Ramon Gabriel Teixeira Rosa

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.

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.

Nonlinear characterization of fs-laser written Gorilla Glass waveguides

In this work, we performed the nonlinear characterization of Gorilla Glass waveguides produced by fs-laser microfabrication with different writing parameters. Their nonlinear refractive index n2 was determined by the dispersive-scan technique. We found that the waveguides n2 values are lower than the one for the pristine material and they depend on the writing parameters. Raman spectroscopy measurements showed important structural modifications related to non-bridging oxygens (NBOs) on the fs-laser irradiated glass, which may lead to the reduction of the third-order polarizability.

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

Tissue slides analysis using red, green, and blue LEDs as microscope light source

The optical microscopy is one of the most powerful tool in the analysis of biological systems. The usual transmitted light microscope uses a white light lamp as source, what sometimes does not bring optimal results, making it necessary to introduce filters to change some illumination properties like the color temperature or the color itself. There is, of course, an intrinsic limitation on the use of filters that is the lack of an analogical control on the illumination properties and a practical limitation that depends on the number of available filters. To address this need, we developed an illumination system based on (Red, Green and Blue) RGB LEDs, were the microscope operator can control the intensity of each one independently and manually. This paper details the developed system and describes the methods used to compare quantitatively the images acquired while using the standard white light illumination and the images obtained with the developed system. To quantify the contrast, we calculated the relative population standard deviation for the intensities of each channel of the RGB image. This procedure allowed us to compare and understand the major advantages of the developed illumination system. All analysis methods have shown that a contrast enhancement can be obtained under the RGB LEDs light. The presented illumination allowed us to visualize the structures in different samples with a better contrast without the need of any additional optical filters.

Image correlation based method for the analysis of collagen fibers patterns

The collagen fibers are one of the most important structural proteins in skin, being responsible for its strength and flexibility. It is known that their properties, like fibers density, ordination and mean diameter can be affected by several skin conditions, what makes these properties a good parameter to be used on the diagnosis and evaluation of skin aging, cancer, healing, among other conditions. There is, however, a need for methods capable of analyzing quantitatively the organization patterns of these fibers. To address this need, we developed a method based on the autocorrelation function of the images that allows the construction of vector field plots of the fibers directions and does not require any kind of curve fitting or optimization. The analyzed images were obtained through Second Harmonic Generation Imaging Microscopy. This paper presents a concise review on the autocorrelation function and some of its applications to image processing, details the developed method and the results obtained through the analysis of hystopathological slides of landrace porcine skin. The method has high accuracy on the determination of the fibers direction and presents high performance. We look forward to perform further studies keeping track of different skin conditions over time.

Portable fluorescence microendoscope system for smartphones and its applications

A portable microscope/microendoscope will be presented in this article. The system was specially designed for Smartphones and taking into account its simplicity, will be able to bring this technology to almost every doctor’s office. It is worth mentioning its flexibility of use, that allows several modes since all the components are interchangeable (the illumination LED, the lens, the optic filters, etc) resulting in different applications, from medical applications until other areas (for example, the inspection of non-accessible pieces of plane engines). In addition, the system has a double platform, working as a conventional microscope or as a fiberoptic microendoscope. In situ and cell smear interrogation of oral mucosa, using a proflavine as dye will be presented. The price of the system does not exceed US

Modification of collagen fiber after PDT in porcine skin models by two photons microscopy analysis

350, plus the price of the fiber bundle (around US

Optical fluorescence in dentistry

500) turning it onto a high resolution affordable system.