Significance: Multispectral imaging enables mapping of chromophore content changes in skin neoplasms, which helps to diagnose a pathology. Different types of light sources can be used for the imaging. Design of laser-based illuminators is more complicated and, consequently, they are more expensive than LED-based illuminators. On the other hand, spectral line illumination has the advantage of less complicated calculations, since only the discrete maximum wavelengths need to be considered. Spectral band and spectral line approaches for multispectral skin diagnostics have not been compared so far. This can help to evaluate the accuracy and effectiveness of both approaches.
Aim: To compare two specific illumination modalities—spectral band and spectral line illumination—from the point of performance for mapping of in vivo skin chromophores.
Approach: Three spectral images of the same skin malformations were captured by a smartphone RGB camera with two different add-on illuminators comprising LED emitters and laser emitters, respectively. Five types of benign skin neoplasms were included in our study. Concentrations of skin melanin, oxy- and deoxy-hemoglobin at image pixel groups were calculated using the Beer–Lambert law.
Results: Skin chromophore maps and statistical analysis of mean concentrations’ changes in the neoplasms compared to the surrounding skin are presented and discussed. The data of the laser emitters led to significantly higher (∼10 times) increase of the oxy-hemoglobin values in vascular neoplasms and much lower deoxy-hemoglobin values, if compared to the data obtained by the LED emitters.
Conclusions: Analysis of the obtained chromophore distribution maps and concentration variations in neoplasms led to conclusion that the spectral line illumination approach is more appropriate for this application. Considering only the peak wavelengths of illumination spectral bands leads to essentially different results if compared to those obtained by spectral line illumination and may cause misinterpretations in the clinical assessment of skin neoplasms.
A brief review on the activities of Biophotonics Laboratory at UL Institute of Atomic Physics and Spectroscopy, following the previous Biophotonics Riga - 2017 conference, is presented. Twelve recent research projects are considered, including two EC Horizon-2020 projects, six European Regional Development Fund projects and four projects funded by the Latvian Council of Science. The projects are generally aimed at development of new optical methods and technologies for noninvasive in-vivo skin assessment to facilitate early diagnostics of skin malformations (including cancers), sepsis and cutaneous blood microcirculation features. Most of the projects explore novel approaches of camera-based biomedical imaging for clinical diagnostics and recovery monitoring.
Skin chromophore maps can be used for assessment of various skin malformations and early cancer diagnostics. Commercially available devices are bulky and expensive.
We present two portable proof-of-concept device prototypes for multi-spectral laser line imaging with three (448 nm, 532 nm and 659 nm) and four (450 nm, 523 nm, 638 nm and 850 nm) wavelength laser illumination. Laser modules and special optics that ensure uniform light distribution over the region of interest have been exploited.
Skin chromophore maps were calculated using Beer-Lambert law, considering light scattering properties in the skin and including photon path length evaluated from the directly measured photon-time-of-flight signals. Chromophore concentrations in the lesion are compared to the surrounding healthy skin.
In vivo measurement results were compared with the results obtained from agar-based multi-layered skin phantoms which mimic vascular and pigmented skin lesions.
An experimental method for remitted photon path length measurements in scattering media has been developed and tested on human skin and skin neoplasms, skin phantoms and cell cultures. The photon time-of-flight (PTOF) measurement method was used in this study, where the photon travel time was converted into path length. Remitted light signals were obtained using a picosecond broadband laser and a set of narrowband interference filters in spectral rang 520 – 760 nm. Five different distances of 1, 8, 12, 16 and 20 mm between the source and detector fibers were used. Measurements were performed at different wavelengths and distance combinations; they were taken from human skin and skin malformations, agar-based phantoms with different concentrations of intralipid and hemoglobin, and from cell cultures (DC3F, B16/F10). Parameters related to the remitted photon mean path length will be presented and analyzed.
Agar-based skin phantoms with different thicknesses and hemoglobin concentration were evaluated for diagnostics of skin lesions by RGB imaging. Scattering properties of the phantoms were simulated using intralipid, absorption properties – using lyophilized powder of human hemoglobin. RGB images of phantoms were captured by self-developed laboratory made devices. The algorithm for calculation of chromophore concentrations are based on Beer-Lambert law and includes the photon path length evaluated from the measured photon-time-of-flight signals. Optical properties and chromophore concentration maps of phantoms obtained from RGB images were analyzed. The influence of chromophore concentration on scattering and absorption, photon path length and chromophore maps are discussed.
To explore challenges for further improvement of diagnostic performance, a project aimed at development of technology for tri-modal skin imaging by combining multispectral, fluorescence lifetime and Raman band imaging was initiated. In this study, each of the mentioned imaging modalities has been preliminary tested and updated. Four different multispectral imaging devices were tested on color standards. Picosecond laser-excited fluorescence lifetime imaging equipment was examined on ex-vivo skin samples. Finally, a new Raman spectroscopy setup with 785 nm laser was launched and tested on cell cultures and ex-vivo skin. Advantages and specific features of the tri-modal skin imaging are discussed.
Skin-remitted picosecond laser pulses have been detected at variable input-output fiber distances (8 … 20 mm) in the spectral range 520-800 nm, with subsequent analysis of the pulse shape changes. Transfer functions representing the temporal responses of remitted photons to infinitely narrow δ-pulse excitation have been calculated. Parameters related to the photon path length in skin – input-output pulse peak delays, pulse FWHM, travel times of the “initial” photons and distributions of the remitted photon path lengths – are presented and analyzed. The measurement results are in general agreement with the photon propagation model expectations
This paper presents the results of statistical clinical data, combining two diagnostic methods. A combination of two skin imaging methods – diffuse reflectance and autofluorescence – has been applied for skin cancer diagnostics. Autofluorescence (AF) and multispectral diffuse reflectance images were acquired by custom made prototype with 405 nm, 526 nm, 663 nm and 964 nm LEDs and RGB CMOS camera. Parameter p’ was calculated from diffuse reflectance images under green, red and infrared illumination, AF intensity (I’) was calculated from AF images exited at 405nm wavelength. Obtained results show that criterion p` > 1 gives possibility to discriminate melanomas and different kind of keratosis from other lesions, and criterion I` < 0.2 gives the possibility to discriminate melanomas from keratosis.
The recent advancements of three laser-based diagnostic technologies developed at the Riga group are briefly reviewed: (i) RGB imaging of cw-laser excited skin autofluorescence intensity and photobleaching rate distributions, (ii) ps-laser excited skin autofluorescence and diffuse reflectance kinetics analysis, (iii) snapshot RGB skin chromophore mapping under triple-laser illumination. These techniques have passed preliminary laboratory and clinical tests which have demonstrated a promising potential for further implementation in portable devices for routine clinical applications. Operation principles, set-up schemes and some clinical results obtained by the above-mentioned techniques are discussed in this paper.
KEYWORDS: Luminescence, Absorption, Fluorescence resonance energy transfer, Molecules, Proteins, Pollution control, Microscopy, Image processing, Resonance energy transfer, Molecular energy transfer
It is widely known that not all of the treated cells survive after introduction of exogenous molecules via any physical method. Therefore, it is important to develop methods that would allow simultaneous evaluation of both molecular delivery efficiency and cell viability. This study presents Förster resonance energy transfer (FRET)-based method that allows molecular transfer and cell viability evaluation in a single measurement by employing two common fluorescent dyes, namely, ethidium bromide and trypan blue. The method has been validated using cell sonoporation. The FRET-based method allows the efficiency evaluation of both reversible and irreversible sonoporation in a single experiment. Therefore, this method could be used to reduce time, labor, and material cost while improving the accuracy of evaluations.
The feasibility of smartphones for in vivo skin autofluorescence imaging has been investigated. Filtered autofluorescence images from the same tissue area were periodically captured by a smartphone RGB camera with subsequent detection of fluorescence intensity decreasing at each image pixel for further imaging the planar distribution of those values. The proposed methodology was tested clinically with 13 basal cell carcinoma and 1 atypical nevus. Several clinical cases and potential future applications of the smartphone-based technique are discussed.
Experimental methodology for parallel measurements of in-vivo skin autofluorescence (AF) lifetimes and photobleaching dynamic has been developed and tested. The AF lifetime decay distributions were periodically collected from fixed tissue area with subsequent detection of the fluorescence intensity decrease dynamic at different time gates after the pulse excitation. Temporal distributions of human in-vivo skin AF lifetimes and bleaching kinetics were collected and analyzed by means of commercial time-correlated single photon counting system.
The autofluorescence lifetime of healthy human skin was measured using excitation provided by a picosecond diode laser operating at a wavelength of 405 nm and with fluorescence emission collected at 475 and 560 nm. In addition, spectral and temporal responses of healthy human skin and intradermal nevus in the spectral range 460 to 610 nm were studied before and after photobleaching. A decrease in the autofluorescences lifetimes changes was observed after photobleaching of human skin. A three-exponential model was used to fit the signals, and under this model, the most significant photoinduced changes were observed for the slowest lifetime component in healthy skin at the spectral range 520 to 610 nm and intradermal nevus at the spectral range 460 to 610 nm.
The effect of ultrasound exposure on bleomycin fluorescence and pharmacological properties is studied. Bleomycin was treated by ultrasound for 7 min. Bleomycin fluorescence was measured during ultrasound exposure by means of fiber-optic spectrometry. Cell colony test was used to evaluate blemycin cytotoxity before and after ultrasound exposure.
The impact of visible cwlaser irradiation on skin autofluorescence lifetimes was investigated in spectral range from 450 nm to 600 nm. Skin optical provocations were performed during 1 min by 405 nm low power cw laser with power density up to 20 mW/cm2. Autofluorescence lifetimes were measured before and immediately after the optical provocation.
Influence of low power laser irradiance on healthy skin using diffuse reflectance spectroscopy and multispectral
imaging was studied. Changes of diffuse reflectance spectra in spectral range from 500 to 600 nm were observed after
405 nm, 473 nm and 532 nm laser provocation, leading to conclusion that the content of skin hemoglobin has changed.
Peaks in spectral absorbance (optical density) curves corresponded to well-known oxy-hemoglobin absorbance peaks at
542 and 577 nm.
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