Optical Imaging

blackeningfourAI and Robotics

Oct 19, 2013 (3 years and 7 months ago)

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DI DO
YOVA


LABORATORY OF BI OMEDI CAL OPTI CS AND APPLI ED
BI OPHYSI CS


SCHOOL OF ELECTRI CAL AND COMPUTERS
ENGI NEERI NG

NATI ONAL TECHNI CAL UNI VERSI TY OF
ATHENS



LABORATORY OF BIOMEDICAL OPTICS AND
APPLIED BIOPHYSICS




OPTICAL IMAGING














CONFOCAL LASER SCANNING MICROSCOPY


IMAGING AT THE CELLULAR LEVEL

TISSUE IMAGING


AFM AND SHG MICROSCOPY


IMAGING OF BIOMOLECULES



TISSUE IMAGING


3D BINOCULAR MACHINE VISION SYSTEM

FLUORESCENCE MOLECULAR IMAGING




Imaging 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 LEVEL

IN 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 Level

Monitoring early events of cellular response to oxidative stress



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
λ
exc:647nm,
λ
em:680
nm

Fibroblast incubated with
MitoTracker Green
λ
exc:488nm,

λ
em: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 H
2
DCFDA.

Fibroblasts incubated with ZnPc
+

H
2
DCFDA

(after oxidation by
ROS produces DCF)

Pseudocolored image

Mitochondrial membrane potential
(ΔΨ
m
)

decrease

after
ZnPc photosensitization + JC
-
1
.

0 min

1 min

3 min

8 min

15 min

30 s

Resting
ΔΨ
m

=
1
4
0

mV




ΔΨ
m

=
90



5

mV after oxidative stress

0 min

2 min

3 min

5 min

30 s

10 min

Resting

pHi = 7.45


0.03


Δ
pHi=0.40


0.08 after oxidative stress

I
ntracellular pH changes after

ZnPc photosensitization

using the membrane permeable
(BCECF
-
AM) probe .

Spatiotemporal global Ca
2+

oscillations evoked by
ZnPc

photosensitization

monitored by Fluo
-
3 (pseudocolored images).

30 s

1 min

2 min

4 min

30 s

1 min

2 min

0 min

Time course experiment of intracellular Ca
2+
concentration.
Resting [Ca
2+

]


60nM

[Ca
2+

]


0.25
μ
M after oxidative stress












Development

of

animal

models
.



Research

related

to

small

animals

optical

imaging





TISSUE OPTICAL IMAGING






Non
-
melanoma carcinomas in SKH
-
1 mice


NMSC 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
μ
m.

Penetration
depth 1490
μ
m


PDT in DERMATOLOGY

OPTICAL 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 Gauging
Small Tumors

3D Binocular Machine Vision System for Gauging
Small Tumors

Animal model for NMSC

Normal

area

Tumour

area

3
-
D Binocular Machine Vision System for Gauging Small
Tumors

3
-
D Binocular Machine Vision System for Gauging Small
Tumors



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 IMAGING

in 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?

PROSTATE 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 IMAGING

One of the most challenging problems in medical imaging is to

see 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 and


become diffuse.

FLUORESCENCE 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 Imaging

PROSTATE CANCER

In our Laboratory we use:


Fluorescence probes for labeling prostate tumours at:


λexc = 680nm


λ
em = 700nm


Free
-
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 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 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 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 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 Solution

Input

Intensity adjustment

Denoising

Segmentation


The data fitting procedure provides the
initial fluorophore distribution.

THE SYSTEM