The interaction of light within tissue has been used to recognize disease since the mid-1800s. The recent developments of small light sources, detectors, and fiber optic probes provide opportunities to quantitatively measure these interactions, which yield information for diagnosis at the biochemical, structural, or (patho)physiological level within intact tissues. However, because of the strong scattering properties of tissues, the reemitted optical signal is often influenced by changes in biochemistry (as detected by these spectroscopic approaches) and by physiological and pathophysiological changes in tissue scattering. One challenge of biomedical optics is to uncouple the signals influenced by biochemistry, which themselves provide specificity for identifying diseased states, from those influenced by tissue scattering, which are typically unspecific to a pathology. In this review, we describe optical interactions pursued for biomedical applications (fluorescence, fluorescence lifetime, phosphorescence, and Raman from cells, cultures, and tissues) and then provide a descriptive framework for light interaction based upon tissue absorption and scattering properties. Finally, we review important endogenous and exogenous biological chromophores and describe current work to employ these signals for detection and diagnosis of disease.

Quantitative optical spectroscopy for tissue diagnosis

Cells contain molecules, which become fluorescent when excited by UV/Vis radiation of suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of cells and is called auto-fluorescence to be distinguished from fluorescent signals obtained by adding exogenous markers. The majority of cell auto-fluorescence originates from mitochondria and lysosomes. Together with aromatic amino acids and lipo-pigments, the most important endogenous fluorophores are pyridinic (NADPH) and flavin coenzymes. In tissues, the extracellular matrix often contributes to the auto-fluorescence emission more than the cellular component, because collagen and elastin have, among the endogenous fluorophores, a relatively high quantum yield. Changes occurring in the cell and tissue state during physiological and/or pathological processes result in modifications of the amount and distribution of endogenous fluorophores and chemical-physical properties of their microenvironment. Therefore, analytical techniques based on auto-fluorescence monitoring can be utilized in order to obtain information about morphological and physiological state of cells and tissues. Moreover, auto-fluorescence analysis can be performed in real time because it does not require any treatment of fixing or staining of the specimens. In the past few years spectroscopic and imaging techniques have been developed for many different applications both in basic research and diagnostics.

Cell and tissue autofluorescence research and diagnostic applications

Color measuring systems and methods are disclosed. Perimeter receiver fiber optics are spaced apart from a central source fiber optic and receive light reflected from the surface of the object being measured. Light from the perimeter fiber optics pass to a variety of filters. The system utilizes the perimeter receiver fiber optics to determine information regarding the height and angle of the probe with respect to the object being measured. Under processor control, the color measurement may be made at a predetermined height and angle. Various color spectral photometer arrangements are disclosed. Translucency, fluorescence and/or surface texture data also may be obtained. Audio feedback may be provided to guide operator use of the system. The probe may have a removable or shielded tip for contamination prevention.

Apparatus and method for measuring optical characteristics of an object

New diagnostic tools are needed for the characterization of dental caries in the early stages of development. If carious lesions are detected early enough, they can be arrested without the need for surgical intervention. The objective of this study was to demonstrate that polarization sensitive optical coherence tomography (PS-OCT) can be used for the imaging of early caries lesions and for the monitoring of lesion progression over time. High-resolution polarization resolved images were acquired of natural caries lesions and simulated caries lesions of varying severity created over time periods of 1 to 14 days. Linearly polarized light was incident on the tooth samples and the reflected intensity in both orthogonal polarizations was measured. PS-OCT was invaluable for removing the confounding influence of surface reflections and native birefringence necessary for the enhanced resolution of the surface structure of caries lesions. This study demonstrated that PS-OCT is well suited for the imaging of interproximal and occlusal caries, early root caries, and for imaging decay under composite fillings. Longitudinal measurements of the reflected light intensity in the orthogonal polarization state from the area of simulated caries lesions linearly correlated with the square root of time of demineralization indicating that PS-OCT is well suited for monitoring changes in enamel mineralization over time.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-7/issue-04/0000/Imaging-caries-lesions-and-lesion-progression-with-polarization-sensitive-optical/10.1117/1.1509752.pdf

Imaging caries lesions and lesion progression with polarization sensitive optical coherence tomography

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of δ-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407–420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm−2. Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm−2 and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm−2 the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407–420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light

A device for detecting dental caries has an illumination device which emits radiation of a pre-determined wavelength towards a tooth. A filter accepts radiation returned by the tooth in a pre-determined spectral range. The accepted radiation is evaluated for caries detection. The illumination device has at least one light guide, by means of which the radiation can be supplied to the tooth. The illumination device emits radiation in the spectral range from 360 to 580 nm. The filter accepts the returned radiation in the spectral range above 620 nm. The device can be used in a flexible manner and enables a low level of caries to be identified.

Device for detecting dental caries

The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NAD(P)H fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior-probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the time range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component as most pronounced for the intact strain DL1. Time-resolved fluorescence seems therefore to be an appropriate method to probe the function of the respiratory chain and-in the further step-to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

Carious regions of human teeth were investigated by means of remission-, fluorescence excitation- and fluorescence emission spectroscopy as well as time-resolved fluorescence measurements in the ns region. A detection of dental caries is possible with all of these spectroscopic methods. The peaks in measured remission- and fluorescence excitation spectra of carious regions correspond with the absorption bands of metal free porphyrins. The emission spectra and fluorescence decay kinetics indicate that protoporphyrin IX may be the main chromophore. HPLC measurement verify the existence of proto- (main component), copro- and probably Zn-protoporphyrin.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria

Human skin shows a strong autofluorescence in the red spectral region with main peaks around 600, 620, and 640 nm caused by the porphyrin production of the gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of photoproducts with fluorescence bands around 670 nm and decay times of about 1 and 5 ns. The photoproduct formation is connected with an increased absorption in the red spectral region. The endogenous fluorescent porphyrins act as photosensitizers. Photodestruction of Propionibacteria acnes by visible light appears therefore to be a promising therapy. The photodynamic activity of the photoproducts is lower than that of protoporphyrin IX.

Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin

Certain bacteria are able to synthesize metal-free fluorescent porphyrins and can therefore be detected by sensitive autofluorescence measurements in the red spectral region. The porphyrin-producing bacterium Propionibacterium acnes, which is involved in the pathogenesis of acne vulgaris, was localized in human skin. Spectrally resolved fluorescence images of bacteria distribution in the face were obtained by a slow-scan CCD camera combined with a tunable liquid crystal filter. The structured autofluorescence of dental caries and dental plaque in the red is caused by oral bacteria, like Bacteroides or Actinomyces odontolyticus. `Caries images' were created by time-gated imaging in the ns-region after ultrashort laser excitation. Time-gated measurements allow the suppression of backscattered light and non-porphyrin autofluorescence. Biopsies of oral squamous cell carcinoma exhibited red autofluorescence in necrotic regions and high concentrations of the porphyrin-producing bacterium Pseudomonas aerigunosa. These studies suggest that the temporal and spectral characteristics of bacterial autofluorescence can be used in the diagnosis and treatment of a variety of diseases.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma

Karsten Koenig

Papers

Device for detecting dental caries

Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria

Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin

In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma