DIDO YOVA

LABORATORY OF BIOMEDICAL OPTICS AND APPLIED BIOPHYSICS

SCHOOL OF ELECTRICAL AND COMPUTERS ENGINEERINGNATIONAL TECHNICAL UNIVERSITY OF ATHENS

LABORATORY OF BIOMEDICAL OPTICS AND LABORATORY OF BIOMEDICAL OPTICS AND APPLIED BIOPHYSICSAPPLIED BIOPHYSICS

OPTICAL IMAGINGOPTICAL IMAGING

CONFOCAL LASER SCANNING MICROSCOPYCONFOCAL LASER SCANNING MICROSCOPY

IMAGING AT THE CELLULAR LEVELIMAGING AT THE CELLULAR LEVELTISSUE IMAGINGTISSUE IMAGING

AFM AND SHG MICROSCOPYAFM AND SHG MICROSCOPY

IMAGING OF BIOMOLECULESIMAGING OF BIOMOLECULES

TISSUE IMAGINGTISSUE IMAGING 3D BINOCULAR MACHINE VISION SYSTEMFLUORESCENCE MOLECULAR IMAGING

Imaging at the Cellular LevelImaging at the Cellular Level

Various imaging technologies are developing to understand and optimize PDT process.

New developments in microscopy are providing crucial information and essential approaches for understanding the structure and function of cells and molecules.

Combined with:

Recent developments in computing

and Molecular probes

Offer great promise for delivery of vital new information.

IMAGING AT THE CELLULAR LEVELIMAGING AT THE CELLULAR LEVELIN PDTIN PDT

The mechanism of tumor destruction by PDT is very complex and is still under investigation. Photoactivation initiates photochemical reactions generating highly cytotoxic reactive oxygen species (ROS) The initial insult is a form of oxidative stress which triggers a variety of events contributing to the inactivation of cancerous cells.

A very interesting problem is to image the cascade of events of induced oxidative stress at the cellular level.

Imaging at the Cellular LevelImaging at the Cellular LevelMonitoring early events of cellular response to oxidative Monitoring early events of cellular response to oxidative

stressstress

We investigated the cascade of early intracellular phenomena evoked by oxidative stress in real time at the single cell level. Oxidative stress was induced by photosensitization of ZnPc in Human Fibroblasts using the 647 nm laser line, using a dose that did not lead to apoptosis or necrosis.

By :• Confocal Laser Scanning Microscopy• Vital Fluorescent Probes• Photosensitive Molecules• Advanced Image Analysis and Processing

Fibroblasts coincubated with ZnPc and MitoTracker Green.Fluorescence image of ZnPc λexc:647nm, λem:680 nm

Fibroblast incubated with MitoTracker Green λexc:488nm, λem:522 nm

Merged image of the red and green fluorescence. By advanced colocalization algorithm, ZnPc is above 85% localized in the mitochondria.

Detection of intracellular ROS (Reactive Oxygen Species) generated by ZnPc photosensitization using

H2DCFDA.

Fibroblasts incubated with ZnPc + H2DCFDA (after

oxidation by ROS produces DCF)

Pseudocolored image

Mitochondrial membrane potential (ΔΨm) decrease

after ZnPc photosensitization + JC-1.

0 min0 min 1 min1 min

3 min3 min 8 min8 min 15 min15 min

30 s30 s

Resting ΔΨm =140 mV ΔΨm =90 5 mV after oxidative stress

0 min0 min 2 min2 min

3 min3 min 5 min5 min

30 s30 s

10 min10 min

Resting pHi = 7.45 0.03

ΔpHi=0.40 0.08 after oxidative stress

Intracellular pH changes after ZnPc photosensitization using the membrane

permeable (BCECF-AM) probe .

Spatiotemporal global Ca2+ oscillations evoked by ZnPc photosensitization monitored by Fluo-3 (pseudocolored

images).

30 s30 s 1 min1 min 2 min2 min

4 min4 min 30 s30 s 1 min1 min 2 min2 min

0 min0 min

Time course experiment of intracellular Ca2+concentration. Resting [Ca2+ ] 60nM

[Ca2+ ] 0.25μM after oxidative stress

Development of animal models.

Research related to small animals optical imaging

TISSUE OPTICAL IMAGINGTISSUE OPTICAL IMAGING

Non-melanoma carcinomas in SKH-1 mice

NMSC ANIMAL MODELNMSC ANIMAL MODEL

Typical series of confocal images obtained horizontally, at 0, 20, 40 and 60 μm from skin surface, of a healthy hairless mouse 1 hour after topical

application of AlClPc. Images were acquired with excitation at 647 nm and emission at 680 nm.

Confocal image obtained from a cross-section of a non-melanoma skin carcinoma topically applied with AlClPc for 1 hour. Images were acquired

with excitation at 647 nm and emission at 680nm. The yellow line indicates the penetration depth. Scale bar: 100 μm.

Penetration depth 1490 μm

PDT in DERMATOLOGY PDT in DERMATOLOGY OPTICAL IMAGING MONITORINGOPTICAL IMAGING MONITORING

Answers to be given:Accurately imaging tumors smaller than 1

cm.

As PDT is a repeatable technique to monitor tumour shrinkage, after each PDT treatment, will facilitate the optimization of therapy.

3-D Binocular Machine Vision System for 3-D Binocular Machine Vision System for Gauging Small TumorsGauging Small Tumors

3D Binocular Machine Vision System for 3D Binocular Machine Vision System for Gauging Small TumorsGauging Small Tumors

Animal model for NMSCAnimal model for NMSC

Normalarea

Tumourarea

3-D Binocular Machine Vision System for Gauging 3-D Binocular Machine Vision System for Gauging Small TumorsSmall Tumors

3-D Binocular Machine Vision System for Gauging Small 3-D Binocular Machine Vision System for Gauging Small TumorsTumors

Successful reconstruction and gauging of tumours smaller than 1 cm maximum diameter via a fully automated software package.

Surface rendering and gauging tool for skin tumours imaging and following of their shrinkage after PDT treatment.

Prospects of other medical applications like in burn depth estimation, by introducing an articulated arm.

Useful in a variety of other 3-D gauging applications like in archeology.

FLUORESCENCE MOLECULAR IMAGINGFLUORESCENCE MOLECULAR IMAGINGin PDTin PDT

Non-invasive monitoring of molecular targets is particularly relevant to photodynamic therapy (PDT), including the delivery of photosensitizer in the treatment site and monitoring of molecular and physiological changes following treatment.

WHAT ABOUT DEEP SEATED TUMORS?WHAT ABOUT DEEP SEATED TUMORS?

PROSTATE CANCER ANIMAL MODELPROSTATE CANCER ANIMAL MODEL

Palpable tumors appear 2 weeks after inoculation. Once they are formed, they grow rapidly.

Tumors reach the appropriate size (thickness 4 – 6 mm) approximately 3 – 5 weeks after the inoculation.

Animals survive up to 100 days after injection.

Tumors 9 weeks post inoculation

FLUORESCENCE MOLECULAR IMAGINGFLUORESCENCE MOLECULAR IMAGING

One of the most challenging problems in medical imaging is tosee a tumour embedded in tissue which is a diffusive medium. Light in the range of ~650 nm – ~950 nm can penetrate up

to several centimeters into tissue because of the low photon absorption in this region of the spectrum, enabling imaging at greater depths.

Tissue autofluorescence is very low in this spectral region as

well.However, these photons are highly scattered into tissue andbecome diffuse.

FLUORESCENCE MOLECULAR IMAGINGFLUORESCENCE MOLECULAR IMAGING

Progress has been enabled by:

The development of new probes that emit at the near IR region and they have increased photostability and selectivity.

Development of new imaging modalities.

Fluorescence Molecular ImagingFluorescence Molecular ImagingPROSTATE CANCER PROSTATE CANCER

In our Laboratory we use:Fluorescence probes for labeling prostate tumours

at: λexc = 680nm λem = 700nmFree-space, non-contact geometry for excitation

(red diode laser) and detection of light

Direction of excitation and detection from the same side of the tissue

Inverse ProblemInverse Problem

Forward problem: image x data y. Inverse problem: data y image x.

The inverse problem is ill-posed because the solution is non-unique and does not depend continuously on the data.

FORWARD SOLVERFORWARD SOLVER

‣Discretization scheme๏Use of the Delaunay Triangulation

Method.๏Construction of Triangulation Matrix.

‣Fluorophore distribution mapping๏Use of the Super-Ellipsoid Models.๏Mapping of the absorption coefficient

based on interior/exterior position determination relative to the Super-Ellipsoid surface.

‣Finite Elements๏Application of the Galerking Finite

Element Method.๏Definition of the Spatial and Angular

distribution basis functions.

INVERSE SOLVERINVERSE SOLVER

‣Data fitting process๏Intensity adjustment.๏Simulated and acquired

image coordinates correlation.๏Feature extraction.๏Image registration.

‣Image fine-tuning process๏Least squares method.๏Levenberg-Marquardt

optimization.๏Database update.

DA•3072 elements•8 sec

RTE•3072 elements•8 directions•1.5 h

Coupled RTE-DA•3072 elements•8 directions•24 min

This configuration was chosen to match the corresponding properties of Indocyanine Green (ICG) dye. The absorption and isotropic scattering properties of 1% Liposyn solution were chosen to mimic the background of the phantom. The excitation source had been simulated as a point source (Dirac function).

Fluorescence Molecular ImagingFluorescence Molecular Imaging

The three figures represent the photon density magnitude of the excitation light (top row, marked as a) and the emission light (bottom row, marked as b) at the y = 0 plane. The outcomes are from 3D experiments. The least squares relative residual was in the order of 10-14 for both DA and RTE and in the order of 10-13 for the coupled model.

Inverse Problem SolutionInverse Problem Solution

Input Intensity adjustment

Denoising Segmentation

The data fitting procedure provides the initial fluorophore distribution.

THE SYSTEMTHE SYSTEM