Laser-induced autofluorescence for medical diagnosis

Abstract

The naturally occurring autofluorescence of cells and tissues is based on biomolecules containing intrinsic fluorophores, such as porphyrins, the amino acids tryptophan and tyrosine, and the coenzymes NADH, NADPH, and flavins. Coenzymes fluoresce in the blue/green spectral region (fluorecence lifetimes: 0.5-6 ns) and are highly sensitive indicators of metabolic function. Steadystate and time-resolved blue-green autofluorescence is, therefore, an appropriate measure of the function of the respiratory chain as well as of cellular and tissue damage. Autofluorescence in the yellow/red spectral region is based mainly on endogenous porphyrins and metalloporphyrins, such as coproporphyrin, protoporphyrin (fluorescence lifetime of porphyrin monomers: >10 ns), and Zn-protoporphyrin (2 ns). Various pathological microorganisms such asPropionibacterium acnes, Pseudomonas aeruginosa, Actinomyces odontolyticus, Bacteroides intermedius, andSaccharomyces cerevisiae are able to synthesize large amounts of these fluorophores and can therefore be located. This permits fluorescence-based detection of a variety of diseases, including early-stage dental caries, dental plaque, acne vulgaris, otitis externa, and squamous cell carcinoma. The sensitivity of noninvasive autofluorescence diagnostics can be enhanced by time-gated fluorescence measurements using an appropriate time delay between ultrashort laser excitation and detection. For example, videocameras with ultrafast shutters, in the nanosecond region, can be used to create "caries images" of the teeth. Alternatively, autofluorescence can be enhanced by stimulating protoporphyrin biosynthesis with the exogenously administered porphyrin precursor 5-aminolevulinic acid (ALA). The fluorophore protoporphyrin IX (PP IX) is photolabile and photodynamically active. Irradiation of PP IX-containing tissue results in cytotoxic reactions which correlate with modifications in fluorescence due to photobleaching and singlet oxygen-dependent photoproduct formation. Therefore, on-line autofluorescence measurements during the phototreatment can yield information on the efficiency of ALA-based photodynamic therapy.

Micro-computerized tomography assessment of fluorescence aided caries excavation (FACE) technology: comparison with three other caries removal techniques

“…35 Porphyrins and metalloporphyrins typically have absorption maxima between 398 and 421 nm and emission maxima between 530 and 633 nm. 24 Because porphyrins are produced by bacterial metabolic activity, the red fluorescence intensity reflects the intensity and extent of the caries-affected tissues. Spectrographic studies have shown that carious dentine fluoresces more intensely in the red portion of the visible spectrum than healthy dentine.…”

On the red fluorescence emission of <i>Aggregatibacter actinomycetemcomitans</i>

“…Still, the question is if the fluorescence is due to the porphyrin content or some other constituent. However, it has been shown that the spectra from red fluorescing bacteria is very similar to the emission spectra of porphyrins [3,4,16].…”

“…There are only a few publications so far about bacteria associated with periodontal disease and their ability to emit red fluorescence thus, and it is of interest to investigate which periodontopathogens that are able to emit red fluorescence since according to the literature this property could be used for diagnosis and treatment [3,4,16]. To our knowledge there are no specific studies on the fluorescence of A. actinomycetemcomitans.…”

“…This red fluorescence emission has been suggested to originate from porphyrins that are synthesized by and present in various microorganisms [2][3][4].…”

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

“…It should be noted that this linear relationship is a calibration curve for PA14/EFGP only. Other strains of PA or other bacterial species would most likely yield a different calibration curve that would have to be obtained prior to any quantification experiments, especially if the stains are not tagged with a fluorophore similar to EFGP, and quantification relies on only the autofluorescence of NADH [55]. Thus, this method has limitations at high CFU counts, where fluorescence intensity may not correlate to a linear curve.…”