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Autofluorescence imaging of basal cell carcinoma by smartphone RGB camera


Abstract and Figures

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.
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Autofluorescence imaging of basal cell
carcinoma by smartphone RGB
Alexey Lihachev
Alexander Derjabo
Inesa Ferulova
Marta Lange
Ilze Lihacova
Janis Spigulis
imaging of basal
cell carcinoma by
smartphone RGB camera
Alexey Lihachev,a,*Alexander Derjabo,b
Inesa Ferulova,aMarta Lange,aIlze Lihacova,aand
Janis Spigulisa
aUniversity of Latvia, Institute of Atomic Physics and Spectroscopy,
Biophotonics Laboratory, Raina Boulevard 19, Riga LV-1586, Latvia
bRiga East University Hospital, Oncology Centre of Latvia, Hipokrata
Street 4, Riga LV-1079, Latvia
Abstract. 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 distri-
bution of those values. The proposed methodology was
tested clinically with 13 basal cell carcinoma and 1 atypical
nevus. Several clinical cases and potential future applica-
tions of the smartphone-based technique are discussed.
©2015 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10
Keywords: autofluorescence; photobleaching; RGB imaging;
Paper 150558LRR received Aug. 20, 2015; accepted for publication
Nov. 12, 2015; published online Dec. 11, 2015.
1 Introduction
Incidences and mortality from skin cancer are still increasing.1
Depending on the melanin concentration, skin tumors are
broadly classified into two typesmalignant melanomas (MM)
and nonmelanoma skin cancers (NMSC). MM is the most
aggressive skin cancer modality with death rate 80% of all
fatal skin cancer cases.1The most common NMSC are basal cell
carcinoma (BCC, about 80% of new cases) and squamous cell
carcinoma (SCC, about 20% of new cases) derived from the
basal and squamous cells of the epidermis, respectively.2BCC
is characterized by very slow growth tendency, low mortality
rate, and high risk of recurrence, while SCC is more aggressive
and associated with the risk of metastasis.3,4
Early detection and removal of skin cancers can significantly
increase the survival time. Noninvasive methods in primary
oncological diagnostics of skin tumors are still topical for
dermatologists and oncologists worldwide. One of those is
skin autofluorescence (AF) imaging and spectroscopy, based
on differences of AF specific information (intensity, spectral
shape, and lifetime) in the tumor and surrounding normal
The feasibility of AF spectroscopy for BCC diagnostics and
differentiation has been studied extensively over this most recent
decade. AF spectra from BCC lesions excited in UV/blue region
(337 to 450 nm) were broadly characterized by decreased
fluorescence intensity in comparison with surrounding healthy
skin,8most often attributed to the shift in the levels of NADH/
NAD+ (reduced form and oxidized form of nicotinamide
adenine dinucleotide) and reduced elastin and collagen, affected
by malignant process. In some cases, especially in late tumor
growing stages, a weak red fluorescence peak of the endogenous
porphyrins has been observed.8
Tissue AF usually shows the photobleaching effect,9which
may be helpful in biomedical applications.1014 Under continu-
ous wave (cw) excitation, skin AF intensity mainly drops during
the first 15 to 20 s, followed by a slow decrease. Photobleaching
kinetics can be well described by empirical double-exponential
function with subsequent extraction of time constants τ1and τ2
that characterize the rate of fast and slow phases of the AF
decrease.9Our previous research has demonstrated that each
skin pathology as well as healthy skin has its own specific
AF intensity decrease kinetics depending on excitation, locali-
zation, melanin content, and blood perfusion. Furthermore, the
analysis of AF decrease kinetics during the first 15 to 20 s of cw
laser excitation seems to be most suitable for clinical
Currently available smartphones equipped with high-resolu-
tion RGB cameras in combination with good light sensitivity
and color representation mainly satisfy the required technical
properties for adequate image acquisition.17 This technology
may become a useful diagnostic tool for dermatologists and
oncologists thanks to wide accessibility, convenient use, and
low cost.18,19 However, the ability to switch off the embedded
automatized settings such as exposure time, white balance, and
ISO is crucial for the skin parametric imaging. Our latest studies
have shown that smartphones such as Galaxy and Nexus are
suitable for mapping of skin chromophores.17
So far use of smartphones in skin pathology diagnostics has
been mainly related to dermatoscopyspecifically magnified
image acquisition under white or color illumination with sub-
sequent analysis based on ABCD rules, fractal image analysis
or other algorithms established in dermatoscopy.2024 In this
paper, we present a smartphone-compatible technique for
acquisition and analysis of 405 nm light-emitting diode (LED)
excited skin autofluorescence images.
2 Materials and Methods
2.1 Experimental Setup
For parametric mapping of skin AF intensity decrease rates, a
sequence of AF images under continuous 405 nm LED (model
LED Engin LZ1-00UA00-U8, spectral band half-width 30 nm)
excitation for 20 s at a power density of 20 mWcm2with a
frame rate 0.5 frswas recorded and analyzed. Four battery-
powered violet LEDs were placed within a cylindrical light-
shielding wall that also ensured fixed distance (60 mm) between
the smartphone camera and evenly irradiated a spot (diameter
40 mm) of the examined tissue. A long pass filter (>515 nm)
was placed in front of smartphone camera to prevent detection of
the LED emission. The recorded RGB images were further sep-
arated to exploit R- and G-images for imaging of skin tissue AF
*Address all correspondence to: Alexey Lihachev, E-mail: aleksejs.lihacovs@ 1083-3668/2015/$25.00 © 2015 SPIE
Journal of Biomedical Optics 120502-1 December 2015 Vol. 20(12)
JBO Letters
in the red and green spectral bands, respectively. Due spectral
cutoff by 515-nm long pass filter B band images in further cal-
culations are not used. The Samsung Galaxy Note 3 smartphone
comprising integrated CMOS RGB image sensor with resolu-
tion of 13 MP was used for image acquisition. All images
were taken using the following settings: ISO100, white bal-
ancedaylight, focusmanual, exposure timefixed 200 ms.
2.2 Image Processing
In order to visualize the skin AF intensity decrease rates during
the photobleaching, the following image processing expression
was applied:
where NðCÞrepresents normalized AF intensity decrease map
for each pixel (or pixel group) during the excitation period,
It0½CAF image at the excitation start moment, It½CAF
image after 20 s of continuous excitation. Ccolor component
of the RGB imagered (R), green (G), and blue (B), respec-
tively. The values of RGB components were defined from the
image data by a special program developed in MATLAB®.
Overall 50 patients with 150 different skin neoplasms
(or suspicious) were inspected in the clinic. For the detailed
image analysis 13 BCC and 1 atypical nevus were selected.
This study was approved by the Ethics Committee of the
Institute of Experimental and Clinical Medicine, University of
Latvia. All involved volunteers were informed about the study
and signed required consent.
3 Results and Discussion
A total of 10 solid and 3 ulcerating BCCs were selected for the
study. In all BCC cases (confirmed by cytological examination)
the AF images showed lowered AF intensity in malignant tissue
as compared with the healthy surrounding skin, which may be
attributed to decreased levels of fluorophores and increased
blood perfusion caused by the malignancy process.2,5,7AF spec-
tra from in vivo BCC under 405 nm excitation are characterized
by broad (450 to 750 nm) emission spectrum with maximum in
green spectral region (510 to 530 nm). In comparison with sur-
rounding healthy skin, the intensity of AF from malignant tissue
usually are lower, while the shape of the spectrum remains
unchanged. Moreover, the intensity of AF is strictly correlated
with the tumor pigmentation, specifically, the higher the pig-
mentation, the lower is the intensity of fluorescence.2,8In all
BCC cases, the G-band (corresponding to the AF maximum)
AF intensity images in comparison with R-band images showed
the more pronounced intensity contrast within the tumor tissue
Fig. 1 Images of ulcerating basal cell carcinoma (BCC). Filtered AF color image (a) at excitation start
moment, (b) the corresponding G-band image, and (c) normalized AF intensity decrease map. The
pseudo color scale represents (b) the G-band intensity range and (c) the normalized AF intensity variation
Fig. 2 Images of solid BCC. Filtered AF color image (a) at excitation start moment, (b) the corresponding
G-band image, and (c) normalized AF intensity decrease map. The pseudo color scale represents (b) the
G-band intensity range and (c) the normalized AF intensity variation range.
Journal of Biomedical Optics 120502-2 December 2015 Vol. 20(12)
JBO Letters
and surrounding healthy skin. Moreover, G-band AF decrease
maps showed more structured compositions at tumor area in
comparison with R-band AF decrease maps. In the cases of
solid BCCs the AF images showed clearly bordered tumor
areas with relatively low AF intensity in comparison with sur-
rounding healthy skin. Whereas the ulcerating BCCs can be
characterized by the high AF intensity in the ulcerating part sur-
rounded by clearly bordered tissue emitting relatively low AF
intensity. Furthermore, in all BCC cases the normalized AF
intensity decrease maps showed high AF intensity decrease
rates at the tumor areas with low AF intensity in comparison
with the surrounding healthy skin and the internal ulcerating
Figure 1represents images of ulcerating BCC: (a) filtered AF
image at the excitation start moment, AF intensity G-band
image (b), and parametric map of normalized AF intensity
decrease rates in the green band (c). AF intensity image
[Fig. 1(b)] shows high AF intensity in the ulcerating part (path-
ology center), surrounded by clearly bordered tissue emitting
relatively low AF intensity. Furthermore, the AF intensity
decreasing is more intensive at the external tumor area expo-
sures in comparison with the surrounding healthy skin and the
internal ulcerating area [Fig. 1(c)].
Another case of solid BCC is presented in Fig. 2. G-band AF
intensity image [Fig. 2(b)] shows relatively low intensity within
the tumor area with clear margins between tumor and surround-
ing healthy skin. The tumor area also shows higher AF intensity
decrease rate [Fig. 2(c)] in comparison with the surrounding
healthy skin. Besides, the images presented in Fig. 2reveal a
small area with lowered fluorescence located at five oclock
from the main tumor. The low AF intensity and high AF inten-
sity decrease rate in that skin area similarly indicates to cancer-
ous process, which is probably determined by multicentric
tumor growing process.
In addition, one atypical nevus was selected for the study
(Fig. 3). The nevus before surgical excision was suspected as
melanoma; histological analysis of the removed tissue samples
had confirmed three different types of tissue cells within the
lesion area. Specifically, the upper part of the pathology mostly
prevailed by intradermal nevus, the middle part by dysplastic
nevus, and the lower part by junctional nevus. Normalized AF
decrease distribution map [Fig. 3(c)], on the other hand, showed
the fastest intensity decrease in the lower (junctional nevus) and
upper side (intradermal nevus), while the middle part (dysplastic
nevus) of lesion photobleached slower. The observed different
AF photobleaching rates most probably are associated with dif-
ferent tissue fluorophore concentration, melanin content, locali-
zation, and metabolic state.
4 Conclusions
Smartphone AF imaging has shown potential for remote pri-
mary evaluation of cancerous or suspicious skin tissues. The
proposed noninvasive technique and method adequately (with
respect to the available literature data) represented planar distri-
bution of AF intensities in malignant and healthy tissues. More-
over, the temporal analysis of AF intensity during the photo-
bleaching showed a potential to be used as an additional indi-
cator for demarcation of suspicious tissues. It may find clinical
implementation, e.g., for primary evaluation of BCC, such as
determination of precise excision margins prior to surgery,
adequate selection of treatment method (in the case of multicen-
tric growing process the nonsurgical methods are more desirable
for the patients), as well as in selective application of immune
response modulators for BCC therapy. The proposed excitation
band around 405 nm covers the absorption maxima of several
porphyrines and may find applications in photodynamic diag-
nostics of superficial nonmelanoma lesions. The most intriguing
result of this research was the fact that AF photobleaching rate
map showed quantitative correlation with the histology tests in
the case of atypical dysplastic nevus. To explain, one can
assume that specific tissue fluorophores might have individual
bleaching kinetics features, which eventually could provide
information on fluorophore concentration and environmental
factors. The increased photobleaching rates in the tumor area
most probably indicate different fluorophore content composi-
tion affected by the tumor growing process, e.g., destruction of
collagen elastin cross-links along with decrease in NADH lev-
els. Undoubtedly, this phenomenon requires additional studies
to clarify the exact mechanism of uneven photobleaching of skin
fluorophores under continuous optical excitation.
This work was supported by the European Regional Develop-
ment Fund project Innovative technologies for optical skin
diagnostics(No. 2014/0041/2DP/
Fig. 3 Color filtered AF image of skin atypical nevus (a) at the excitation start moment, (b) the corre-
sponding G-band image and (c) normalized AF intensity decrease map. The color scale at (b) image
represents G-band intensity range and at (c) imagerange of normalized AF intensity variations.
Journal of Biomedical Optics 120502-3 December 2015 Vol. 20(12)
JBO Letters
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... The lights penetrate to different layers of the skin with irradiating power density of 20 mW/cm 2 . Images were collected with a color CMOS 5 megapixel IDS camera (MT9P006STC, IDS uEye UI3581LE-C-HQ, Obersulm, Germany) fixed at 60 mm distance from the illuminated skin with a field of view of 2 × 2 cm 2 [35]. A long pass filter (T515 nm > 90%) was inserted in front of the camera to block 405 nm excitation illumination. ...
... The acquired images were automatically transferred to a cloud server, as described earlier [50]. The description of this prototype device can be found in [35,51]. To avoid the decrease in the induced AF intensity caused by photobleaching process [35], all images were taken during the first second of LED exposure. ...
... The description of this prototype device can be found in [35,51]. To avoid the decrease in the induced AF intensity caused by photobleaching process [35], all images were taken during the first second of LED exposure. A black marker was applied next to the lesions to improve image alignment (area: 0.125 cm 2 ). ...
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... Autofluorescence imaging under narrow band light emitting diode (LED) excitation uses narrow spectral light of different wavelengths to determine the distribution of the endogenous fluorophores of the skin. Using 405 nm wavelength illumination, skin autofluorescence occurs that is mainly attributed to keratins [8,9]. Nonlinear microscopy (NLM) is a promising method for label-free imaging, mostly used in brain research. ...
... A newly developed LED device was used, as described previously [8,14]. A set of autofluorescence images under continuous 405 nm LED excitation was recorded. ...
... Light absorption and emission depends greatly on the fluorophore and chromophore content and distribution of the skin, and thus biological processes that affect these can consequently change the optical properties of the tissue [21]. Alteration of tissue autofluorescence has been of particular interest in skin cancer research and diagnostics [8,9,14,22]. However, a better characterization of the optical properties of skin tissue components by studying genetic disorders could provide valuable data for noninvasive optical diagnosis of skin diseases. ...
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... As the next step, smartphone-compatible technique for acquisition and analysis of violet LED excited skin fluorescence intensity and AFPB rate distribution images has been developed and clinically tested [39]. Design of the prototype device is illustrated on Figure 9. ...
... More details on the skin fluorescence imager design and its test results are provided in [17,39]. ...
... Overall 50 patients with 150 different skin neoplasms were inspected with the smartphone based fluorescence imager. For more detailed image analysis 13 basal cell carcinomas (BCC) and 1 atypical nevus were selected [39]. In order to visualize the skin AF intensity decrease rates during the photo-bleaching, the following image processing expression was applied: ...
Optical tissue imaging has several advantages over the routine clinical imaging methods, including non-invasiveness (does not change the structure of tissues), remote operation (avoids infection) and ability to quantify the tissue condition by means of specific image parameters. Dermatologists and other skin experts need compact (preferably pocket-size), self-sustained and easy-to-use imaging devices. The operational principles and designs of ten portable in-vivo skin imaging prototypes developed at the Biophotonics Laboratory of Institute of Atomic Physics and Spectroscopy, University of Latvia during the recent five years are presented in this paper. Four groups of imaging devices are considered. Multi-spectral imagers offer possibilities for distant mapping of specific skin parameters, so facilitating better diagnostics of skin malformations. Autofluorescence intensity and photobleaching rate imagers show a promising potential for skin tumor identification and margin delineation. Photoplethysmography video-imagers ensure remote detection of cutaneous blood pulsations and can provide real-time information on cardiovascular parameters and anesthesia efficiency. Multimodal skin imagers perform several of the above-mentioned functions by taking a number of spectral and video images with the same image sensor. Design details of the developed prototypes and results of clinical tests illustrating their functionality are presented and discussed.
... This device was designed to measure skin diffuse reflectance images by using these four different LED illuminations fixed at 35 mm distance, arranged circularly in the ring and covered by a matt plate diffusor to deliver uniform illumination. Images were collected with a color CMOS 5-megapixel IDS camera (MT9P006STC, IDS uEye UI3581LE-C-HQ, Obersulm, Germany) fixed at 60 mm distance from the illuminated skin [36]. The acquired images were automatically transferred to a cloud server for further data processing and analysis [37]. ...
... A long-pass filter (T515 nm > 90%) was inserted in front of the camera, allowing it to capture G, R, and IR spectral channels. The detailed description of this prototype device has been previously published [36,38]. The LED-based multispectral images were analyzed with ImageJ v1.46 software (NIH, Bethesda, MD, USA) [39]. ...
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... It would be more effective to predict skin health if all this information can be extracted and segmented directly from the fluorescence images using image processing or machine learning. A. Lihachev et al. [23,24] used the fluorescence images to differentiate seborrheic keratosis, basal cell carcinoma, nevi and melanoma. K. Tsuchida et al. [25] found out the positive correlation between oxidative stress and skin porphyrins, which also indicated skin porphyrins are related to skin aging. ...
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The multi-spectral-line imaging concept, which was recently implemented for the snapshot mapping of three main skin chromophores—melanin, oxy-hemoglobin, and deoxy-hemoglobin, is further explored for the snapshot capturing of four spectral line images at wavelengths of 450, 523, 638, and 850 nm, with the consecutive acquiring of a 405 nm excited fluorescence image. A corresponding laser-based prototype device was designed and assembled. Processing of the mentioned five images enables obtaining distribution maps of four skin chromophores within the malformation and comparing their mean fluorescence intensity with that of the surrounding healthy skin. This set of information is helpful for dermatologists, cosmetologists, oncologists, and other healthcare professionals to quantify the diagnosis of skin malformations (including cancers) and to follow up the recovery process after therapy. This paper describes the design of the developed proof-of-concept prototype device and initial test results.
With the development of science and technology, smartphone has become a necessary communication tool for everyone. It not only has the corresponding communication function but also can be used for data recording and processing. Thus, it is of great advantage to develop a smartphone-based sensor. This chapter is focused on the introduction of three common detection strategies by using a smartphone as a portable sensing device: spectral detection, imaging image analysis and particle counting analysis. Finally, a conclusion and prospects are provided to extend the application of smartphone-based devices.
This chapter is dedicated to the specification of hyperspectral imaging devices based on acousto-optical tunable filters and their application for the detection and recognition of skin cancer. The basic concepts related to this technology are detailed. In contrast to conventional optical imaging modalities, HSI can capture much more information from the investigated tissue, differentiate and identify different substances by their spectral signature due to light reflection, absorption, and scattering across the visual spectral range. The applicability of hyperspectral imagers for endoscopic, microscopic, and macroscopic studies, as well as image reconstruction is demonstrated for different HSI schemes. A large part of the chapter reveals the possibility of featured hyperspectral imagers to capture in vivo high-resolution images of skin tissues in visible spectrum. The details of skin hyperspectral image analysis are presented along with the accuracy of skin neoplasms detection.
This book provides an in-depth description and discussion of different multi-modal diagnostic techniques for cancer detection and treatment using exact optical methods, their comparison, and combination. Coverage includes detailed descriptions of modern state of design for novel methods of optical non-invasive cancer diagnostics; multi-modal methods for earlier cancer diagnostic enhancing the probability of effective cancer treatment; modern clinical trials with novel methods of clinical cancer diagnostics; medical and technical aspects of clinical cancer diagnostics, and long-term monitoring. Biomedical engineers, cancer researchers, and scientists will find the book to be an invaluable resource. • Introduces optical imaging strategies; • Focuses on multimodal optical diagnostics as a fundamental approach; • Discusses novel methods of optical non-invasive cancer diagnostics.
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RGB (red-green-blue) technique for mapping skin chromophores by smartphones is proposed and studied. Three smartphones of different manufacturers were tested on skin phantoms and in vivo on benign skin lesions using a specially designed light source for illumination. Hemoglobin and melanin indices obtained by these smartphones showed differences in both tests. In vitro tests showed an increment of hemoglobin and melanin indices with the concentration of chromophores in phantoms. In vivo tests indicated higher hemoglobin index in hemangiomas than in nevi and healthy skin, and nevi showed higher melanin index compared to the healthy skin. Smartphones that allow switching off the automatic camera settings provided useful data, while those with "embedded" automatic settings appear to be useless for distant skin chromophore mapping. © 2015 Society of Photo-Optical Instrumentation Engineers (SPIE).
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The main results obtained during the last five years in the field of laser-excited in-vivo human skin photobleaching effects are presented. The main achievements and results obtained, as well as methods and experimental devices are briefly described. In addition, the impact of long-term 405-nm cw low-power laser excitation on the skin autofluorescence lifetime is experimentally investigated.
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The importance of dermatological noninvasive imaging techniques has increased over the last decades, aiming at diagnosing nonmelanoma skin cancer (NMSC). Technological progress has led to the development of various analytical tools, enabling the in vivo/in vitro examination of lesional human skin with the aim to increase diagnostic accuracy and decrease morbidity and mortality. The structure of the skin layers, their chemical composition, and the distribution of their compounds permits the noninvasive photodiagnosis of skin diseases, such as skin cancers, especially for early stages of malignant tumors. An important role in the dermatological diagnosis and disease monitoring has been shown for promising spectroscopic and imaging techniques, such as fluorescence, diffuse reflectance, Raman and near-infrared spectroscopy, optical coherence tomography, and confocal laser-scanning microscopy. We review the use of these spectroscopic techniques as noninvasive tools for the photodiagnosis of NMSC.
Background Lately, various smartphone applications have been introduced as diagnostic self-monitoring tools in the evaluation of pigmented moles, but most of these techniques have not been evaluated systematically. Objectives The purpose of this study was to evaluate prospectively the sensitivity and specificity of a recently developed smartphone application using fractal image analysis for the risk evaluation algorithm in the diagnosis of malignant melanoma compared to clinical diagnosis and histopathological result. Methods Consecutive patients with melanocytic lesions were recruited and clinical and dermoscopical diagnosis was documented by two dermatologists independently. Imaging and analysis with the smartphone application was performed prior to excision of lesions. The findings were compared to the histological results as gold standard. ResultsOf 195 included lesions histopathological analysis revealed 40 melanomas, 42 dysplastic nevi and 113 benign nevi. The sensitivity of the diagnosis melanoma by fractal image analysis using smartphone images was 73%, the specificity was 83% compared to a sensitivity of 88% and specificity of 97% regarding the clinical diagnosis by the dermatologists. Conclusion The smartphone application using fractal analysis might be a promising tool in the pre-evaluation of pigmented moles by laypersons, while it is to date inferior to the diagnostic evaluation by a dermatologist.
A short review will be presented of the recent clinical achievements in the field of skin autofluorescence and exogenous fluorescence tumor detection. Photosensitizers used for exogenous photodetection of cutaneous lesions will be discussed from the point of view of their photodynamic diagnostic properties and their advantages and drawbacks associated with clinical applications. A survey on various results of detection and differentiation of the fluorescent data observed from malignant cutaneous lesions will be summarized and an optimization of the skin cancer detection techniques based on fluorescence diagnostics will be analyzed and discussed. A short presentation will be given of own experimental results and clinical experience acquired in the past decade in the autofluorescence diagnostics of different benign, dysplastic, and malignant skin neoplasia. The origins of the fluorescence spectra, their peculiarities, the feasibility of clinical tumor detection, and differentiation needs will also be discussed.
Connective tissues are complex structures which contain collagen and elastin fibers. These fiber based structures have a great influence on material mechanical properties and need to be studied at the microscopic scale. Several microscopy techniques have been developed in order to image such microstructures; among them are two-photon excited fluorescence microscopy and second harmonic generation. These observations have been coupled with mechanical characterization to link microstructure kinematic to macroscopic material parameter evolution. In this study, we present a new approach to measure local strain in soft biological tissues using a side effect of fluorescence microscopy: photobleaching. Controlling the loss of fluorescence induced by photobleaching, we create a pattern on our sample that we can monitor during mechanical loading. The image analysis allows computing 3D displacements of the patterns at various loading levels. Then, local strain distribution is derived using the finite element discretization on a four nodes element mesh created from our photobleached pattern. Photobleaching tests on human liver's capsule have revealed that this technique is non-destructive and has not any impact on mechanical properties. This method is likely to have other applications in biological material studies, considering that all collagen-elastin fibers based biological tissues possess autofluorescence properties, and thus can be photobleached.
Carotenoids are important substances for human skin due to their powerful antioxidant properties in reaction of neutralization of free radicals and especially reactive oxygen species, including singlet oxygen. Concentration of carotenoids in the skin could mirror the current redox status of the skin and should be investigated in vivo. Optical methods are ideally suited for determination of carotenoids in mammalian skin in vivo as they are both noninvasive and quick. Four different optical methods could be used for in vivo measurement of carotenoids in the human or animal skin: (1) resonance Raman spectroscopy; (2) Raman microscopy; (3) reflection spectroscopy; (4) skin color measurements. The advantages, shortcomings, and limitations of the above-mentioned optical methods are discussed.
Here we present a method for improving Raman spectroscopy signal-to-noise ratio (SNR) based on fluorescence photobleaching. Good SNR is essential to obtain biochemical information about biological tissues. Subtracting high levels of tissue autofluorescence background is a major challenge in extracting weak Raman signals. We found that pre-exposure to laser light significantly reduces tissue autofluorescence, but minimally affects Raman signals, allowing subsequent acquisition of high-SNR Raman spectra. We demonstrated this method with in vivo Raman spectral measurements of human skin. This method will benefit clinical skin Raman measurements of body sites with high autofluorescence background such as the forehead and nose.
The study of biological systems in their real environmental conditions is crucial to decipher the true image of structures and processes underlying their functionality. In this regard, development of non-invasive optical techniques that do not require labelling, such as the investigation of tissue endogenous fluorescence, is particularly important and, as reflected in the increasing number of contributions published recently on this subject, was recognized by many leading groups. Multi-spectral and lifetime detection of fluorescence provides an effective experimental tool to discriminate between multiple naturally-occurring fluorophores in living tissues. At the same time, however, data analysis allowing us to understand the spectral, temporal and spatial information gathered, describing individual molecules involved in the autofluorescence of intact biological systems, represents a tough scientific challenge that has not yet been fully resolved. In this review, we discuss the latest advances in technologies that record and assess spectrally-resolved fluorescence lifetime data as well as their biological and clinical applications. We show how these methods provide efficient sensing of molecules correlated with changes in the mitochondrial metabolic redox state in pathological conditions and/or of cell ultrastructures in diseased tissue, based on the presence of oxidation/reductionsensitive fluorophores and/or cell-specific chromophores. Future directions are also outlined. (© 2009 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
Background and Objectives Laser-induced fluorescence spectroscopy is a non-invasive technique previously used for detection of cancer in a variety of organ systems. The objective of this study was to determine whether in vivo laser-induced fluorescence spectroscopy alone at the visible excitation wavelength of 410 nm could be used to detect non-melanoma skin cancers.Study Design/Materials and Methods The system consisted of a nitrogen/dye laser tuned at 410 nm, an optical multichannel analyzer, and a fiber optic probe for excitation of tissue and collection of fluorescence emission. Two hundred and seventy nine measurements were performed from normal and abnormal tissues in 49 patients. Patients were classified as having either skin types I, II, or III. Biopsy of the abnormal tissues were then performed. Each measurement was assigned as either normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), pre-cancerous, or benign. Total emission photon count was used as the discriminating index. A threshold value was calculated to separate normal tissue indices from indices of cancer tissues. The classification accuracy of each data point was determined using the threshold value.ResultsCancers were classified 93, 89, and 78% correctly in patients with skin types I, II, and III, respectively. Normal tissues were classified 93, 88, and 50% correctly in patients with skin types I, II, and III, respectively. Using the same threshold, pre-cancerous spectra were classified 78 and 100% correctly in skin types I and III, respectively. Benign lesions were classified 100, 46, and 27% correctly in patient with skin types I, II, and III, respectively.Conclusions In vivo laser induced fluorescence spectroscopy at 410 nm excitation and using the intensity of emission signal is effective for detection of BCC, SCC, and actinic keratosis, specially in patients with light colored skin. Lasers Surg. Med. 31:367–373, 2002. © 2002 Wiley-Liss, Inc.