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Non-invasive biomedical research and diagnostics enabled by innovative compact lasers
Abstract
For over half a century, laser technology has undergone a technological revolution. These technologies, particularly semiconductor lasers, are employed in a myriad of fields. Optical medical diagnostics, one of the emerging areas of laser application, are on the forefront of application around the world. Optical methods of non- or minimally invasive bio-tissue investigation offer significant advantages over alternative methods, including rapid real-time measurement, non-invasiveness and high resolution (guaranteeing the safety of a patient). These advantages demonstrate the growing success of such techniques.In this review, we will outline the recent status of laser technology applied in the biomedical field, focusing on the various available approaches, particularly utilising compact semiconductor lasers. We will further consider the advancement and integration of several complimentary biophotonic techniques into single multimodal devices, the potential impact of such devices and their future applications. Based on our own studies, we will also cover the simultaneous collection of physiological data with the aid a multifunctional diagnostics system, concentrating on the optimisation of the new technology towards a clinical application. Such data is invaluable for developing algorithms capable of delivering consistent, reliable and meaningful diagnostic information, which can ultimately be employed for the early diagnosis of disease conditions in individuals from around the world.
... Lasers have become a powerful tool in basic biological research and medicine [1]. A massive variety of laser applications exists in ophthalmology [2], dermatology [3], and surgery [4,5]. ...
... The chemical effects in biological media are commonly divided into two groups: (1) Changes in the water molecules create reactive oxygen species (ROS), subsequently affecting organic molecules. OH* and H2O2 oxygen species have been shown to cause cell damage [26]; the process of their formation following the ionization and dissociation of water molecules is described in detail [27]. ...
The evolution of laser technologies and the invention of ultrashort laser pulses have resulted in a sharp jump in laser applications in life sciences. Developmental biology is no exception. The unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of transparent material without disrupting the surrounding tissues makes ultrashort lasers a versatile tool for precise microsurgery of cells and subcellular components within structurally complex and fragile specimens like embryos as well as for high-resolution imaging of embryonic processes and developmental mechanisms. Here, we present an overview of recent applications of ultrashort lasers in developmental biology, including techniques of noncontact laser-assisted microsurgery of preimplantation mammalian embryos for oocyte/blastomere enucleation and embryonic cell fusion, as well as techniques of optical transfection and injection for targeted delivery of biomolecules into living embryos and laser-mediated microsurgery of externally developing embryos. Possible applications of ultrashort laser pulses for use in Assisted Reproductive Technologies are also highlighted. Moreover, we discuss various nonlinear optical microscopy techniques (two-photon excited fluo-rescence, second and third harmonic generation, and coherent Raman scattering) and their application for label-free non-invasive imaging of embryos in their unperturbed state or post-laser-induced modifications.
... MTS is the functional element of an organ and is a structural and functional complex composed of specialized parenchyma cells, cells of the connective tissue suspended in a noncellular matrix, blood and lymphatic microvessels, and fiber nerve endings, which are combined into a single system by regulatory mechanisms [4]. Violations in the MTS play a key role in the pathogenesis of various diseases complications (for example, rheumatological and endocrinological [5][6][7], dermatological [8], and otolaryngological [9] ones). Even during minimally invasive surgical operations, it is necessary to assess the state of MTS, for example, in case of organ ischemia, etc. [10]. ...
This work demonstrates some results of the successful experience of using wearable devices for multimodal optical diagnostics of microcirculatory-tissue systems both in clinical practice and in the conditions of a Space experiment. The multimodal approach based on simultaneously applying a minimum of 2 optical diagnostic methods, for example, laser Doppler flowmetry and fluorescence spectroscopy, allows analysing of blood flow and metabolic processes in biological tissue more systematically. Combining several wearable devices into a distributed diagnostic system allows placing them simultaneously on symmetrical parts of the body (such as opposite limbs) thus providing a possibility to study the symmetry of the measured signals. The obtained data on the state of the microcirculatory-tissue systems of the human body recorded with wearable multimodal devices make it possible to assess the relationship and dynamics of oxygen delivery and utilization by tissues more comprehensively and reliably in a variety of diagnostic tasks.
... The modeling of laser-tissue interaction is challenging because it is absorbing as well as scattering in nature due to its constituents such as water, hemoglobin, melanin RBCs, cell membrane, etc. [3,5]. So, Various mathematical models such as Beer-Lambert's law, diffusion approximation theory, radiative transfer equation (RTE), etc., have been developed to model the laser-tissue interaction. ...
The laser has been widely used in medical fields. One application of the laser is laser-based photo-thermal therapy, wherein the short-pulsed laser is generally used to destroy the cancerous cells. The efficacy of the laser-based photo-thermal therapy can be improved if we minimize the thermal damage to the surrounding healthy tissue. So, it is essential to understand the laser-tissue interaction and thermal behavior of biological tissue during laser-based photo-thermal therapy. The light propagation through the biological tissue is generally mathematically modeled by the radiative heat transfer equation (RTE). The RTE has been solved using the discrete ordinate method (DOM) to determine the intensity inside the laser-irradiated biological tissue. Consequently, the absorbed photon energy act as the source term in the Fourier/non-Fourier model-based bio-heat transfer equation to determine the temperature distribution inside the biological tissue subjected to short-pulse laser irradiation. The non-Fourier model-based bio-heat transfer equation is numerically solved using the finite volume method (FVM). The numerical results have been compared with the analytical results obtained using the finite integral transform (FIT) technique. A comparative study between the Fourier and non-Fourier heat conduction models has also been carried out.
... The laser-based technique takes much less time to acquire high-quality spectral signals [13]. Fluorescence spectroscopy (FS) is an excellent tool for non-invasively obtaining valuable biochemical information related to the metabolic properties and structural components of the extracellular matrix in tissue [13,14]. This technique is rapidly expanding due to its safety, relative cost-effectiveness, and efficiency [15][16][17]. ...
The diagnosis and treatment of non-melanoma skin cancer remain urgent problems. Histological examination of biopsy material—the gold standard of diagnosis—is an invasive procedure that requires a certain amount of time to perform. The ability to detect abnormal cells using fluorescence spectroscopy (FS) has been shown in many studies. This technique is rapidly expanding due to its safety, relative cost-effectiveness, and efficiency. However, skin lesion FS-based diagnosis is challenging due to a number of single overlapping spectra emitted by fluorescent molecules, making it difficult to distinguish changes in the overall spectrum and the molecular basis for it. We applied deep learning (DL) algorithms to quantitatively assess the ability of FS to differentiate between pathologies and normal skin. A total of 137 patients with various forms of primary and recurrent basal cell carcinoma (BCC) were observed by a multispectral laser-based device with a built-in neural network (NN) “DSL-1”. We measured the fluorescence spectra of suspected non-melanoma skin cancers and compared them with “normal” skin spectra. These spectra were input into DL algorithms to determine whether the skin is normal, pigmented normal, benign, or BCC. The preoperative differential AI-driven fluorescence diagnosis method correctly predicted the BCC lesions. We obtained an average sensitivity of 62% and average specificity of 83% in our experiments. Thus, the presented “DSL-1” diagnostic device can be a viable tool for the real-time diagnosis and guidance of non-melanoma skin cancer resection.
... Experimental studies were carried out using a multifunctional laser non-invasive certified diagnostic system "LAKK-M" (SPE "LAZMA" Ltd., Moscow, Russia), the appearance of which is presented in Fig. 1b [15][16][17]. The system is designed for research and diagnostics in various fields of biomedicine by simultaneously using the methods of pulse oximetry, laser Doppler flowmetry (LDF), tissue reflectance oximetry, and fluorescence spectroscopy. ...
The article presents the results of studies of the melanin effect on recorded signals in laser Doppler flowmetry (LDF) and tissue reflectance oximetry (TO). By investigating the properties of recorded microvascular blood flow and skin oxygenation signals, we have gained new knowledge about race-related differences in the formation of these signals. The effect of the skin melanin content on the intensity of the recorded signals in the LDF and TO methods was evaluated, and the limitations in the use of these methods in representatives of different ethnic groups were shown.
... Biologists traditionally depend on the histology of excised tissues at different time points to study any dynamic process. With the advent of deep tissue sensing techniques, non-invasive in-vivo sensing has caught up with many clinical scientists [5][6][7]. Chick embryo chorioallantoic membrane (CAM) model is a prevalent animal model and readily available for tissue engineering [8,9]. Surface displacement measurement has been done for heterogenous CAM samples using 2D and 3D photoacoustic imaging [10]. ...
There is a strong need for non-invasive detection of normal tissue from diseased
one and a better understanding of the factors involved in the infection’s growth. Continuous
monitoring of tissue samples at different time points is highly desirable. We demonstrate using
the photoacoustic spectral response technique (PASR) for in situ analysis in a developing chicken
embryo as a model (CAM) for anti-angiogenesis and vascular development. The photoacoustic
technique is an emerging modality that is based on the acoustic detection of optical absorption of
biological samples. The detected PA signals and their spectral response were used as a signature
to determine the vasculature development pathology. Continuous monitoring of vascular growth
and an anti-drug (Cisplatin) effect on vasculature has been done. PASR was investigated for the
10th day, 11th day, and 12th day control and inoculated egg samples. It shows that the dominant
frequency of the PA spectral response for 10th day control and inoculated eggs lies between
0.45–0.52 MHz, whereas for 11th day and 12th day control eggs lie at 0.61 ± 0.152 MHz and
0.67 ± 0.001 MHz, respectively. The inoculated 11th and 12th day eggs lie at 0.35 ± 0.156 MHz
and 0.16 ± 0.004 MHz, respectively. PASR could monitor the change in growth within a span of
one day, which was not possible through the conventional imaging approach. This would open
up a potential diagnostic technique for continuous monitoring of CAM assays.
... The evolution of proper diagnostic techniques for monitoring skin abnormalities is currently one of the most challenging areas of research. Nearly all examination and diagnostic methods for measuring the progress of skin disease are based on visualisation [1]. Abnormal changes in the skin layers structure cause optical properties alteration of healthy and diseased tissue. ...
Recent studies have displayed that the biological tissue, especially skin, alters the polarization state of the incident light. Using this property will enable the study of abnormalities and diseases that change not only the light intensity but also its polarization state. This paper briefly considers spatial speckle-correlometry and polarimetry method for measuring changes of polarization state of the light scattered from a biological tissue. This technique provides the possibility to have the most comprehensive information on the optical and polarization properties of the skin sector containing scar structures and other abnormalities and diseases. Practical application of the developed approach is shown in the research.
... L AST decade advance in development of the compact efficient and reliable lasers in visible and especially in near infrared spectrum ranges significantly accelerated the laser applications in the different fields of the biology and medicine [1]. ...
The mechanisms underlining the cell adaptive and/or activating oxidative stress, called low level light or photobiomodulation therapies (PBMT), still remain unclear for the near-infrared spectrum range (750-3000 nm), especially for the 1265-1270 nm range (highest absorption by molecular oxygen). It is most probable that the mitochondria may also appear to be the main target for these wavelengths. It is known that mitochondria can generate ROS under visible and 800-1060 nm spectrum range irradiation, which in turn control voltage-dependent anion channels (VDAC). Here we investigated cellular damage regarding VDAC activity, level of oxidative stress, malondialdehyde content, cell viability, mitochondrial potential and mass, GSH level, mitochondrial and nuclear DNA damage in the cancer cell culture exposed to low-level laser irradiation at 1265 nm. We used a continuous wave laser with output power 4 mW; the energy densities employed were 0.3-9.45 J/cm
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. We observed that the laser radiation at 1265 nm can induce the oxidative stress, enhance apoptosis, and disturb mitochondrial functioning at the energy density of 9.54 J/cm
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. In addition, inhibition of VDAC enhances the observed effects. It has been shown that the laser irradiation at 1265 nm damages mitochondrial DNA but does not affect the nuclear DNA. The performed experiments bring us to the conclusion that the laser irradiation at 1265 nm can affect cells through mitochondrial damage and the inhibition of VDAC enhances effects of PBMT.
With the advancement of medical science, new healthcare methods have been introduced. Biomedical signals have provided us with a deep insight into the working of the human body. Invasive biomedical signaling and sensing involve inserting sensors inside the human body. Non-invasive biomedical signals such as electroencephalogram (EEG), electromyogram (EMG), electrocardiogram (ECG), electrooculogram (EOG), phonocardiogram (PCG), and photoplethysmography (PPG) can be acquired by placing sensors on the surface of the human body. After the acquisition of these biomedical signals, further processing such as artifact removal and feature extraction is required to extract vital information about the subject’s health and well-being. In addition to conventional signal processing and analysis tools, advanced methods that involve machine and deep learning techniques were introduced to extract useful information from these signals. There are several applications of non-invasive biomedical signal processing, including monitoring, detecting, and estimating physiological and pathological states for diagnosis and therapy. For example, detection and monitoring of different types of cancer, heart diseases, blood vessel blockage, neurological disorders, etc. In addition, biomedical signals are also used in brain control interfaces (BCI), Neurofeedback and biofeedback systems to improve the mental and physical health of the subjects.
Investigations of optical solitons have always been a hot topic due to their important scientific research value. In recent years, ultrafast lasers based on two-dimensional materials such as saturable absorbers (SAs) have become the focus of optical soliton research. In this work, various soliton operations are demonstrated in Er-doped fiber lasers (EDFLs) based on SAs. First, a low-threshold passively mode-locked EDFL with traditional soliton output is constructed, and the pump threshold is as low as 10.1 mW. Second, by adjusting the net dispersion of the cavity, stable dissipative soliton operation can also be obtained. Traditional soliton mode-locked operation with controllable Kelly sidebands from first order to fourth order is realized by adjusting the pump power in a double-ended pumped structure, and the SNR is as high as 55 dB. All results prove that used as SA material has great potential and wide application prospects in investigating optical soliton operations in mode-locked fiber lasers with both normal and anomalous dispersion.
To date, Ho
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-doped fluoride fibers have been investigated as a viable route for deep-red fiber lasers. However, high output performance deep-red fiber lasers around 750 nm have been proven challenging to achieve due to limitations in both the excitation source and pumping mechanism. In this study, we use a home-made 640 nm pump source to develop a deep-red laser with a high pumping rate, and obtain a compact watt-level high efficiency deep-red laser output. Population inversion is achieved by strong excited state absorption in a Ho
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-doped ZBLAN fiber. We realize a maximum 1.46 W deep-red fiber laser operating at 752.6 nm, and its slope efficiency is as high as 53%. The improved rate equations are used to describe the quasi-four-level system, and the experimental results agree well with the simulations. Moreover, with the development of blazed grating, we demonstrate a tunable deep-red fiber laser with a tuning range of 13.7 nm, from 746.5 to 760.2 nm, which is the widest tuning range of Ho
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-doped fiber laser at deep-red wavelengths.
The current study is associated with the numerical investigation of the thermal behavior of cylindrical-shaped biological tissue during laser-based photo-thermal therapy. The light-tissue interaction has been modeled using the transient radiative transfer equation (TRTE). The TRTE in a cylindrical coordinate system is solved using the modified discrete ordinate method to obtain the intensity distribution inside the biological tissue. The solution of TRTE is coupled with Pennes bio-heat transfer equation (BHTE) to understand the thermic response of biological tissue. The Pennes BHTE is solved using the finite volume method (FVM). The two different types of optical inhomogeneity (absorption and scattering inhomogeneity) are considered in the current study. The inhomogeneity's optical and thermophysical properties may be the same/different from the homogeneous biological tissue. This conjugate heat transfer problem (CHTP) is solved using the harmonic mean technique. First, the present result is verified with the data available in the literature. Subsequently, the effect of inhomogeneity's optical properties on the temperature distribution is investigated. A comparative study between the same and different thermophysical properties of the biological tissue and inhomogeneity is performed. This study will help to accurately model the thermal characteristics of the laser-irradiated biological tissue having different thermophysical properties than the inhomogeneity.
The development of noninvasive methods for every application requires knowledge of the detection principles. In the case of diabetes, progress requires the cooperation of both medical professionals and engineering specialists, including biologists, biotechnologists, chemists, physicists, electronics experts, and computer scientists. In addition, the limitations of the developments of such methods are determined by the biological or technological limits and therefore knowledge of the biological compounds related to diabetes, such as glucose, insulin, glucagon, and glycated hemoglobin as well as the knowledge of the possible detection methods that can be realized in a noninvasive manner (e.g., optical detection, bio-impedance measurements) is necessary. In this chapter, the basics of the biological compounds related to diabetes and measurement techniques are presented and discussed with references.
Herein, we report on the growth and characterization of La0.78Y0.32Yb0.04Sc2.86(BO3)4 (Yb:LYSB) crystal as bifunctional laser and nonlinear optical (NLO) medium. High quality Yb:LYSB crystal with non-congruent melting has been grown by the Czochralski technique. Structural, linear and NLO properties, and near-infrared (NIR) laser emission performances of the grown crystal are presented. The absorption cross-section at 980 nm for σ-polarization was evaluated to be 0.6 × 10⁻²⁰ cm², and emission cross-section around 1080 nm in π-polarization and σ-polarization amounted to 3.6 × 10⁻²¹ cm² and 1.33 × 10⁻²¹ cm², respectively. The spectroscopic investigations revealed the existence of two different Yb³⁺ centers in the LYSB crystal matrix. Efficient laser emission at 1028 nm was obtained with a c-cut 4 at.% Yb:LYSB uncoated sample with a thickness of 3.5 mm. Employing the pump at 971.5 nm with a fiber-coupled diode laser, the Yb:LYSB medium yielded laser pulses with 1.5 mJ energy at 4.25 mJ energy of the absorbed pump pulse, corresponding to an overall optical-to-optical efficiency of 0.35. The laser operated with a high slope efficiency of 0.57. The refractive indexes were measured and the NLO properties of Yb:LYSB crystal for second harmonic generation of 1028 nm laser radiation were determined. The results of this work indicate Yb:LYSB as a promising crystal to design new laser sources in the NIR and green (∼514 nm) spectral ranges based on direct laser emission and self-frequency doubling processes, respectively.
We report significant improvement in the performance of TEM00 alexandrite laser operation by employing high power fibre-coupled red diode pumping, novel cavity design, and active direct Shack-Hartmann wavefront sensor measurement of pump-induced lensing. We demonstrate 12.7 W of laser power in low-order (M2∼5) mode operation from a compact double-end-pumped cavity, and with novel cavity design, a record power of 7.4 W in TEM00 operation with excellent beam quality (M2≤1.1). With single-end pumping, laser power of 4.7 W (M2∼1.3) was achieved with slope efficiencies as high as 54.9 %; a record efficiency for red-diode-pumped alexandrite. Using a birefringent filter, continuous laser wavelength tuning from 725-808 nm is achieved in diffraction-limited TEM00 mode, with laser power of 4.7 W at 765 nm, and >1 W across 730-805 nm, which is a higher tunable power than any other directly diode-pumped vibronic laser, to the best of our knowledge.
Angular measurements in optics of biological tissues are used for different applied spectroscopic task for roughness surface control, define of refractive index and for research of optical properties. Purpose of the research is investigation of the reflectance of biologic tissues by the ellipsoidal reflector method under the variable angle of the incident radiation.
The research investigates functional features of improved photometry method by ellipsoidal reflectors. The photometric setup with mirror ellipsoid of revolution in reflected light was developed. Theoretical foundations of the design of an ellipsoidal reflector with a specific slot to ensure the input of laser radiation into the object area were presented. Analytical solution for calculating the angles range of incident radiation depending on the eccentricity and focal parameter of the ellipsoid are obtained. Also created the scheme of image processing at angular photometry by ellipsoidal reflector.
The research represents results of experimental series for samples of muscle tissues at wavelengths 405 nm, 532 nm, 650 nm. During experiment there were received photometric images on the equipment with such parameters: laser beam incident angles range 12.5–62.5°, ellipsoidal reflector eccentricity 0.6, focal parameter 18 mm, slot width 8 mm.
The nature of light scattering by muscle tissues at different wavelengths was represented by graphs for the collimated reflection area. The investigated method allows qualitative estimation of influence of internal or surface layers of biologic tissues optical properties on the light scattering under variable angles of incident radiation by the shape of zone of incident light.
The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.
This is the second section of the review-tutorial paper describing fundamentals of tissue optics and photonics. As the first section of the paper was mostly devoted to description of biological tissue structures and their specificity related to interactions with light [1], this section 3 describes light-tissue interactions themselves that caused by tissue dispersion, scattering, and absorption properties, including light reflection and refraction, absorption, elastic, and quasi-elastic scattering. The major tissue absorbers and modes of elastic scattering, including Rayleigh and Mie scattering, will be presented.
The influence of various melanin concentrations on the endogenous fluorescence intensity of biological tissue has been experimentally studied, and the fluorescence signals have been modeled by the Monte Carlo method. The modeling is based on a four-layer optical model of the skin, using known optical parameters of skin with various melanin concentrations. The fluorescence spectra obtained by the Monte Carlo method agrees with the results of the experimental investigations.
This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry.
We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra.
In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.
Skin blood microcirculation and the metabolism activity of tissue were examined on the patients with type 2 diabetes. Laser Doppler flowmetry (LDF) with 1064 nm laser light source and fluorescence spectroscopy (FS) with excitation light of 365 nm and 450 nm have been used to monitor the blood perfusion and the content of coenzymes NADH and FAD. Concluding, the proposed combined LDF and tissue FS approach allows to identify the significant violations in the blood microcirculation and metabolic activity for type 2 diabetes patients.
Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a “generic” computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system “LAKK-M” was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.
Detection, characterization, and staging constitute the fundamental elements in the endoscopic diagnosis of gastrointestinal diseases, but histology still remains the diagnostic gold standard.
New developments in endoscopic techniques may challenge histopathology in the near future. An ideal endoscopic technique should combine a wide-field, “red flag” screening technique with an optical contrast or microscopy method for characterization and staging, all simultaneously available during the procedure. In theory, biophotonic advances have the potential to unite these elements to allow in vivo “optical biopsy.”
These techniques may ultimately offer the potential to increase the rates of detection of high risk lesions and the ability to target biopsies and resections, and so reduce the need for biopsy, costs, and uncertainty for patients. However, their utility and sensitivity in clinical practice must be evaluated against those of conventional histopathology.
This review describes some of the most recent applications of biophotonics in endoscopic optical imaging and metrology, along with their fundamental principles and the clinical experience that has been acquired in their deployment as tools for the endoscopist. Particular emphasis has been placed on translational label-free optical techniques, such as fluorescence spectroscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon and multi-photon microscopy, second harmonic generation (SHG) and third harmonic generation (THG) imaging, optical coherence tomography (OCT), diffuse reflectance, Raman spectroscopy, and molecular imaging.
This review gives an overview of the developments in the analysis of drugs of abuse and other illicit substances by Raman spectroscopy for forensic purpose. The review covers the brief overview of basic principle and instrumentation of Raman spectroscopy along with selected and recent applications for characterization of drugs of abuse using this technique. These applications show the potential value of Raman spectroscopy in the qualitative and quantitative analysis of trace amounts of drugs of abuse and other illicit substances on different matrices such as cloth, currency notes, fiber etc., without extensive sample preparation in a non-destructive manner.
Abstract: Traumatic brain injury (TBI) is a growing health concern effecting civilians and military personnel. Research has yielded a better understanding of the pathophysiology of TBI, but effective treatments have not been forthcoming. Near-infrared light (NIR) has shown promise in animal models of both TBI and stroke. Yet, it remains unclear if sufficient photonic energy can be delivered to the human brain to yield a beneficial effect. This paper reviews the pathophysiology of TBI and elaborates the physiological effects of NIR in the context of this pathophysiology. Pertinent aspects of the physical properties of NIR, particularly in regards to its interactions with tissue, provide the background for understanding this critical issue of light penetration through tissue. Our recent tissue studies demonstrate no penetration of low level NIR energy through 2 mm of skin or 3 cm of skull and brain. However, at 10–15 W, 0.45%–2.90% of 810 nm light penetrated 3 cm of tissue. A 15 W 810 nm device (continuous or non-pulsed) NIR delivered 2.9% of the surface power density. Pulsing at 10 Hz reduced the dose of light delivered to the surface by 50%, but 2.4% of the surface energy reached the depth of 3 cm. Approximately 1.22% of the energy of 980 nm light at 10–15 W penetrated to 3 cm. These data are reviewed in the context of the literature on low-power NIR penetration, wherein less than half of 1% of the surface energy could reach a depth of 1 cm. NIR in the power range of 10–15 W at 810 and 980 nm can provide fluence within the range shown to be biologically beneficial at 3 cm depth. A companion paper reviews the clinical data on the treatment of patients with chronic TBI in the context of the current literature.
Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied. The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.
Copyright © 2015 IPEM. Published by Elsevier Ltd. All rights reserved.
Raman spectroscopy is an optical technique based on inelastic scattering of light by vibrating molecules and can provide chemical fingerprints of cells, tissues or biofluids. The high chemical specificity, minimal or lack of sample preparation and the ability to use advanced optical technologies in the visible or near-infrared spectral range (lasers, microscopes, fibre-optics) has recently led to an increase in medical diagnostic applications of Raman spectroscopy. The key hypothesis underpinning this field is that molecular changes in cells, tissues or biofluids, that are either the cause or the effect of diseases, can be detected and quantified by Raman spectroscopy. Furthermore, multivariate calibration and classification models based on Raman spectra can be developed on large "training" datasets and used subsequently on samples from new patients to obtain quantitative and objective diagnosis. Historically, spontaneous Raman spectroscopy has been known as a low signal technique requiring relatively long acquisition times. Nevertheless, new strategies have been developed recently to overcome these issues: non-linear optical effects and metallic nanoparticles can be used to enhance the Raman signals, optimised fibre-optic Raman probes can be used for real-time in-vivo single-point measurements, while multimodal integration with other optical techniques can guide the Raman measurements to increase the acquisition speed and spatial accuracy of diagnosis. These recent efforts have advanced Raman spectroscopic to the point where the diagnostic accuracy and speed are compatible with clinical use. This paper reviews the main Raman spectroscopy techniques used in medical diagnostics and provides an overview of various applications.
Copyright © 2015. Published by Elsevier B.V.
Fluorescent spectroscopy (FS) is becoming more widely used in chemistry, biology, in various fields of medical technology and medicine in general. Many purulent wounds, burns and other destructive inflammatory processes are accompanied by changes in the fluorescent activity of the tissues, which occurs due to a misbalance in accumulation of natural fluorophores: FAD, NADH, lipofuscin, porphyrins, structural proteins, etc. The study of redox ratio (RR), characterizing the metabolic processes, is important in the assessment of the metabolic activity ofmicrocirculatory-tissue systems (MTS). However, one of the big problems of the FS method is still the correct interpretation of the data and the development of practical methods for its application in clinical medicine. To solve this problem and create new diagnostic criteria, we propose to evaluate the adaptive capacity of MTS using indicators of links between nutritive blood flow and redox ratio during a physiological rest and functional load (occlusion test). As is known, these parameters (RR and nutritive blood flow) characterize the metabolic activity of tissues.We have performedan experimental study of the relationship between the RR, defined by FS, and nutritive blood flow, defined by the methods of laser Doppler flowmetry. Preliminary results in the study of a complex approach to diagnosis of the state of biological tissue were obtained. A positive relationship between the nutritive blood flow in the microcirculatory channel and RR of skin tissue is observed.The speed of change of metabolism in the phase of occlusion and reperfusion and duration of phase of recovery may be the criteria for adaptive capabilities of MTS, which has practical significance for physiology and medicine.
The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.
Diseases of urinary bladder are a common healthcare problem world over. Diagnostic precision and predicting response to treatment are major issues. This study aims to create an optical cross-sectionional model of a bladder, capable of visually representing the passage of photons through the tissue layers. The absorption, transmission and reflectance data, along with the derived transmission coefficients (of scattering and absorption) were obtained from literature analysis and were used in the creation of a " generic " cross-section optical property model simulating the passage of thousands of photons through the tissue at different wavelengths. Fluorescence spectra of diagnostically relevant biomarkers excited by the UV and blue wavelengths were modelled on the basis of the Monte-Carlo method. Further to this, fluorescence data gathered by the " LAKK-M " system from pig bladders was applied to the model for a specific representation of the photon passage through the tissues. The ultimate goal of this study is to employ this model to simulate the effects of different laser wavelength and energy inputs to bladder tissue and to determine the effectiveness of potential photonics based devices for the diagnosis of bladder pathologies. The model will aid in observing differences between healthy and pathological bladder tissues registered by photonics based devices.
Scanning laser ophthalmoscopes (SLOs) are able to achieve superior contrast and axial sectioning capability compared to fundus photography. However, SLOs typically use monochromatic illumination and are thus unable to extract color information of the retina. Previous color SLO imaging techniques utilized multiple lasers or narrow band sources for illumination, which allowed for multiple color but not “true color” imaging as done in fundus photography. We describe the first “true color” SLO, handheld color SLO, and combined color SLO integrated with a spectral domain optical coherence tomography (OCT) system. To achieve accurate color imaging, the SLO was calibrated with a color test target and utilized an achromatizing lens when imaging the retina to correct for the eye’s longitudinal chromatic aberration. Color SLO and OCT images from volunteers were then acquired simultaneously with a combined power under the ANSI limit. Images from this system were then compared with those from commercially available SLOs featuring multiple narrow-band color imaging.
Collagen is an endogenous fluorophore that accounts for about 70% of all proteins of human skin, so it can be an optical marker for structural abnormalities in tissues registered by laser fluorescent diagnostics in vivo. Using the examples of such abnormalities as scars, scleroderma and basal cell carcinoma, this study shows the differences between coefficients of fluorescent contrast kf(λ) of abnormalities from the ones for healthy tissues at fluorescent excitation wavelength 360–380 nm. It is shown that scars and dysplasia are characterized by reduced values of kf(λ) for collagen. Due to high turbidity and phase heterogeneousness as well as variation of parameters of blood microcirculation and concentrations of other related chromophores, there is no mathematical model that precisely calculates the concentration of collagen in tissues only with the use of the value of fluorescent signal intensity. So, probably, the best marker of the pathological process is a comprehensive representation of kf(λ) for all endogenous fluorophores, i.e., for all used visible wavelengths. In this case identification of abnormal tissues is quite possible by detecting some deviations of coefficients kf(λ) for the optically identical and symmetrical regions of the human body.
Background and objective:
The formation of reactive oxygen species (ROS) is associated with cardiovascular disease (CVD). High dietary cholesterol can significantly alter the delicate balance between pro-oxidation and antioxidant defences leading to reactive oxygen species formation in the vasculature, without significant structural changes in tissue composition. We aimed to establish a methodology for the noninvasive assessment of skin fluorescent biomarkers in mice.
Materials and methods:
C57/black/6 wild-type (WT; n = 25) male mice were subdivided to receive normal rodent chow (n = 11) or a high cholesterol diet (2% cholesterol; n = 14) for 20 weeks. Skin autofluorescence measurements were made on the backs of anaesthetized (1.5-2% isoflurane in oxygen) mice. A laser probe was used to make simultaneous measurements of: collagen, elastin, nicotinamide pyridoxine, flavins, lipofuscin and β-carotene. Results are expressed as group mean in arbitrary units (AU) ± standard error (SE). Hearts were excised and weighed (mg); cardiac hypertrophy was measured by ratio [heart weight (mg)/bodyweight (g) ± SE]. Student's t-test was used for statistical significance analysis (p ≤ 0.05).
Results:
There were no significant differences between cholesterol- and chow-fed animals for collagen (34 ± 5AU vs. chow 34 ± 4 AU, p = 0.51) and elastin (66 ± 6 AU vs. chow 82 ± 7 AU, p = 0.11). Significant differences were evident for nicotinamide adenine dinucleotide (92 ± 7 AU vs. chow 118 ± 7 AU, p = 0.01), pyridoxine (56 ± 4 AU vs. chow 73 ± 4 AU, p = 0.01), flavins (44 ± 3 AU vs. chow 57 ± 4 AU, p = 0.01), lipofuscin (35 ± 3 AU vs. chow 46 ± 3 AU, p = 0.01) and β-carotene (19 ± 2 AU vs. chow 25 ± 2 AU, p = 0.01). Cholesterol-fed animals had significantly heavier hearts (7 ± 0.3 ratio vs. chow 5 ± 0.1 ratio, p = 0.001).
Conclusion:
Cholesterol feeding induced cardiovascular disease as noted by cardiac hypertrophy in wild-type mice. A reduction was observed in pyridoxine, nicotinamide adenine dinucleotide, flavins, lipofuscin and β-carotene, which are established risk factors for cardiovascular disease. We report no significant changes in structural proteins collagen and elastin, suggesting no generalized tissue restructuring, which might otherwise explain the observed pathological differences.
Multi-functional laser non-invasive diagnostic systems allow the study of a number of microcirculatory parameters, including index of blood microcirculation (Im) (by laser Doppler flowmetry, LDF) and oxygen saturation (StO2) of skin tissue (by tissue reflectance oximetry, TRO). This research aimed to use such a system to investigate the synchronization of microvascular blood flow and oxygen saturation rhythms under normal and adaptive change conditions. Studies were conducted on eight healthy volunteers of 21–49 years. These volunteers were observed between one and six months, totalling 422 basic tests (3 min each). Measurements were performed on the palmar surface of the right middle finger and the lower forearm's medial surface. Rhythmic oscillations of LDF and TRO were studied using wavelet analysis. Combined tissue oxygen consumption data for all volunteers during 'adaptive changes' increased relative to normal conditions with and without arteriovenous anastomoses. Data analysis revealed resonance and synchronized rhythms in microvascular blood flow and oxygen saturation as an adaptive change in myogenic oscillation (vasomotion) resulting from exercise and possibly psychoemotional stress. Synchronization of myogenic rhythms during adaptive changes may lead to increased oxygen consumption as a result of increased microvascular blood flow velocity.
The metrological basis for optical non-invasive diagnostic devices is an unresolved issue. A major challenge for laser Doppler flowmetry (LDF) is the need to compare the outputs from individual devices and various manufacturers to identify variations useful in clinical diagnostics. The most common methods for instrument calibration are simulants or phantoms composed of colloids of light-scattering particles which simulate the motion of red blood cells based on Brownian motion. However, such systems have limited accuracy or stability and cannot calibrate for the known rhythmic components of perfusion (0.0095-1.6 Hz). To solve this problem, we propose the design of a novel technique based on the simulation of moving particles using an electromechanical transducer, in which a precision piezoelectric actuator is used (e.g., P-602.8SL with maximum movement less than 1 mm). In this system, Doppler shift is generated in the layered structure of different solid materials with different optical light diffusing properties. This comprises a fixed, light transparent upper plane-parallel plate and an oscillating fluoroplastic (PTFE) disk. Preliminary studies on this experimental setup using the LDF-channel of a “LAKK-M” system demonstrated the detection of the linear portion (0-10 Hz with a maximum signal corresponding to Doppler shift of about 20 kHz) of the LDF-signal from the oscillating frequency of the moving layer. The results suggest the possibility of applying this technique for the calibration of LDF devices.
Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.
Photodynamic therapy (PDT) is a technique developed to treat the ever-increasing global incidence of cancer. This technique utilises singlet oxygen ((1)O2) generation via a laser excited photosensitiser (PS) to kill cancer cells. However, prolonged sensitivity to intensive light (6-8 weeks for lung cancer), relatively low tissue penetration by activating light (630 nm up to 4 mm), and the cost of PS administration can limit progressive PDT applications. The development of quantum-dot laser diodes emitting in the highest absorption region (1268 nm) of triplet oxygen ((3)O2) presents the possibility of inducing apoptosis in tumour cells through direct (3)O2 → (1)O2 transition. Here we demonstrate that a single laser pulse triggers dose-dependent (1)O2 generation in both normal keratinocytes and tumour cells and show that tumour cells yield the highest (1)O2 far beyond the initial laser pulse exposure. Our modelling and experimental results support the development of direct infrared (IR) laser-induced tumour treatment as a promising approach in tumour PDT.
Objective:
The purpose of this study was to investigate the safety and efficacy of two novel light sources for large area and full body application, providing polychromatic, non-thermal photobiomodulation (PBM) for improving skin feeling and appearance.
Background data:
For non-thermal photorejuvenation, laser and LED light sources have been demonstrated to be safe and effective. However, lasers and LEDs may offer some disadvantages because of dot-shaped (punctiform) emission characteristics and their narrow spectral bandwidths. Because the action spectra for tissue regeneration and repair consist of more than one wavelength, we investigated if it is favorable to apply a polychromatic spectrum covering a broader spectral region for skin rejuvenation and repair.
Materials and methods:
A total of 136 volunteers participated in this prospective, randomized, and controlled study. Of these volunteers, 113 subjects randomly assigned into four treatment groups were treated twice a week with either 611-650 or 570-850 nm polychromatic light (normalized to ∼ 9 J/cm(2) in the range of 611-650 nm) and were compared with controls (n=23). Irradiances and treatment durations varied in all treatment groups. The data collected at baseline and after 30 sessions included blinded evaluations of clinical photography, ultrasonographic collagen density measurements, computerized digital profilometry, and an assessment of patient satisfaction.
Results:
The treated subjects experienced significantly improved skin complexion and skin feeling, profilometrically assessed skin roughness, and ultrasonographically measured collagen density. The blinded clinical evaluation of photographs confirmed significant improvement in the intervention groups compared with the control.
Conclusions:
Broadband polychromatic PBM showed no advantage over the red-light-only spectrum. However, both novel light sources that have not been previously used for PBM have demonstrated efficacy and safety for skin rejuvenation and intradermal collagen increase when compared with controls.
The devising of a general engineering theory of multifunctional diagnostic systems for non-invasive medical spectrophotometry is an important and promising direction of modern biomedical engineering. We aim in this study to formalize in scientific engineering terms objectives for multifunctional laser non-invasive diagnostic system (MLNDS). The structure-functional model as well as a task-function of
generalized MLNDS was formulated and developed. The key role of the system software for MLNDS general architecture at steps of ideological-technical designing has been proved. The basic principles of
block-modules composition of MLNDS hardware are suggested as well.
A scientific approach to the formulation of medical and technical requirements (MTRs) for noninvasive spectrophotometric diagnostic devices using optical technologies such as laser Doppler flowmetry and absorption spectroscopy is proposed. The theoretical modeling framework, metrological certification, and testing of these devices are still in the early stages of development. The theoretical estimation of the received signal levels for wavelengths between 514 and 940 nm is highly dependent on the blood volume level in the subject tissue. The proposed approach allows, in particular, the calculation of technical and metrological performance constraints of the instruments, such as the ranges of the sensitivity and power-related signal-to-noise ratios for different spectral channels and different biomedical (biochemical and physiological) parameters. Substantiation of specialized MTRs for the noninvasive spectrophotometric diagnostic devices can enable them to develop to the level of standardized measurement techniques.
Mid-infrared quantum cascade lasers are semiconductor injection lasers whose active core implements a multiple-quantum-well structure. Relying on a designed staircase of intersubband transitions allows free choice of emission wavelength and, in contrast with diode lasers, a low transparency point that is similar to a classical, atomic four-level laser system. In recent years, this design flexibility has expanded the achievable wavelength range of quantum cascade lasers to similar to 3-25 mu m and the terahertz regime, and provided exemplary improvements in overall performance. Quantum cascade lasers are rapidly becoming practical mid-infrared sources for a variety of applications such as trace-chemical sensing, health monitoring and infrared countermeasures. In this Review we focus on the two major areas of recent improvement: power and power efficiency, and spectral performance.
The integration of multiple optical techniques within a single diagnostic device is used to address the difficulties in standardising measurement of cutaneous blood micro-dynamics caused by high variability. We demonstrate the benefits of simultaneous assessment of blood relative volume (Vb), microcirculation index (Im) and tissue oxygen saturation (StO2), during long-term examination of healthy volunteers. Consequently, five rhythmic components: endothelial, neurogenic, myogenic, breath and heart pulses were established showing high variability up to 30 – 50% as well as in initial parameters around 16%. All rhythmic components were synchronous with some latency between Im and StO2 in the myogenic component supports the hypothesis of strong correlation between peripheral hemodynamics and oxygen utilisation in tissues.
Estimation of regional tissue oxygenation (rStO2) by near infrared spectroscopy enables non-invasive end-organ oxygen balance monitoring and could be a valuable tool in intensive care. However, the diverse absolute values and dynamics of different devices, and overall poor repeatability of measurements are a problem. The aim of the present study is to test the hypothesis that INVOS 5100C, FORE-SIGHT and NONIN EQUANOX 7600 have similar properties concerning absolute values, repeatability, and sensitivity to changes in rStO2. To test repeatability the sensors were repositioned 20 times during hemodynamic steady state on the adult forearm. Afterwards six vascular occlusions by inflation of an upper arm cuff were done to achieve low oxygenation in the forearm. Absolute values were compared by repeated-measures ANOVA, repeatability was estimated by the within-subject standard deviation, Sw, and response to changing oxygenation by the down slope of rStO2 during vascular occlusion in the respective arm. 10 healthy adults, 21-29 years old, with double skinfolds on the forearm less than 10 mm participated. The median rStO2 was 70.7 % (interquartile range (IQR) 7.7 %), 68.4 % (IQR 8.4 %), and 64.6 % (IQR 4.8) with INVOS, NONIN, and FORE-SIGHT, respectively, the median rate of decline was 13.2 %/min (IQR 9.6), 22.8 %/min (IQR 18.0), and 10.8 %/min (IQR 6.0), and the same-site repeatability was 2.9 % (95 % CI 2.4-3.3), 4.6 % (CI 3.9-5.3), and 2.0 % (CI 1.7-2.3). INVOS gave significantly higher steady state values than FORE-SIGHT, and NONIN had the steepest decline in rStO2, but the poorest repeatability. Two measures of signal-to-noise were similar among devices. This suggests that good repeatability comes at the expense of low sensitivity to changes in oxygenation. Values of rStO2 on the forearm from INVOS, NONIN and FORE-SIGTH cannot be used interchangeably.
The urgency of BCC study affecting maxillofacial area and neck is not only caused by high prevalence of this disease, but also insufficient efficiency of existing treatment methods which lead to full or partial recovery only in 60-80% of cases. We analyzed the results of 198 BCC cases cryosurgical treatment. 33 (16,6%) patients showed continued tumor growth. It has been hypothesized that the behavior and character of microcirculation changes during patient's testing have to correlate with damaging rate of tumors that will allow to develop indications for surgical treatment with local destruction - cryosurgery or cryolaser treatment. We have tested the new group of 33 patients with primary and recurrence types of basal cell carcinoma (BCC) by means of Laser Doppler Flowmetry, Tissues Reflectance Oximetry, Laser Fluorescence Diagnostics before operation. It was shown that the microcirculatory data indicates the presence of cryoresistance.
Background
Any innovation which facilitates the early detection of neoplastic changes in upper aerodigestive tract mucosa has potential to greatly improve survival and quality of life in persons prone to develop malignancies in this area. One technology that has shown great promise during initial investigations is fluorescence spectroscopy. Fluorescence spectroscopy evaluates the physical and chemical properties of tissue by analyzing the intensity and character of light emitted in the form of fluorescence. This technology has been investigated for the noninvasive detection of malignancy in various sites including the gastrointestinal tract, lung, breast, and cervix.
Methods
This article reviews the recent work investigating the capabilities of fluorescence spectroscopy to discriminate between normal and neoplastic mucosa in the oral cavity. Also discussed are potential applications for the detection and diagnosis of premalignant and malignant lesions of the upper aerodigestive tract, and some of the obstacles to overcome to make this technology feasible. © 1998 John Wiley & Sons, Inc. Head Neck 20: 556–562, 1998.
Most researchers agree that biological confocal microscopy was jump-started by the confocal design first published by White and Amos in 1985 in the Journal of Cell Biology. As a result, this remains a relatively young field. Yet the use of the technique has grown phenomenally since those early efforts, with new users joining the ranks daily. The publication of Basic Confocal Microscopy reflects the burgeoning need to train new students, technologists, and faculty wishing to use confocal microscopy in their research. A direct outgrowth of the authors’ five-day intensive course in the subject begun in 2005, this book covers the basics and includes all the information required to design, implement, and interpret the results of, biological experiments based on confocal microscopy. Concise yet comprehensive, the volume begins by covering the core issues of fluorescence, specimen preparation and labeling, before moving on to address the analog-to-digital conversion of specimen data gathered using confocal microscopy. Subsequent chapters detail the practicalities of operating confocal microscopes, providing all the information necessary to begin practicing confocal microscopy as well as optimizing the material obtained. The final block of chapters examine 3-dimensional analysis and the reconstruction of data sets, outline some of the ethical considerations in confocal imaging, and then supply a number of resources that the authors have found useful in their own work. Once readers have mastered the information this book presents, the resources found in its pages will be an excellent guide to continued learning about the more advanced forms of confocal microscopy.
This concise, user-oriented and up-to-date desk reference offers a broad introduction to the fascinating world of medical technology, fully considering today’s progress and further development in all relevant fields. The Springer Handbook of Medical Technology is a systemized and well-structured guideline which distinguishes itself through simplification and condensation of complex facts. This book is an indispensable resource for professionals working directly or indirectly with medical systems and appliances every day. It is also meant for graduate and post graduate students in hospital management, medical engineering, and medical physics.
We report on low threshold current density (<400 A cm⁻²) injection lasing in (Al x Ga1-x)0.5In0.5P-GaAs-based diodes down to the green spectral range (<570 nm). The epitaxial structures are grown on high-index (611)A and (211)A GaAs substrates by metal-organic vapor phase epitaxy and contain tensile-strained GaP-enriched insertions aimed at reflection of the injected nonequilibrium electrons preventing their escape from the active region. Extended waveguide concept results in a vertical beam divergence with a full width at half maximum of 15 for (611)A substrates. The lasing at the wavelength of 569 nm is realized at 85 K. In an orange-red laser diode structure low threshold current density (190 A cm⁻²) in the orange spectral range (598 nm) is realized at 85 K. The latter devices demonstrated room temperature lasing at 628 nm at ∼2 kA cm⁻² and a total power above 3 W. The red laser diodes grown on (211)A substrates demonstrated a far field characteristic for vertically multimode lasing indicating a lower optical confinement factor for the fundamental mode as compared to the devices grown on (611)A. However, as expected from previous research, the temperature stability of the threshold current and the wavelength stability were significantly higher for (211)A-grown structures.
This topical review presents an overview on novel concepts for light emitting diodes (LEDs) and lasers for the near infrared to the THz regime. GaSb-based quantum well lasers are shown to be a promising concept for laser from the near to mid infrared. The GaSb-based edge-emitting lasers offer low thresholds for wavelengths ranging from about 2 to 3.7 μm. However, the development of vertical-cavity surface-emitting lasers and other advanced laser concepts is lagging behind due to material issues and complicated process technology. InP-based type-II quantum wells are an innovative concept for sources emitting in the wavelength range from 2 to 4 μm. This concept combines extended long wavelength emission with the reliable process technology of the already well-established InP-based lasers. Based on this, we present LEDs up to 3.5 μm wavelength, surface emitting lasers at 2.5 μm wavelength and edge emitting lasers up to 2.7 μm. For longer wavelengths, the so-called GaSb- and InAs-based interband cascade lasers can be used operating up to about 7 μm. The mid infrared range between 3 and 20 μm is also covered by quantum cascade lasers (QCL), which are dominating especially in the longer wavelength range above 7 μm. The far infrared reaching to the THz regime is exclusively covered by QCL. While for decades the only available semiconductor laser source for the far infrared and THz range was the direct THz QCL, recent progress demonstrated THz emission in nonlinear mid infrared QCLs. These devices are emitting THz by a nonlinear frequency conversion process, which allows operation at room temperature and beyond. Tunable THz lasers were demonstrated using both monolithic tuning mechanisms and an external cavity approach.
An important field of application of lasers is biomedical optics. Here, they offer great utility for diagnosis, therapy and surgery. For the development of novel methods of laser-based biomedical diagnostics careful study of light propagation in biological tissues is necessary to enhance our understanding of the optical measurements undertaken, increase research and development capacity and the diagnostic reliability of optical technologies. Ultimately, fulfilling these requirements will increase uptake in clinical applications of laser based diagnostics and therapeutics. To address these challenges informative biomarkers relevant to the biological and physiological function or disease state of the organism must be selected. These indicators are the results of the analysis of tissues and cells, such as blood. For non-invasive diagnostics peripheral blood, cells and tissue can potentially provide comprehensive information on the condition of the human organism. A detailed study of the light scattering and absorption characteristics can quickly detect physiological and morphological changes in the cells due to thermal, chemical, antibiotic treatments, etc [1-5]. The selection of a laser source to study the structure of biological particles also benefits from the fact that gross pathological changes are not induced and diagnostics make effective use of the monochromatic directional coherence properties of laser radiation.
Endothelial dysfunction is directly linked to preeclampsia, a maternal hypertensive condition that is life threating for both the mother and the baby. Epidemiological studies show that women with a history of pre-eclampsia have an elevated risk for cardiovascular disease. Here we report a new non-invasive diagnostic test for preeclampsia in mice that allows us to non-invasively assess the condition of the animals during the experiment and treatment in established models of preeclampsia. A laser-based multifunctional diagnostics system (LAKK-M) was chosen to carry out non-invasive analysis of multiple parameters. The device was used to simultaneously record the microcirculatory blood flow and oxygen saturation, as well as fluorescence levels of endogenous fluorophores. Preliminary experiments were conducted on adenoviral (Ad-)-mediated overexpression of sFlt-1 (Ad-sFlt-1) to mimic preeclampsia-like symptoms in mice. The recorded data displayed the ability of the LAKK-M diagnostics device to detect significant differences in perfusion measurements between the control and Ad-sFlt-1 treatment. Preliminary results provide a potential avenue to employ these diagnostics technology to monitor and aid in maintaining control of live animal conditions throughout the experiment and treatment.
Raman spectroscopy (RS) has potential for disease classification within the gastrointestinal tract (GI). A near-infrared (NIR) fiber-optic RS system has been developed previously. This study reports the first in vivo Raman spectra of human gastrointestinal tissues measured during routine clinical endoscopy. This was achieved by using this system with a fiber-optic probe that was passed through the endoscope instrument channel and placed in contact with the tissue surface. Spectra could be obtained with good signal-to-noise ratio in 5 s. The effects on the spectra of varying the pressure of the probe tip on the tissue and of the probe-tissue angle were determined and shown to be insignificant. The limited set of spectra from normal and diseased tissues revealed only subtle differences. Therefore, powerful spectral-sorting algorithms, successfully implemented in prior ex vivo studies, are required to realize the full diagnostic potential of RS for tissue classification in the GI.
We present the results of research and development in photodynamic therapy and fluorescence diagnostics performed by the Center of Natural Research, General Physics Institute, Russian Academy of Sciences, in collaboration with several research and medical institutes. The main attention is focused on physical aspects of the problem. We describe schemes and the principle of operation of devices employed by our group in clinical and experimental studies. The problems of interaction of laser radiation with biological tissues are considered. The assessment of an outlook for using different photosensitizers is based on their spectral-fluorescent properties, their ability to excite molecular oxygen, and their stability to irradiation with light. Some results of clinical applications of the developed devices and methods are presented.
The lasers developed in FAST-DOT are mainly targeted toward compact sources of ultra-short pulses. As such, they utilize semiconductor quantum dots (QDs) and semiconductor laser technology. The real strength of these lasers is their compact size, potentially low production cost, and good performance. The performance that FAST-DOT lasers can achieve is not sufficient to compete directly in terms of pulse duration or peak power with the Ti:sapphire lasers currently used in many applications that can produce shorter pulses and higher peak powers, but with a high cost and complex system. However, the performance that has been obtained with FAST-DOT lasers in terms of average power, peak power, pulse duration, pulse energy, and wavelength is high enough to make them excellent sources for some applications where the ultrahigh performance of a Ti:sapphire laser is not necessary, and the lower cost and smaller footprint would be a major benefit.
Diagnostic techniques based on optical spectroscopy have the potential to link biochemical and morphological properties of tissues to individual patient care. In particular, these techniques are fast, noninvasive, and quantitative. Furthermore, they can be used to elucidate key tissue features, such as cellular metabolic rate, vascularity, intravascular oxygenation, and alterations in tissue morphology. These tissue features can be interpreted to shed light on a variety of clinical problems, such as precancerous and cancerous growth and atherosclerosis. The goal of this article is to review development and application of optical spectroscopy in the ultraviolet (UV) and visible (VIS) spectral regions, as a diagnostic tool in clinical applications. Particular emphasis is placed on steady-state UV/VIS fluorescence spectroscopy for the detection of precancers and cancers, in vivo.
Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD (“redox ratio”) can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant
difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.
Fluorescence lifetime imaging (FLIM) is a key fluorescence microscopy technique to map the environment and interaction of fluorescent probes. It can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, because FLIM is independent of the local fluorophore concentration and excitation intensity. One prominent FLIM application relevant for biological concerns is the identification of FRET to study protein interactions and conformational changes, but FLIM is also used to image viscosity, temperature, pH, refractive index and ion and oxygen concentrations, all at the cellular level, as well as cell and tissue autofluorescence. The basic principles and recent advances in the application of FLIM, FLIM instrumentation, data analysis, molecular probe and FLIM detector development will be discussed.
form only given. In the last two decades optical coherence tomography (OCT) has established itself as a unique non-invasive, optical medical diagnostic imaging modality, enabling unprecedented in vivo cross-sectional tomographic visualization of internal microstructure in a variety of biological systems. Ophthalmology has been the most successful and commercially most active medical field for OCT so far, but several other OCT applications, e.g. in cardiology, dentistry, gastroenterology or dermatology, are on the verge of expanding their market comparable to or larger than that of ophthalmology.Especially in the last decade ultrabroad bandwidth light sources as well as spectral/frequency domain OCT detection technology enabled three-dimensional ultrahigh resolution OCT with unprecedented axial resolution, approaching resolution levels of conventional histopathology, enabling optical biopsy of biological tissue. Furthermore emerging swept source laser technologies and parallel or full-field OCT techniques enabled multiple millions of A-scan rates per second, allowing large area OCT scans with high definition sampling, investigation of dynamic processes or four-dimensional (3D over time) imaging. In addition, extensions of OCT are under development that should provide enhanced contrast or non-invasive depth resolved functional imaging of the investigated tissue, including extraction of birefringent, spectroscopic, blood flow or physiologic tissue information. These extensions of OCT should not only improve image contrast, but should also enable the differentiation and early detection of pathologies via localized functional state. Recently OCT has also been combined with different complementary imaging technologies (photoacoustics, CARS, multi-photon microscopy, fluorescent imaging, ultrasound, adaptive optics) to hybrid/multi-modal approaches to compensate fundamental limits of OCT in order to significantly enhance its performance towards molecular imaging.
Many patients with angina and signs of myocardial ischemia on stress testing have no significant obstructive epicardial coronary disease. There are many potential coronary and non-coronary mechanisms for ischemia without obstructive epicardial coronary disease, and prominent among these is coronary microvascular and/or endothelial dysfunction. Patients with coronary microvascular and/or endothelial dysfunction are often at increased risk of adverse cardiovascular events, including ischemic events and heart failure despite preserved ventricular systolic function. In this article, we will review the diagnosis and treatment of coronary microvascular and endothelial dysfunction, discuss their potential contribution to heart failure with preserved ejection fraction, and highlight recent advances in the evaluation of atherosclerotic morphology in these patients, many of whom have non-obstructive epicardial disease.
Nonlinear system identification and analysis methods are employed to study the low-frequency oscillations present in time-series data obtained from reflectance imagery of microvasculature. Using the method of surrogate data testing the analysis reveals the deterministic nature of these oscillations believed by many to be chaotic. Further investigations by means of nonlinear system identification techniques indicate however that the underlying dynamics can described by a periodically driven nonlinear dynamical model exhibiting quasiperiodic behavior.
Light-emitting diodes (LEDs) fabricated from gallium nitride (GaN) have led to the realization of high-efficiency white solid-state lighting. Currently, GaN white LEDs exhibit luminous efficacy greater than 150 lm W−1, and external quantum efficiencies higher than 60%. This has enabled LEDs to compete with traditional lighting technologies, such as incandescent and compact fluorescent (CFL) lighting. Further improvements in materials quality and cost reduction are necessary for widespread adoption of LEDs for lighting. A review of the unique polarization anisotropy in GaN is included for the different crystal orientations. The emphasis on nonpolar and semipolar LEDs highlights high-power violet and blue emitters, and we consider the effects of indium incorporation and well width. Semipolar GaN materials have enabled the development of high-efficiency LEDs in the blue region and recent achievements of green laser diodes at 520 nm.
To study the diagnostic potential of fluorescence spectroscopy and its comparison with different screening methods, including Pap smear and colposcopy, in detecting early cervical neoplasia.
The study was conducted on patients with gynecological complaints. A full gynecological workup of the patients was done along with Pap smear and colposcopy. Cervical biopsy was done in suspected cases and fresh tissue was sent to IIT for spectroscopy.
There is a definite increase in NADH fluorescence (67.4 %) and a decrease in collagen fluorescence (74 %) in dysplastic tissues. When epithelial fluorescence and stromal fluorescence are considered together, diagnostic accuracy is increased to 96.5 %.
The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, fast, and cost-effective alternative to the conventional smear test which needs trained personnel for its interpretation. Research is still continuing to obtain a statistically significant cutoff value from in vitro studies and then use them for in vivo study.
We measured the fluorescence spectra of the whole blood, the red blood cell (RBC) and the hemoglobin using 457.9-nm Ar+ laser excitation. It was found that the fluorescence spectra of the whole blood and the RBC have much similarities in the intensity, the emission peaks and the emitting region, and abundant peaks can be found. But for the hemoglobin, fluorescence could only be found in the wavelength range 580-650 nm. It was concluded that in the wavelength range of 650-850 nm, the fluorescence spectra were emitted by the new fluorophores generated by the breakdown of some weak bonds on the RBC membrane, such as the C-C bond and the C-N bond. In the wavelength range of 590-650 nm, the fluorescence spectra are mainly emitted by the hemoglobin, but the hemoglobin solution of cracked RBC has a strong quencher effect on the fluorescence spectrum. The experimental result and the theoretical analysis are meaningful for the medical diagnostics and the therapy.