Instrumentational Setup

Some instruments Some instruments we may encounterwe may encounter

Basics on PDT, Basics on PDT, PDDPDD

Electromagnetic waves

Optical Power

Infrared Light

Detector Types

Luminescent Sources

Spectral range of visible light

LongerWavelengh

ShorterWavelengh

All colours have a specific wavelengthThe wavelength increases as the colours approach the red end of the spectrum

Spectroscope setupSpectroscope setup

Principle of fluorescence spectroscopy

Principle of fluorescence

∆E > 0

Fluorescence photon

ExcitationPhoton

∆E < 0

Fluorescent molecule

Pp IX Spectrum measured on Peritoneal Nodule

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16

550 600 650 700 750

Measured DataCorrected Data

Fluo

resc

ence

em

issi

on [a

.u.]

Wavelength [nm]

Excitation Spectra of some Bio-molecules

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1

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200 250 300 350 400 450 500

Fluo

resc

ence

Inte

nsity

[a.u

.]

Wavelength [nm]

Porphyrins (Hp)FlavinsElastinCollagen

Tryptophan Lipo-PigmentsPyridoxineNADH

Emission Spectra of some Bio-molecules

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Fluo

resc

ence

Inte

nsity

[a.u

.]

Wavelength [nm]

Porphyrins (Hp)

FlavinsElastinCollagen

Tryptophan Lipo-PigmentsPyridoxine

NADH

Fluorescence emission spectra of cervical tissues

Patient with CIN II, CIN III and HPV infection

λEx=330 nm

Ramanujam & al. Spectroscopic diagnosis of CIN, Gynecologic Oncology 52, 31-38 (1994)

Why is there a difference between neoplasic lesions vs. healthy tissue

The diagnostic basis of spectroscopy is not yet understood at the biochemical level

Possible explanations:Attenuators

– Increase in Oxy-haemoglobin attenuation

Relative contribution of tissue fluorophores

– Decrease in contribution of collagen fluorescence– Increase in the contribution of NADH

Architectural effectOther ...

Parameters of fluorescence spectroscopy

Color of fluorescencephotons ?

FluorescenceSpectroscopy

Excitationwavelength?

?

Autofluorescence (ENT)

Carcinoma of the left vocal cord,precancerous lesion right vocal cord

(bacterial growth)

Early Tumor Detection without Marker Substance

K. Malzahn, C. Arens, H. GlanzK. Malzahn, C. Arens, H. Glanz

JustusJustus--LiebigLiebig--University GiessenUniversity Giessen

Principle ofPrinciple of PDTPDT

HEALTHY TISSUE

LESION

PHOTOSENSITIZER ADMINISTRATION(systemic or topical)

PS PS

PSPS

PSPS

PS

PS

PS

t∆LIGHT

SOURCE

t∆

"SELECTIVE" ILLUMINATION“SELECTIVE” DESTRUCTION

Photophysical Processes Photophysical Processes in:in:Fluorescence Fluorescence detectiondetection Photodynamic TherapyPhotodynamic Therapy

S00

S11 T 11

FluorescenceFluorescence630 nm630 nm

700 nm700 nm630 nm630 nm

400 nm400 nm

Ene

rgy

Ene

rgy

= 1ns= 1nsττ

PhosphorescencePhosphorescence

= 10µs= 10µsττ

S22ISCISCICIC

collision collision energyenergytransfer **

2211OOtransfer

= 250 ns= 250 nsττd = 45 nmd = 45 nm∆∆

OO33

22

AbsorptionAbsorption

Singlet Singlet OxygenOxygenproductionPorphyrins spectroscopy productionPorphyrins spectroscopy

Possible Effects of PDT

5 ALA +

Emission of photons

ROSOxigen Species

Light

Functional Tumorsuppressor genes

Loss of tumorsuppressor genes

Reactive

NECROSIS APOPTOSIS

•• TheThe PropertiesProperties of the Tumorof the Tumor-- Leaky VasculatureLeaky Vasculature-- Compromised LymphaticCompromised Lymphatic drainagedrainage-- Large Large interstitial spaceinterstitial space-- DecreasedDecreased pH valuepH value

((reduces solubilityreduces solubility ofof porphyrins porphyrins

aggregationaggregation + + proteinprotein association)association)

MechanismsMechanisms of Selective Tumor Uptake and of Selective Tumor Uptake and LocalizationLocalization ofof Exogenous PhotosensitizersExogenous Photosensitizers

•• TheThe PropertiesProperties of the Tumorof the Tumor (Cont.)(Cont.)-- Elevated numbersElevated numbers ofof lowlow--density protein receptorsdensity protein receptors-- PresencePresence ofof macrophagesmacrophages

((taketake upup large large amountsamounts ofof HPD)HPD)-- High amountHigh amount ofof newly synthesized collagennewly synthesized collagen

((that binds porphyrinsthat binds porphyrins))-- High amountHigh amount ofof LipidLipid

((thatthat hashas a a high affinityhigh affinity for for lipophiliclipophilic dyesdyes))-- Membrane Membrane potentialspotentials ofof malignant cellsmalignant cells

MechanismsMechanisms ofof Selective Tumor Uptake and Selective Tumor Uptake and LocalizationLocalization ofof Exogenous PhotosensitizersExogenous Photosensitizers

PDT PDT withwith second second generationgeneration PSPS

5 5 -- ALA / ALA / PpIXPpIX

•• Absorption Absorption atat 635 nm (635 nm (BlueBlue light light also usedalso used))

•• Dose 30 Dose 30 -- 60 mg/kg 60 mg/kg orallyorally (20% for (20% for topicaltopical administrations)administrations)

•• D / L D / L intervalinterval 4 4 -- 18 h18 h

•• Light dose 10 Light dose 10 -- 150 J/cm2150 J/cm2

•• Skin Skin photosensitizationphotosensitization 24 24 -- 48 h48 h

Fluorescence contrast

Autofluorescence,Endogenous Fluorophores:Flavins, Porphyrins, NADH, etc

Endogenously induced:ALA-PPIXFluorophores ?

Exogenous:Photophrin®, mTHPC

Penetration depth of light in tissue in relation to the wavelength

W avelength [ nm ]

mm

400 600 800 1000 1200 1400 1600 1800 2000

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Photodynamic TherapyPhotodynamic Therapy

Light Light parametersparameters

•• WavelengthWavelength

•• DrugDrug -- light light intervalinterval

•• IrradianceIrradiance

•• DurationDuration ofof irradiationirradiation

•• Total light doseTotal light dose

Early Tumor DetectionPDD / AF

Urology

Pneumology

Neurosurgery

Combined Diagnosis System

Rigid TelescopesFiberscopesOP - Microscopes

White LightALA-ModeAutofluorescence-Mode

Fluorescence Observation (PPIX)

Neurosurgery

Special Fluorescence MicroscopeStorz AG/ Carl Zeiss

Early Tumor Detection with Marker Substance

High grade Glioma

Stummer, ReulenStummer, ReulenMunichMunich--GroßhadernGroßhadern

Fluorescence imaging and spectroscopic system used for fluorescence photodetection of cervical lesions after topical

application of 5-ALA or h-ALA

Hillemanns & al. Photodetection of Intraepithelial Neoplasia, Cancer 88, 2275-92 (2000)

Fluorescence image of the cervix after h-ALA application

White light Fluorescence

Fluorescence image and white light image of the cervix uteri after the application of 3% acetic acid. Application of 10mg h-ALA in 10ml 0.9% NaCl solution on the cervix during 3 hrs.

Conclusion

Existing instrumentation in clinical practiceIn certain disciplines may need adaptation or developmentMuch clinical development and research is done in the field of photosensitizersAutofluorescence diagnostic procedures becomemore interesting