko, Combination of a cryo-AFM with an ultramicrotome for serial section cryo-tomography of soft materials - Proceedings 10th Multinational Congress on Microscopy 2011 SEP 4-9, 2011; Urbino, ITALY. pp.707-7087) N. B. Matsko, J. Wagner, A. Efimov, I. Haynl, S. Mitsche, W. Czapek, B. Matsko, W. Grogger, F. Hofer, Self-Sensing and –Actuating Probes for Tapping Mode AFM Measurements of Soft Polymers at a Wide Range of Temperatures, Journal of Modern Physics, 2011, 2, pp. 72-788) A. Alekseev, A. Efimov, K. Lu, J. Loos. Three-dimensional electrical property reconstruction of conductive nanocomposites with nanometer resolution, Advanced Materials, Vol. 21, 48 (2009), рр. 4915 – 49199) A. Efimov, V. Sevastyanov, W. Grogger, F. Hofer, and N. Matsko. Integration of a cryo ultramicrotome and a specially designed cryo AFM to study soft polymers and biological systems, MC2009, Vol. 2: Life Sciences, p. 25, Verlag der TU Graz 2009.10) A. E. Efimov, A. G: Tonevitsky, M. Dittrich & N. B. Matsko. Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples. Journal of Microscopy, Vol. 226, Pt 3, June 2007, pp. 207–217

Revenue Generation

Company Revenue

Model

MarketsScientific & industry

customers specialized in:

1. Biomaterials

2. Polymers

3. Pharmaceuticals

4. Liquid crystals

5. Nanoemulsions and nanoliquids

6. Other soft/hydrated materials

Products:1. CryoUMT/AFM

system

2. CryoUMT/Raman/AFM system

(next stage of development)

Service:Analytical services with use of our nanotomography technology

World market estimation*

* According to report by Future Markets, Inc. 2011

We develop unique product valuable for many users of AFM/SEM/TEM in rapidly growing Biomedical, Polymers and Nanomaterials market

segments

We plan to achieve 10 M$ market share in 5 years

AFM/SEM/TEM (total), M$ AFM/SEM/TEM,Biomed+Polymers+Nanomaterials, M$

2010 328,44 97,6

2015 (prognosis)

588,69 217,39

Companies and institutions expressed interest in the technology • Dow Chemicals (Dow Benelux BV, polymers )

• FELMI/ZFE Graz (Austria, microscopy center)

• Boeckeler Instruments (USA, equipment for microscopy preparation and ultramicrotomy)

• Anton Paar (Austria, analytical equipment)

• Boston Scientific (USA, drug translucent stents)

• Weizmann Institute (Israel, biomaterials)

• Carmel Olefins (Israel, polymers)

• Nike IHM (USA, multilayer polymers for the shoes)

• Cornell University (USA, nanofibers for textile)

• Shell International (Netherlands, Dr. R. Haswell, fuel membranes)

• General Motors, Rochester Lab (USA, fuel membranes)

• Xerox Inc. (USA, 3-D structure of ink and paint particles)

• Nova Nordisk (USA, polymers)

• Nazarbaev University (Kazakhstan, microscopy center)

• Institute of Biophysics, Chinese Academy of Sciences (structural cell biology)

SNOTRA LLC.

We are looking for partners:

Customers for our devices and analytical servicesPartners for collaborative research projectsPartners for commercialization and market entryInvestors for the company and technology

SNOTRA LLC.

SNOTRA LLC. as a participant of Skolkovo Foundation is applying for a grant funding.

Terms of financing of R&D projects by Skolkovo Foundation (stage 1):

Total project budget: up to 1 Million Euro for 3 years Co-investment: 25% = 250 000 Euro

Granted financing: 750 000 Euro

SNOTRA LLC. is looking for co-investors for this grant application and may offer a share in the company.

SNOTRA LLC.

Contact us:General Director, CEO: Dr. Anton Efimov,E-mail: antefimov@gmail.comPhone: +7(916)1642475Mail: SNOTRA LLC., Shcherbakovskaya str.,3,

Moscow, 105318 Russia