characterization of the heart tissues by the time resolved ......seminaras ryga, 2012.03.30,...

Seminaras Ryga, 2012.03.30, R.Rotomskis

Characterization of the heart

tissues by the time resolved

fluorescence spectroscopy

J.Venius1, S.Bagdonas2, R.Rotomskis1,2

1Biomedical Physics Laboratory, Institute of Oncology, Vilnius

University, P. Baublio 3b, Vilnius LT-08406, 2Biophotonics group of

Laser Research Center, Faculty of Physics, Vilnius University,

Sauletekio 9, bldg. 3, Vilnius LT-10222, Lithuania


Optical Biopsy

Transmitted

Light

Pump Light Output Light

Scattered Light

Tissue


Many of the intrinsic fluorophores in biological tissues

have the overlapping absorption spectra


Fluorescence reflects the general composition of the tissue

and depends mostly on the present fluorophores;


500 550 600 650 700 7500,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4 Navikinis audinys

Irs tantis navikinis audinys

Nekrozė

Kraujagys lė

Kraujos ruva

Inte

nsyvu

ma

s,

sn

t.vn

t.

, nm

11 pav. Fluorescencijos

registravimo in vivo optinė ir

elektrinė grandinė.

Many of the intrinsic fluorophores in biological tissues

have the overlapping spectra, it is difficult to identify the

selective excitation region for the fluorescence-based

discrimination between two different tissues.


6

1) Bloody tissue 2) Necrosis; 3) Blood vessel; 4) Cancerous tissue; 5) Necrotic cancer

Naviko preparatų

histologijos įvertinimas:

550 600 650 700 750

0

100

200

300

400

500

600

Z

,nm

Flu

ore

sce

ncija

,sn

t.vn

t.

The fluorescence

of cancerous tissue

2

3 1

2

2 4

1

3

2

1 5

z


600 650 700 7500

1

Wavelength (nm)

Cancerous tissue

Cancer imaging


Problem

Sinusinis mazgas

Atrioventrikulinis mazgas

De inš ė šaka

Kairė šaka

The muscular origin

makes it complicated to

distinguish HCS from the

surrounding tissues,

therefore, there is an

immense necessity to

visualize HCS during the

operation time.

During the heart surgery there is a possibility to harm the

conduction system of the heart (HCS), which may cause

dangerous obstruction of the heart functionality.


The specimens prepared from a human heart samples of 2-3

centimetres in size were obtained at autopsy (12–24 h post

mortem) by the pathologist, were fixed in a 10% neutral buffered

formalin solution immediately after excision and kept in the dark at

4ºC prior to the measurements

The specimen that contained Heart

Conductiv System (HCS) was

prepared from the left branch of His

bundle. The specimen for Myocardium

(MC) was prepared from the ventricles

and the Connective Tissue (CT)

specimen was prepared from the

aorta. The location of particular tissue

was marked on each specimen by the

pathologist. Steady state fluorescence

spectra of HCS, CT and MC have

been measured from multiple places

on three prepared specimens. In total

20 fluorescence spectra were

registered from each type of the

specimen.


250 275 300 3250,00

0,02

0,04

0,06

0,08

Myocardium

Conduction

system

Pancreas

Ab

so

rba

nce

, o

.u.

Wavelength, nm

250 275 300 3250,0

0,5

1,0

1,5

2,0

Conduction system

Pancreas

Tyr

Trp

Wavelength, nm


11

300 400 500 600 700 800

0,3

0,6

0,9

Flu

ore

scen

cijo

s in

rensy

vum

as (

s.v.)

Bangos ilgis (nm)

Trp 10-3 M

K

ŠLS

MK

Fluorescence spectra of heart

Trp triptofan aqueouss solution (10-3 M) exc.=255 nm.

(ŠLS – heart conduction system, MK – myocardium, K – pancreas).


12

Fluorescence measurement

13 pav. Savosios fluorescencijos

registravimas ex vivo.

FL

Š

L1 L2

F

Š

Š

M

S

11 pav. Fluorescencijos

registravimo in vivo optinė ir

elektrinė grandinė.

B)

Savosios fluorescencijos

registravimas in vivo (A) ir

ex vivo (B).

A)


13

žadinimo = 365nm

Spektrų

registravimo

sritis

Filtrų pralaidumo

charakteristikos


14

0 10 20 30 40 50 60

250

300

350

400

450

500

550 Nenormuoti spektrai

Intensyvumas,

s.v.

Spektro numeris

1,8 cm

6 mm

Nenormuoti spektrai

0 1 2 3 4 5 6

250

300

350

400

450

500

550 Nenormuoti spektrai

Inte

nsyvum

as, s.v

.

Atstumas, mm

Miokardas

Laidžioji sistema

Inte

nsyvu

ma

s,s

.v.

, nm

400 500 600 700 800

0

500

1000

1500

2000

Miokardas

Laidžioji sistema

Inte

nsyvu

ma

s,s

.v.

, nm

Miokardas

Laidžioji sistema

Inte

nsyvu

ma

s,s

.v.

, nm

400 500 600 700 800

0

500

1000

1500

2000

400 500 600 700 800

0

500

1000

1500

2000


S2000

440 460 480 500 520 540

0

20

40

60

3,0 2,7

2,4 2,1

1,8 1,5

1,2 0,9

0,6 0,3

d (mm)

Bangos ilgis (nm)

Flu

ore

scen

cijo

s in

ten

syv

um

as (

s.v.

)

Myocardium

Heart conduction

system

Fluorescence

microscopy of

heart tissue


16

ŠLS atskyrimo nuo JA ir MK modelis.

ŠLS + JA

ŠLS + MK

• Žadinimo bangos ilgis

330 nm ;

• Vaizdas fiksuojamas

400 nm – 450 nm srityje

350 400 450 500

0

200

400

600

800

1000

Flu

ore

scen

cijo

s in

tensy

vum

as (

s.v.)

Bangos ilgis (nm)

JA

SLS MK

B

ŠLS + JA

• Žadinimo bangos ilgis

280 nm ;

• Vaizdas fiksuojamas

320 nm – 360 nm srityje

300 350 400 450 500 550

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

Flu

ore

scen

cijo

s in

ten

syv

um

as (

s.v

.)

Bangos ilgis, nm

JA

SLS

MK

A

ŠLS + MK

ŠLS

Atlikus dviejų

nuotraukų

sankirtos

veiksmą,

išryškėja tik ŠLS.


A B

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0,8

1,0

1,2

1,4 Registravimo taškas

Ra

ud(4

60

)

Atstumas, mm

Myocardium (Raud(460) > 1,2)

Conductive system (1,2 ≤ Raud(460) ≥ 0,9)

Connective tissue (Raud(460) less 0,9)

Conductive system


18

Tarpskilvelinė pertvara

Vaizdas paprastoje

šviesoje Vaizdas apšvietus 365 nm

bangos ilgio šviesa

Laidžioji sistema

Kompiuteriu sumodeliuotas

ŠLS vaizdas


350 400 450 500 5500

5

10

15

20

25

Flu

ore

sce

ncijo

s inte

nsyvu

mas, s.v

.Bangos ilgis, nm

350 400 450 500 5500

5

10

15

20

25

Flu

ore

sce

ncijo

s in

ten

syvu

ma

s, s.v

.

Bangos ilgis, nm

350 400 450 500 5500

5

10

15

20

25

Flu

ore

sce

ncijo

s in

ten

syvu

ma

s, s.v

.

Bangos ilgis, nm

Connective

tissue

Myocardium Hearth

conduction

system

Heart autofluorescence spectra


The prototype application

in surgery of heart


FLIM (Fluorescence Lifetime Imaging Microscopy)


CdSe/ZnS-COOH uptake NIH3T3 cells

Confocal FLIM 1h


CdSe/ZnS-COOH uptake NIH3T3 cells

FLIM


7 tr sinovija – ALA-Me


7 tr Cartilage – ALA-Me

injected to the knee


27

Tarpskilvelinė pertvara

Vaizdas paprastoje

šviesoje Vaizdas apšvietus 365 nm

bangos ilgio šviesa

Laidžioji sistema

Kompiuteriu sumodeliuotas

ŠLS vaizdas


τ = fluorescence life-time

•

Two exponential decay λmon= 480

nm (HCS)

Three exponential approximation λmon= 480 nm

The time-resolved spectroscopy revealed that at least three

constituents are responsible for the fluorescence of HCS, in

the spectral region of 430 nm – 550 nm under excitation at

405 nm.


Connective tissue

τ1=0.96

τ2=3.53

τ3=10.39

Myocardium τ1=0.8

τ2=3.13

τ3=10.56

The fluorescence decay in the spectral region of

430 nm – 550 nm under excitation at 405 nm

The time-resolved spectroscopy revealed that at least three

constituents are responsible for the fluorescence of HCS, CT and

MC in the spectral region of 430 nm – 550 nm under excitation at

405 nm.


The main fluorophores in the heart tissue were shown to be

collagen and elastin,

however, collagen in the heart tissues is found in several

forms. The cardiac collagen of extracellular matrix consists

of 85% type I collagen. In addition to type I, other fibril

forming collagen types found in the heart are type III and V.

The collagen in a valve, which is composed mostly of the

connective tissue, consists of:

collagen I (74 %),

collagen III (24 %)

and collagen V (2 %).

Therefore, the fluorophores that mostly contribute to the

registered fluorescence decay in all three types of tissue

could be identified as elastin, collagen I and collagen III


HCS MK CT

A1 28 ± 2 27 ± 3 20 ± 4

A2 47 ± 2 46 ± 2 51 ± 4

A3 25 ± 3 27 ± 3 29 ± 5

The average contribution values of the first, the second and

the third constituent to the fluorescence spectra measured

in the spectral region of 430 nm – 550 nm

Elastin

Kolagen I

Kolagen III


HCS MK CT

1 0,7 ± 0,1 0,7 ± 0,1 0,9 ± 0,1

2 3,2 ± 0,3 3,1 ± 0,2 3,5 ± 0,4

3 10,7 ± 1,2 10,6 ± 1 10,7 ± 1,7

Three exponential approximation

The three autofluorescence lifetimes being attributed to HCS

and MC showed no statistically significant difference,

whereas the two shorter (τ1 and τ2) of HCS appeared to be

different from those of CT.

Elastin

Kolagen I

Kolagen III


Conclusions

•The time-resolved spectroscopy revealed that at least three constituents

are responsible for the fluorescence of HCS, CT and MC in the spectral

region of 430 nm – 550 nm under excitation at 405 nm. The lifetimes of

every tissue did not change throughout the measured region, indicating

that the composing fluorophores are the same.

•The three autofluorescence lifetimes being attributed to HCS and MC

showed no statistically significant difference, whereas the two shorter (τ1

and τ2) of HCS appeared to be significantly different from those of CT.

•The fractional components of fluorescence intensity revealed no

significant difference in composition of muscular type tissues HCS and MC.

On the other hand, the relative spectral composition (constituents A1 – A3)

of CT differed significantly from HCS. The observed differences could be

explained by relatively larger amounts of elastin present in HCS and

collagen – in CT.


Thank you

very much

Ričardas

Rotomskis

1Biomedical Physics Laboratory, Institute

of Oncology, Vilnius University, P. Baublio

3b, Vilnius LT-08406,

2Biophotonics group of Laser Research

Center, Faculty of Physics, Vilnius

University, Sauletekio 9, bldg. 3, Vilnius

LT-10222, Lithuania

characterization of the heart tissues by the time resolved ......seminaras ryga, 2012.03.30,...

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