Nano-scale Optical and Hyperspectral Imaging CytoViva, Inc.

Sam Lawrence – Chief Executive Officer

Integrated optical & hyperspectral imaging technology… • Patented high S/N illumination optics, specifically

designed for imaging nano-scale samples

• VNIR hyperspectral imaging system (HSI) enabling

pixel level spectral characterization of the sample being imaged

…specifically designed to advance nano-scale research.

Live oral cancer cells with

aggregating Au nanoparticles

Technology

Overview

VNIR

Spectrophotometer Optical Camera

Optical Image Hyperspectral Data

CytoViva Equipped

Optical

Microscope

Motorized

Stage 150w Halogen Light

Source

CytoViva Dual

Mode

Fluorescence

Module

Current System

Footprint

Functionality Enabling Improved Optical Performance: 1. Pre-aligned Koehler illumination: Precisely focuses

the source light onto the entrance slit of the annular condenser

2. Main feature of critical illumination: Focuses the light precisely on the same plane of the sample as the focal point of the objective; result of pre-aligned Koehler illumination

Patented Optical Illumination System enables nano-scale imaging

Pre-aligned

Koehler Illumination

Critical Illumination

* Optics Letters, Vol. 31, Issue 19, pp. 2855-2857

* Annular illumination produces an improved point spread function. Through design enhancements in the alignment and focus of annular Illumination, CytoViva produces significantly improved optical performance over other comparable techniques including standard darkfield (annular) illumination.

Optical

Imaging

How It Works

• The VNIR spectrophotometer mounts onto a microscope equipped with the CytoViva Optical Illumination System

• It captures the unique reflectance spectra of objects from the microscope field of view (VNIR spectrum from 400nm-1,000nm)

• The complete spectra for each pixel of the

CCD detector is captured (pixel size as small as 128nm)

• Spectral data is reported in high resolution (down to 2.0nm)

• The data is presented as a spectral curve

and as a RGB image

• Detailed quantitative analysis of each object in the field of view can be performed.

Diagram of Diffraction Grating Hyperspectral Imaging System

Hyperspectral

Imaging (HSI)

Comparison of standard darkfield vs. CytoViva Optical Illumination System

Both images represent the identical field of view of 50nm colloidal gold captured under identical conditions: Olympus BX-41, 40X objective, Dage MTX color digital camera at a constant exposure and an X-Cite 120 light source.

CytoViva Optical Illumination System Standard darkfield condenser

50nm AuNP on glass substrate 50nm AuNP on glass substrate

Optical

Imaging

a.) Standard darkfield microscopy

b.) CytoViva Optical Illumination System

S/N Response of CytoViva Optical Illumination System

• Nanoparticle target is

scanned

• Unique spectral signatures of nanoparticle is loaded in a library database

• New sample containing

nanoparticle is scanned

• Using nanoparticle spectral library, nanoparticle is mapped in the new sample

AuNPs mapped (in red )

Library spectra from AuNP

Epithelial cells incubated with AuNPs: hyperspectral image

AuNPs in solution: hyperspectral image

Mapping AuNPs

in live cells

NIST IIT Madras India Dow Chemical

Nanomaterials cell interactions Nanomaterials development Nanoparticle analysis

FDA Taiwan National University Johnson & Johnson

Nanotoxicology Nanomedicine Nanoparticles in composites

NASA Rice University Pfizer Nanocomposites Nanomaterials characterization Pathogen characterization

Argonne National Labs Max Planck Institute Germany Schering-Plough

Nanoparticle development Nanoparticle development Nanoemulsions

NIH-National Cancer Institute University of Sao Paulo Merck

Nano drug characterization Nanomaterials development Nanomedicine NIOSH MD Anderson Cancer Center Proctor & Gamble Nanotoxicology Nanomedicine Emulsions & nanoparticles Lawrence Berkeley Labs Agroscope Zurich Switzerland Novozymes Biofuels development Nanoparticle plant tissue interaction Biofuels development

Sample Clients

and Applications

Nanomaterial Characterization

Examples

200nm Au shells (no PEG) 200nm Au shells (PEG coated) 200nm Au shells (no PEG)

PEG coated NPs are spectrally different from non-PEG coated

Characterizing Drug-Coated

Nanoparticles

AgNPs in Solution

Spectra from individual non-dispersed AgNPs

Aggregating AgNPs in water

Dispersed AgNPs in ionic solution Spectra from individual dispersed AgNPs

Hyperspectral Scan of Toray Fiber Sample #1 No CNT Coating

Hyperspectral Scan of Toray Fiber Sample #3

Specialized GOx CNT Coating

Hyperspectral Scan of Toray Fiber Sample #2

Raw CNT Coating

Hyperspectral measurements of single pixel areas from the three different samples quantitatively confirms different surface chemistry from each sample.

CNT characterization

application

Hyperspectral Image Scan Reference Spectra of GOx coated CNTs

Scanned image and Zoom illustrating GOx coated CNTs adhering to fiber

Overlay image illustrating all areas in red containing the selected spectral profile and location of the GOx functionalized CNTs

CNTs Mapped in Sample

Derived from previously scanned pure GOx coated CNT sample

CNT characterization

application

Nano-Bio Interface

Examples

Hyperspectral image 100x: Algae cell + AgNP

AgNPs in Algae

spectral library combined : Ag from all environments

Mapping in red all pixels matching spectral library Algae cell + AgNP

spectral library: AgNPs inside algae cell: chlorophyll modifies AgNP spectral response

AgNPs in Algae

Spectral signatures collected from the CNT sample and loaded into the

spectral library

Aggregating CNTs on glass slide

MWCNTs inhaled by mouse

in lung tissue

The red areas are the pixels matching the CNT spectral profiles within the spectral library.

Hyperspectral Scanned Image 100X

Carbon Nanotubes inhaled

by mouse in lung tissue

AuNP in Cancer Cells

Hyperspectral image: Rala coated AuNP in cells

Spectral library created from aggregating Rala coated Au particles in cells

Pixels in red indicate presence of aggregating Rala AuNP spectra in cells.

AuNP pixel match classification

Spectral Image: Sample 6

Spectral library

created from

Sample 6

Pixels in red indicate presence of cisplatin in tissue

Mapping bare drug

in tissue

TiO2 Injected in Whole

Animal Organism

Whole animal organism control TiO2 nanoparticles injected in whole animal organisms

TiO2 nanoparticles in solution

Note: This spectra response incorporates characteristics of both samples

CytoViva Inc. is a private company affiliated with Auburn University…

CytoViva technology is utilized in over 300 research laboratories world-wide

U.S.

Canada

Taiwan

Singapore

India

Japan

UK

Korea

Australia South Africa

Global

Presence

Italy Switzerland

Germany

Norway

Netherlands

Brazil

China

CytoViva

3D Optical Imaging