Marcelo Saito Nogueira

Time-resolved fluorescence lifetime for cutaneous melanoma detection

Melanoma is the most aggressive skin cancer type. It is characterized by pigmented lesions with high tissue invasion and metastatic potential. The early detection of melanoma is extremely important to improve patient prognosis and survival rate, since it can progress to the deadly metastatic stage. Presently, the melanoma diagnosis is based on the clinical analysis of the macroscopic lesion characteristics such as shape, color, borders following the ABCD rules. The aim of this study is to evaluate the time-resolved fluorescence lifetime of NADH and FAD molecules to detect cutaneous melanoma in an experimental in vivo model. Forty-two lesions were analyzed and the data was classified using linear discriminant analysis, a sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved. These results show the potential of this fluorescence spectroscopy for melanoma detection.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis.

Time-resolved fluorescence spectroscopy for clinical diagnosis of actinic cheilitis.

Actinic cheilitis is a potentially malignant disorder of the lips. Its first cause is believed to be UV sun radiation. The lesion is highly heterogeneous, making the choice of area to be biopsied difficult. This study exploits the capabilities of time-resolved fluorescence spectroscopy for the identification of the most representative area to be biopsied. A preliminary study was performed on fourteen patients. A classification algorithm was used on data acquired on nine different biopsies. The algorithm discriminated between absent, mild, and moderate dysplasia with a sensitivity of 92.9%, 90.0%, and 80.0%, respectively. The false positive rate for healthy tissue (specificity) was 88.8%.

Portable fluorescence lifetime spectroscopy system for in-situ interrogation of biological tissues

Abstract. Fluorescence spectroscopy and lifetime techniques are potential methods for optical diagnosis and characterization of biological tissues with an in-situ, fast, and noninvasive interrogation. Several diseases may be diagnosed due to differences in the fluorescence spectra of targeted fluorophores, when, these spectra are similar, considering steady-state fluorescence, others may be detected by monitoring their fluorescence lifetime. Despite this complementarity, most of the current fluorescence lifetime systems are not robust and portable, and not being feasible for clinical applications. We describe the assembly of a fluorescence lifetime spectroscopy system in a suitcase, its characterization, and validation with clinical measurements of skin lesions. The assembled system is all encased and robust, maintaining its mechanical, electrical, and optical stability during transportation, and is feasible for clinical measurements. The instrument response function measured was about 300 ps, and the system is properly calibrated. At the clinical study, the system showed to be reliable, and the achieved spectroscopy results support its potential use as an auxiliary tool for skin diagnostics.

Raman spectral post-processing for oral tissue discrimination – a step for an automatized diagnostic system

Most oral injuries are diagnosed by histopathological analysis of a biopsy, which is an invasive procedure and does not give immediate results. On the other hand, Raman spectroscopy is a real time and minimally invasive analytical tool with potential for the diagnosis of diseases. The potential for diagnostics can be improved by data post-processing. Hence, this study aims to evaluate the performance of preprocessing steps and multivariate analysis methods for the classification of normal tissues and pathological oral lesion spectra. A total of 80 spectra acquired from normal and abnormal tissues using optical fiber Raman-based spectroscopy (OFRS) were subjected to PCA preprocessing in the z-scored data set, and the KNN (K-nearest neighbors), J48 (unpruned C4.5 decision tree), RBF (radial basis function), RF (random forest), and MLP (multilayer perceptron) classifiers at WEKA software (Waikato environment for knowledge analysis), after area normalization or maximum intensity normalization. Our results suggest the best classification was achieved by using maximum intensity normalization followed by MLP. Based on these results, software for automated analysis can be generated and validated using larger data sets. This would aid quick comprehension of spectroscopic data and easy diagnosis by medical practitioners in clinical settings.

Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

Assembly and characterization of a fluorescence lifetime spectroscopy system for skin lesions diagnostic

The fluorescence spectra and fluorescence lifetime analysis in biological tissues has been presented as a technique of a great potential for tissue characterization for diagnostic purposes. The objective of this study is to assemble and characterize a fluorescence lifetime spectroscopy system for diagnostic of clinically similar skin lesions in vivo. The fluorescence lifetime measurements were performed using the Time Correlated Single Photon Counting (Becker & Hickl, Berlin, Germany) technique. Two lasers, one emitting at 378 nm and another at 445 nm, are used for excitation with 20, 50 and 80 MHz repetition rate. A bifurcated optical fiber probe conducts the excitation light to the sample, the collected light is transmitted through bandpass filters and delivered to a hybrid photomultiplier tube detector. The fluorescence spectra were obtained by using a portable spectrometer (Ocean Optics USB-2000-FLG) with the same excitation sources. An instrument response function of about 300 ps was obtained and the spectrum and fluorescence lifetime of a standard fluorescent molecule (Rhodamine 6G) was measured for the calibration of the system ((4.1 ± 0.3) ns). The assembled system was considered robust, well calibrated and will be used for clinical measurements of skin lesions.

Lifetime fluorescence for the detection of skin lesions

A lifetime fluorescence spectroscopy system was used to interrogate melanoma skin lesions in an animal model. Fluorescence measurements were taken at 24, 72, and 96 hours after cells inoculation.

Subcellular localization and photodynamic activity of Photodithazine (glucosamine salt of chlorin e6) in murine melanoma B16-F10: an in vitro and in vivo study

Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.

Assessment of oxidative stress and metabolic rates in liver grafts using time-resolved fluorescence spectroscopy

One of the most relevant postoperative problems in liver transplantation is the initial graft dysfunction, which is generated by the oxidative stress due to ischemia and/or reperfusion. This leads to ischemia-reperfusion injury of the transplanted organ. This injury can be severe and, when the primary graft is nonfunctional, an urgent retransplantation is required. In this context, tools to monitor the oxidative stress in liver grafts would improve the surgical decision-making for transplantation, increasing its success rate. In this study, we evaluated the potential of time-resolved fluorescence spectroscopy to measure oxidative stress in liver grafts before transplantation. This was performed in livers after ischemia and reperfusion kept in 0 °C (control group) and or 20 °C (ischemia-reperfusion injury group). Both fluorescence spectra and lifetimes were monitored immediately, each 1 hour for the first 12 hours, and at 24 hours after the removal of the liver. The hepatic tissue was excited by lasers emitting in 378 nm and 445 nm for investigation of possible metabolic rates associated to NAD(P)H, FAD, lipopigments, and lipofuscin molecules. The fluorescence decay curves were fitted to the convolution between the instrument response function with a bi-exponential decay for 378 nm and a tri-exponential decay for 445 nm by using the SPCImage software. For both excitation wavelengths, the relative weights for the first exponential component decreased faster as a function of time for the ischemia-reperfusion injury group compared to the control group. This suggests time-resolved spectroscopy is a promising technique to help clinicians to make decisions before liver transplantation.