Wagnieres et al., Photochem. Photobil. 68(5), 1998

In Vivo Fluorescence Spectroscopy and Imaging for Oncological Applications

Introduction

• Critical status report on the detection and characterisation of premalignant or malignant lesions using in vivo fluorescence spectroscopy and imaging

• Also considered is use of these techniques to guide tissue biopsy and surgical resection

• Approaches to tissue characterisation by light-induced fluorescence spectroscopy (LIFS) and imaging classified according to fluorophore type investigated and principle of instrumentation used. 3 main categories:
1. Endogenous fluorophores (causes autofluorescence)
2. Fluorophores synthesised in tissue after external administration of precursor molecule:
• protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA)
3. Fluorophores administered as exogenous drugs:
• fluorescein
• indocyanin green (ICG)
• photosensitizers used for photodynamic therapy (PDT), eg. hematoporphyrin derivatives (HpD) and tetra(m-hydroxyphenyl)chlorin (mTHPC)

Historical background

• History: First study concerning fluorescence in tumours under illumination with UV/violet light (Policard, 1924). The fluorescence was attributed to endogenous porphyrins

Clinical context

• Often, early tumours arise in a region of transformed mucosa on the surface of hollow organs (e.g. bronchi, GI tract, bladder and female reproductive tract) and are radiologically and endoscopically occult, i.e. not detectable upon gross examination)

• In the GI tract, conventional white-light endoscopy has a low sensitivity for detection of dysplastic lesions.

• Patients at high risk require frequent surveillance, including mutiple biopsies, over a long period to increase the chances of detecting dysplastic or early-stage neoplastic lesions.

• Additional potential applications of fluorescent techniques:
• provide guidance in locating optimum sites for biopsy (7)
• define the surgical margins for tumor resection (73, 105, 106)
• optimisation and monitoring of PDT treatments (21, 94, 107, 108)

• Optical biopsy: A controversial but recurring concept; envisaged that diagnosis based on in situ optical measurements could be performed without tissue removal for histopathological examination
• This paper thinks it is probably an unachievable goal, and detracts from the valuable and realistic uses of fluorescence and other optical techniques.

• Potential clinical advantages:
1. high signal sensitivity, esp. if point measurements are used
2. particularly suited for examination of tissue surfaces
3. flexibility in anatomical sites tt can be investigated, esp. using small diameter optical fibre probes
4. reduction in tissue biopsies
5. ease of use by clinician
6. potential for reduced healthcare costs as a consequence of minimally-invasive nature
7. speed of technique - fast!
8. improved patient outcome

Fluorophores

• Endogenous fluorophores:
• Most associated with structural matrix of tissues (most impt of which are collagen and elastin); fluorescence is as a result of cross-linking between amino acids
• Or they can also be involved in cellular metabolic processes (NADH, flavins)
• Others include aromatic amino acids (tryptophan, tyrosine, phenylalanine), various porphyrins and lipopigments (ceroids, lipofuscin) that are end-products of lipid metabolism
• Each fluorophore has distinct a excitation/emission spectrum

• Any tissue contains a mixture of many fluorophores of different concentrations.
  

• Fluorophores are not uniformly distributed in tissue and vary with depth below the tissue surface. E.g. GI tract has a distinct layered structure (mucosa, submucosa, muscularis), each of which has a different fluorophore composition.

• Early detection of premalignant lesions/cancer using autofluorescence is dependent on the following fields:
1. Fluorophore concentration/Spatial distribution
2. Metabolic status (NADH fluorescent when reduced)
3. Biochemical/Biophysical microenvironment
4. Tissue architecture (mucosal thickening/loss of layer)
5. Wavelength-dependent light attentuation

• In vivo fluorescence signal is dependent on the excitation/emission wavelengths used. E.g.:
1. Fluorescence from aromatic amino acids observed only with UV excitation
1. Depth of penetration of excitation light increases with wavelength

• Exogenous fluorophores developed primarily as PDT (photodynamic therapy) sensitizers

Fluorophore precursors

• 5-Aminolevulinic acid (ALA) - rate limiting precursor in heme biosynthesis. Penultimate step of the synthesis process produces the fluorophore protoporphyrin IX (PpIX)
• PpIX excited in the Soret band around 400 nm, or around 635 nm at the highest Q-band. Typical red emission in 625-725 nm region.

Fluorophore localization and delivery vehicles

• Impt for tumour detection by exogenous fluorescence: Why do certain drugs appear to localize preferentially in tumours?
• tumour pH lower than that or normal tissue
• prescence of plasma lipoproteins, in particular LDL
• tumour vasculature

Physical Principles and Instrumentation

• General principles
• Light sources: arc lamps and CW, intensity-modulated or pulsed lasers. Lasers allow use with standard endoscopes!
  
• Illumination and detection optics: 2 ways - fibre is places in direct contact with tissue (might be afected by pressure at point of contact; or a larger tissue surface area is illuminated (spectral distortion may arise)
• For spectroscopic measurements: fluorescence emission collected via same fibre as used for light delivery
• Detectors: Intensified charge-coupled device (CCD) camera, nonintensified cameras, high-sensitivity photographic film or direct viewing by eye

• Point systems
• Most in vivo instruments employ optical fibres to guide the fluorescence excitation and emission light
• Basic instrument structure: light source, fibre light guides, detector.

• Light attenuation: determines how fast the light intensity decreases with distance from objects.

Clinical applications in the GI tract

• Strong rationale for developing fluorescence diagnostics in the GI tract:
1. Detection of dysplastic and early malignant lesions
2. Differentiating hyperplastic, metaplastic and dysplastic pathologies

• Challenges faced when performing fluorescence spect. or imaging in GI tract:
1. prescence of pre-exsiting benign disease (ulcerative colitis, Barrett's esophagus) that may also have altered fluorescence compared with normal tissue - might interfere/affect results of procedure
2. contamination due to food residues, stoll and mucus
3. the large and motile luminal surface