Inside Cover: Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging (J. Biophotonics 12/2019)

CARS microscopy was employed to monitor the reorganization of intracellular lipids and proteins, as well as cellular transformations, after irradiation with near infrared (NIR) light. NIR light was shown to induce apoptosis in HeLa cells in vitro in a dose‐dependent manner. The progression of apoptosis assessed with CARS microscopy is apparently associated with the generation of reactive oxygen species followed by an excessive formation of lipid droplets and their peroxidation. Further details can be found in the article by Svitlana M. Levchenko, Andrey N. Kuzmin, Artem Pliss, et al. ( e201900179 )

     

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Optimal imaging windows of indocyanine green‐assisted near‐infrared dental imaging with rat model and its comparison to X‐ray imaging

In this study, we used rat animal model to compare the efficiency of indocyanine green (ICG)‐assisted dental near‐infrared fluorescence imaging with X‐ray imaging, and we optimized the imaging window for both unerupted and erupted molars. The results show that the morphology of the dental structures was observed clearly from ICG‐assisted dental images (especially through the endoscope). A better image contrast was easily acquired at the short imaging windows (<10 minutes) for unerupted and erupted molars. For unerupted molars, there is another optimized imaging window (48‐96 hours) with a prominent glow‐in‐the‐dark effect: only the molars remain bright. This study also revealed that the laser ablation of dental follicles can disrupt the molar development, and our method is able to efficiently detect laser‐treated molars and acquire the precise morphology. Thus, ICG‐assisted dental imaging has the potential to be a safer and more efficient imaging modality for the real‐time diagnosis of dental diseases.     

Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs

In this study, the distribution of biomaterials and its molecular mechanism of embryonic development in Japanese medaka fish were analyzed nondestructively and noninvasively without staining using near‐infrared (NIR) imaging. The microscopic NIR imaging system used in this research was a device capable of ultra‐high‐speed imaging; using this system, one can acquire microscopic imaging data in a few seconds. Therefore, the medaka eggs remained alive throughout measurements and were successfully monitored in vivo. The distributions of biomolecules were examined by mapping the intensities of NIR bands resulting from lipids, proteins and water in 2 dimensions (2D). The structures of eyes, lipid bilayer membranes, micelles and water‐structure differences at the interface of different substances constituting different structures on the egg were visualized. Furthermore, insights on the metabolic mechanisms of lipids and membrane functions were drawn from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. These results indicated the potential for NIR imaging in evaluating the biological functions and metabolic systems of cells and embryos.      

Spectral correction for handheld optoacoustic imaging by means of near‐infrared optical tomography in reflection mode

In vivo imaging of tissue/vasculature oxygen saturation levels is of prime interest in many clinical applications. To this end, the feasibility of combining two distinct and complementary imaging modalities is investigated: optoacoustics (OA) and near‐infrared optical tomography (NIROT), both operating noninvasively in reflection mode. Experiments were conducted on two optically heterogeneous phantoms mimicking tissue before and after the occurrence of a perturbation. OA imaging was used to resolve submillimetric vessel‐like optical absorbers at depths up to 25 mm, but with a spectral distortion in the OA signals. NIROT measurements were utilized to image perturbations in the background and to estimate the light fluence inside the phantoms at the wavelength pair (760 nm, 830 nm). This enabled the spectral correction of the vessel‐like absorbers' OA signals: the error in the ratio of the absorption coefficient at 830 nm to that at 760 nm was reduced from 60%‐150% to 10%‐20%. The results suggest that oxygen saturation (SO ₂) levels in arteries can be determined with <10% error and furthermore, that relative changes in vessels' SO ₂ can be monitored with even better accuracy. The outcome relies on a proper identification of the OA signals emanating from the studied vessels.      

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.     

The utility of far‐infrared illumination in oxygenation dynamics as measured with near‐infrared spectroscopy

Near‐infrared spectroscopy (NIRS) is a noninvasive method for measuring the oxygenation in muscle and other tissues in vivo. For quantitative NIRS measurement of oxygenation dynamics, the vessel‐occlusion test was usually applied as physiological intervention. There are several drawbacks of the vessel‐occlusion method that include skin contact, uncomfortable and microcirculation block of patients. Thus, we propose the far‐infrared (FIR) illumination as a new physiological intervention method in this paper. Our preliminary result shows a linear correlation of oxygenation dynamic signals between FIR illumination and arterial‐occlusion test (AOT) that implies the FIR illumination could be applied for hemodynamic response measurement in clinical diagnosis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Inside Cover: Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs (J. Biophotonics 4/2018)

The biomaterial distribution and its molecular mechanism of embryonic development in Japanese medaka fish were visualized without staining using high‐speed near‐infrared imaging. It was a remarkable achievement to visualize the structures of eyes, lipid bilayer membranes, micelles, and water structural variations at the interface of different substances. Furthermore, insights on lipid metabolism and membrane functions were obtained from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. Further details can be found in the article by Mika Ishigaki ( e201700115 )

     

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Label‐free in vivo cellular‐level detection and imaging of apoptosis

Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label‐free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound‐to‐free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.

     

Development and evaluation of realistic optical cell models for rapid and label‐free cell assay by diffraction imaging

Methods for rapid and label‐free cell assay are highly desired in life science. Single‐shot diffraction imaging presents strong potentials to achieve this goal as evidenced by past experimental results using methods such as polarization diffraction imaging flow cytometry. We present here a platform of methods toward solving these problems and results of optical cell model (OCM) evaluations by calculations and analysis of cross‐polarized diffraction image (p‐DI) pairs. Four types of realistic OCMs have been developed with two prostate cell structures and adjustable refractive index (RI) parameters to investigate the effects of cell morphology and index distribution on calculated p‐DI pairs. Image patterns have been characterized by a gray‐level co‐occurrence matrix (GLCM) algorithm and four GLCM parameters and linear depolarization ratio δ_L have been selected to compare calculated against measured data of prostate cells. Our results show that the irregular shapes of and heterogeneity in RI distributions for organelles play significant roles in the spatial distribution of scattered light by cells in comparison to the average RI values and their differences among the organelles. Discrepancies in GLCM and δ_L parameters between calculated and measured p‐DI data provide useful insight for understanding light scattering by single cells and improving OCM.      

Analysis of relation between skin elasticity and the entropy of skin image using near‐infrared and visible light sources

Skin elasticity has been regarded as one of the main indicators of skin condition. Current measurement devices for skin elasticity are mostly expensive for home‐use and should contact the skin surface. As a first step to develop improved methods, we focus on the relation between skin elasticity and the entropy of skin images. Reduced skin elasticity causes wrinkles. It spreads frequency components and increases their randomness in the frequency domain. The randomness is quantified as entropy, which is a measure of the disorder of a system in physics. Therefore, skin elasticity is expected to have a negative relation with entropy. This tendency can be improved by applying penetration depth characteristics according to the wavelength of light. From cheeks and forehead of 12 Korean adults, skin images are acquired with three different light sources (470 nm, 870 nm and broadband light) and skin elasticity is measured. The root mean square error between the measured data and the fitted model is “0.27” (870 nm), “0.49” (broadband light) and “1.42” (470 nm). Furthermore, the results are analyzed by classifying by sex, age and measurement area. This study demonstrates the possibility of developing noncontact home‐use devices to measure skin elasticity in the future.     

Altered near‐infrared spectroscopy response to breath‐holding in patients with fibromyalgia

Fibromyalgia (FM) is a complex syndrome characterized by chronic widespread pain and a heightened response to pressure. Most medical researches pointed out that FM patients with endothelial dysfunction and arterial stiffness. A continuous‐wave near‐infrared spectroscopy (NIRS) system is used in present study to measure the hemodynamic changes elicited by breath‐holding task in patients with FM. Each patient completed a questionnaire survey including demographics, characteristics of body pain, associated symptoms, headache profiles and Hospital Anxiety and Depression Scale. A total of 27 FM patients and 26 health controls were enrolled. In comparison with healthy controls, patients with FM showed lower maximal and averaged change of oxyhemoglobin concentration in both the left (1.634 ±0.890 and 0.810 ±0.525 μM) and the right (1.576 ±0.897 and 0.811 ±0.601 μM) prefrontal cortex than healthy controls (P < .05 for both sides) during the breath‐holding task. In conclusion, FM is associated with altered cerebrovascular reactivity measured by NIRS and breath‐holding task, which may reflect endothelial dysfunction or arterial stiffness. Oxygenated hemoglobin concentration changes of healthy controls and FM patients.      

Head exposure system for a human provocation study to assess the possible influence of UMTS‐like electromagnetic fields on cerebral blood circulation using near‐infrared imaging

A head exposure setup for efficient and precisely defined exposure of human subjects equipped with a near‐infrared imaging (NIRI) sensor is presented. In a partially shielded anechoic chamber the subjects were exposed to Universal Mobile Telecommunications System (UMTS)‐like electromagnetic fields (EMF) by using a patch antenna at a distance of 4 cm from the head. The non‐contact design of the exposure setup enabled NIRI sensors to easily attach to the head. Moreover, different regions of the head were chosen for localised exposure and simultaneous NIRI investigation. The control software enabled the simple adaptation of the test parameters during exploratory testing as well as the performance of controlled, randomised, crossover and double‐blind provocation studies. Four different signals with a carrier frequency of 1900 MHz were chosen for the exposure: a simple continuous wave signal and three different UMTS signals. Furthermore, three exposure doses were available: sham, low (spatial peak specific absorption rate (SAR) = 0.18 W/kg averaged over 10 g) and high (spatial peak SAR = 1.8 W/kg averaged over 10 g). The SAR assessment was performed by measurement and simulation. Direct comparison of measurement and numerical results showed good agreement in terms of spatial peak SAR and SAR distribution. The variability analysis of the spatial peak SAR over 10 g was assessed by numerical simulations. Maximal deviations of ?22% and +32% from the nominal situation were observed. Compared to other exposure setups, the present setup allows for low exposure uncertainty, combined with high SAR efficiency, easy access for the NIRI sensor and minimal impairment of test subjects. Bioelectromagnetics 33:124–133, 2012. © 2011 Wiley Periodicals, Inc.     

Phase dual‐slopes in frequency‐domain near‐infrared spectroscopy for enhanced sensitivity to brain tissue: First applications to human subjects

We present a first in vivo application of phase dual‐slopes (DS?), measured with frequency‐domain near‐infrared spectroscopy on four healthy human subjects, to demonstrate their enhanced sensitivity to cerebral hemodynamics. During arterial blood pressure oscillations elicited at a frequency of 0.1 Hz, we compare three different ways to analyze either intensity (I) or phase (?) data collected on the subject's forehead at multiple source‐detector distances: Single‐distance, single‐slope and DS. Theoretical calculations based on diffusion theory show that the method with the deepest maximal sensitivity (at about 11 mm) is DS?. The in vivo results indicate a qualitative difference of phase data (especially DS?) and intensity data (especially single‐distance intensity [SDI]), which we assign to stronger contributions from scalp hemodynamics to SDI and from cortical hemodynamics to DS?. Our findings suggest that scalp hemodynamic oscillations may be dominated by blood volume dynamics, whereas cortical hemodynamics may be dominated by blood flow velocity dynamics.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Controlling the near‐infrared transparency of costal cartilage by impregnation with clearing agents and magnetite nanoparticles

Penetration depth of near‐infrared laser radiation to costal cartilage is controlled by the tissue absorption and scattering, and it is the critical parameter to provide the relaxation of mechanical stress throughout the whole thickness of cartilage implant. To enhance the penetration for the laser radiation on 1.56 μm, the optical clearing solutions of glycerol and fructose of various concentrations are tested. The effective and reversible tissue clearance was achieved. However, the increasing absorption of radiation should be concerned: 5°C‐8°C increase of tissue temperature was detected. Laser parameters used for stress relaxation in cartilage should be optimized when applying optical clearing agents. To concentrate the absorption in the superficial tissue layers, magnetite nanoparticle (NP) dispersions with the mean size 95 ± 5 nm and concentration 3.9 ± 1.1 × 10¹¹ particles/mL are applied. The significant increase in the tissue heating rate was observed along with the decrease in its transparency. Using NPs the respective laser power can be decreased, allowing us to obtain the working temperature locally with reduced thermal effect on the surrounding tissue.      

Real‐time monitoring prefrontal activities during online video game playing by functional near‐infrared spectroscopy

A growing body of literature has suggested that video game playing can induce functional and structural plasticity of the brain. The underlying mechanisms, however, remain poorly understood. In this study, functional near‐infrared spectroscopy (fNIRS) was used to record prefrontal activities in 24 experienced game players when they played a massively multiplayer online battle arena video game, League of Legends (LOL), under naturalistic conditions. It was observed that game onset was associated with significant activations in the ventrolateral prefrontal cortex (VLPFC) and concomitant deactivations in the dorsolateral prefrontal cortex (DLPFC) and frontal pole area (FPA). Game events, such as slaying an enemy and being slain by an enemy evoked region‐specific time‐locked hemodynamic/oxygenation responses in the prefrontal cortex (PFC). It was proposed that the VLPFC activities during LOL playing are likely responses to visuo‐motor task load of the game, while the DLPFC/FPA activities may be involved in the constant shifts of attentional states and allocation of cognitive resources required by game playing. The present study demonstrated that it is feasible to use fNIRS to monitor real‐time prefrontal activity during online video game playing.