airSEM™ webinar Dr. Dubi Shachal, CEO

December 2016

2

Outline

Technology

Value proposition

Used cases

3

The paradigm shift

Existing Vacuum SEM airSEM Microscope

In contrast to traditional vacuum SEM that requires a vacuum chamber to visualize high resolution images, the airSEM technology allows imaging at

ambient conditions in open air

Vacuum chamber

4

How does it work ?

Field emitter SEM column

Semi- transparent membrane

Sample holder

Detector

airSEM Column:

High resolution Schottky field emitter SEM column

Beam energy range: 10-30kV

Typical beam currents: 0.1-2nA

air gap characteristics

Typical working distance 50-300 µm

Scattering is minimized by introducing He to this region

Membrane thickness is typically few or 10’s of nanometres

Resolution: 5nm@30kV

5

airSEM™ detectors and imaging modes

BSE EDX Surface detector airSTEM

airSEM

Lung tissue section

Upright light microscope

Inverted light microscope

Controlled environment

Correlative image fluorescence/airSEM

6 confidential

What is it good for?

Shorter time to data Minimal sample preparation

7

No Charging

Operating in air provides inherent charge neutralization mechanism overcoming the need for special preparation steps to minimize charging on non-conducting substrates as glass

airSEM™ Vacuum SEM

The images were generated in air as is without any coating

Simplified sample preparation flow (1)

8 confidential

Simplified sample preparation flow (2)

airSEM™ surpasses the steps of fixation, dehydration, drying or coating which in many cases alter the chemistry of the sample and its structure and may introduce artifacts

9 confidential

What is it good for?

Shorter time to data Minimal sample preparation

Simple and rapid application flow

10

Simple application flow (1)

Step 3 Step 1 Step 2

Imaging with optical microscope

High resolution Imaging and localized elemental composition

Sample moving between the two microscopes

Wafer imaged for orientation and for the target area selection (ROI)

Having both microscopes on the same platform enables precise navigation between the various imaging channels

Both imaging and elemental composition using EDX are performed with the airSEM™ on the target area

airSEM™ OM

11

Imaging beyond the common practice used: Rat lung section (Bronchus)

x1000 x4000 X10,000

x1,000

Simple application flow (2)

12 confidential

What is it good for?

Shorter time to data Minimal sample preparation Simple and rapid application flow Correlative microscopy

13 confidential

Correlative imaging

Light microscope

One of the advantages of the airSEM™ imaging station is simplifying the process of correlating images taken with different modalities on the same ROI.

BSE EDX map

Cross section of brakes pad

14 confidential

What is it good for?

Shorter time to data Minimal sample preparation Simple and rapid application flow Correlative microscopy

Enabling imaging of samples/processes Vacuum non compatible samples: liquids/high vapor

pressure materials/explosives

Kayla Nguyen, Justin Richmond-Decker, Megan Holtz, David Muller

airSTEM 30keV

Imaging sulfur nano-particles

For vacuum SEM, surfur nano-particles need to be encapsulted to prevent sublimation. With airSEM, one can directly image such particles

16 confidential

Can we image wet samples?

250nm Au particles imaging on Agar substrate

17 confidential

What is it good for?

Shorter time to data Minimal sample preparation Simple and rapid application flow Correlative microscopy

Enabling imaging of samples/processes Vacuum non compatible samples: liquids/high vapor

pressure materials/explosives

Follow processes on the nanometer scale

18

Value Proposition (V)

Follow chemical/physical processes on the nanometer scale

airSEM enables to follow changes on the nanometer scale triggered by:

Chemistry: gas phase or solution

Temperature

Interaction with light

Pressure

…

Step I Step II Step III

19 confidential

Chemical “bath”

20 confidential

Details

The etching of individual ZnO nano crystals were followed using airSEM

A dedicated sample holder allowing introducing and removing chemicals while keeping the sample in the same location under the microscope was used

After finding the area of interest, the sample was exposed to HCl (0.1%) for 30sec. The solution was removed and imaging was preformed without washing the sample with clean water

Imaging was always performed on a dry sample

This sequence was performed several times

For the last two iterations, prolonged exposure to the solution (10 minutes) were used

21 confidential

22 confidential

23 confidential

Life Science Used Cases

24 confidential

Correlative microscopy on tissue

Efforts to combine fluorescence and EM have been hindered by the divergent and incompatible sample preparation protocols of the two methods. The fixation and staining required to preserve good ultrastructure can destroy protein fluorescence and/or increase the auto-fluorescence background.

Direct correlation ensures minimal sample shrinkage, deformation etc. producing a highly reliable correlation.

The high intensity fluorescent signal shows the localization of collagen fibrils around alveolar cavities

In Courtesy of Inna Solomonov, Dalit Talmi-Frank, Lab of Prof Irit Sagi. Department of Biological regulation

25 confidential

Dual labeling correlative microscopy

Problem I: Carbon coating artefact Problem II: the protein was destroyed during the dehydration process

Objective: to study the role of specific protein in the Extra Cellular Matrix, using dual labeling correlative fluorescence and SEM

Problem: Sample preparation procedures

In collaboration with Yael M. and Prof. Debbie Fass Department of Structural Biology

26 confidential

Immuno-gold labeling imaging

15nm gold nanoparticles

Fibroblasts cells with ECM, fixated and stained using antibodies - Fibronectin labelled with (15nm)

colloidal gold nanoparticles, were imaged with airSEM under ambient condition

In collaboration with Yael M. and Prof. Debbie Fass Department of Structural Biology

27 confidential

airSTEM + fluorescence correlative microscopy Human Carcinoma Cells Fresh leaf

High resolution STEM detection is possible in open air, despite the scattering of the electron beam by the gas molecules, good contrast is achieved for sample detector separations up to 1nm

Avian brain sections

28 confidential

Step 1: Fluorescence imaging Step 2: Fixation Step 3: Water removal Step 4: airSEM imaging

• In Vitro (liquid) live cell Imaging over time, using Inverted confocal fluorescence Microscope in conjunction with airSEM™

• Adding fixative into the cell culture medium

• Remove the liquid but keep the cells in a humid environment so its structure/chemistry is unaltered

• Image with airSEM under humid environment

Collinear imaging of cell culture

airSEM

HNA Confocal microscopy

Petri dish with cell culture in liquid

airSEM airSEM airSEM

Fixative Water removal

A dedicated collinear airSEM™ imaging station allowing imaging fully immersed biological samples using an inverted optical microscope followed by imaging the same location of the sample with the airSEM™ under semi hydrated state

confidential

Thank you !

30

Image gallery Ag nano crystals

lead halide Perovskite Perovskite (FAPbBr3) Metallographic

section

Silica nanoparticles Alumina filter

Paper with ink Zinc Oxide

31 confidential

Cell culture

32 confidential

Tissue sections in natural state lung bronchus

33 confidential

Biological samples under natural state Coccolithophores Butterfly wing Fresh leaf

Sea Sponge Mouse oocyte Powdery mildew