Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations

There is a need to further improve the clinical care of our most vulnerable patients—preterm infants. Novel diagnostic and treatment tools facilitate such advances. Here, we evaluate a potential percutaneous optical monitoring tool to assess the oxygen and water vapor content in the lungs of preterm babies. The aim is to prepare for further clinical studies by gaining a detailed understanding of how the measured light intensity and gas absorption signal behave for different possible geometries of light delivery and receiver. Such an experimental evaluation is conducted for the first time utilizing a specially developed 3‐dimensional‐printed optical phantom based on a geometry model obtained from computer tomography images of the thorax (chest) of a 1700‐g premature infant. The measurements yield reliable signals for source–detector distances up to about 50 mm, with stronger gas absorption signals at long separations and positions related to the lower part of the lung, consistent with a larger relative volume of this. The limitations of this study include the omission of scattering tissue within the lungs and that similar optical properties are used for the wavelengths employed for the 2 gases, yielding no indication on the optimal wavelength pair to use.      

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

Wavelength‐Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS) for noninvasive early cancer detection and tissue hypoxia monitoring

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO₂). Unlike conventional photoacoustic spectroscopy, WM‐DPAS measures simultaneously two signals induced from square‐wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy‐ and oxy‐hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two‐wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM‐DPAS to detect very small changes in total hemoglobin concentration (C_Hb) and oxygenation levels, thereby identifying pre‐malignant tumors before they are anatomically apparent. The non‐invasive nature also makes WM‐DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM‐DPAS can be tuned for different applications such as quick cancer screening and accurate StO₂ quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM‐DPAS theory has been validated with sheep blood phantom measurements.

Sensitivity comparison between conventional single‐ended signal and differential signal.     

Three‐dimensional visualization system for ophthalmic microscopes using visible light and near‐infrared illumination

In this paper, we describe a three‐dimensional visualization system for ophthalmic microscopes that is aimed at microsurgery without the eyepieces. A three‐dimensional visualization system for ophthalmic microscopes using the mixed illumination, which consists of visible light and near‐infrared illumination, is established in order to acquire more exact information of object and reduce the amount of light irradiated to the patients, and its usage in microsurgery without eyepieces is herein described. A custom‐designed stereoscopic three‐dimensional display which is manufactured for the convenience of the surgeons during the long‐time surgery, is connected directly to the camera of the ophthalmic microscope in order to eliminate the discomfort of eyepieces to the surgeon and signal delay between the camera, mounted on the microscope, and display device for surgeon. The main features of the established system are the signal delay‐free for surgeon and the low level of illumination for patient. In particular, it could significantly reduce the amount of light irradiated on a patient's eye via NIR illumination. Upon comparison with the conventional system during clinical ophthalmology trials, this system is confirmed to require almost the same operation time and reduced discomfort and eyestrain during long periods of observation.      

In vivo full‐field functional optical hemocytometer

We present an in vivo lab‐free full‐field functional optical hemocytometer (FFOH) for application to the capillaries of a live biological specimen, based on the absorption intensity fluctuation modulation (AIFM) effect. Because of the absorption difference between the red blood cells (RBCs) and background tissue under low‐coherence light illumination, an endogenous instantaneous intensity fluctuation is generated by the AIFM effect when RBCs discontinuously traverse the capillary. The AIFM effect is used to highlight the RBC signal relative to the background tissue by computing the real‐time modulation depth. FFOH can simultaneously provide a flow video, the flow velocity and the RBC count. Ourexperimental results can potentially be applied to study the physiological mechanisms of the blood circulation systems of near‐transparent live biological samples.      

Bessel‐beam illumination in dual‐axis confocal microscopy mitigates resolution degradation caused by refractive heterogeneities

One of the main challenges for laser‐scanning microscopy of biological tissues with refractive heterogeneities is the degradation in spatial resolution that occurs as a result of beam steering and distortion. This challenge is particularly significant for dual‐axis confocal (DAC) microscopy, which achieves improved spatial‐filtering and optical‐sectioning performance over traditional confocal microscopy through off‐axis illumination and collection of light with low‐numerical aperture (NA) beams that must intersect precisely at their foci within tissues. DAC microscope image quality is sensitive to positional changes and distortions of these illumination‐ and collection‐beam foci. Previous studies have shown that Bessel beams display improved positional stability and beam quality than Gaussian beams when propagating through tissues with refractive heterogeneities, which suggests that Bessel‐beam illumination may enhance DAC microscopy of such tissues. Here, we utilize both Gaussian and Bessel illumination in a point‐scanned DAC microscope and quantify the resultant degradation in resolution when imaging within heterogeneous optical phantoms and fresh tissues. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination.

     

Cone–shell Raman spectroscopy (CSRS) for depth‐sensitive measurements in layered tissue

We report the development of a depth‐sensitive Raman spectroscopy system using the configuration of cone–shell excitation and cone detection. The system uses a 785 nm diode laser and three identical axicons for Raman excitation of the target sample in the form of a hollow conic section. The Raman scattered light from the sample, passed through the same (but solid) conic section, is collected for detection. Apart from its ability of probing larger depths (? few mm), an important attraction of the system is that the probing depths can be varied by simply varying the separation between axicons in the excitation arm. Furthermore, no adjustment is required in the sample arm, which is a significant advantage for noncontact, depth‐sensitive measurement. Evaluation of the performance of the developed setup on nonbiological phantom and biological tissue sample demonstrated its ability to recover Raman spectra of layers located at depths of ?2–3 mm beneath the surface.

     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Light distribution and thermal effects in the rat brain under optogenetic stimulation

Optical brain stimulation gained a lot of attention in neuroscience due to its superior cell‐type specificity. In the design of illumination strategies, predicting the light propagation in a specific tissue is essential and requires knowledge of the optical properties of that tissue. We present the estimated absorption and reduced scattering in rodent brain tissue using non‐destructive contact spatially resolved spectroscopy (cSRS). The obtained absorption and scattering in the cortex, hippocampus and striatum are similar, but lower than in the thalamus, leading to a less deep but broader light penetration profile in the thalamus. Next, the light distribution was investigated for different stimulation protocols relevant for fiber‐optic based optogenetic experiments, using Monte Carlo simulation. A protocol specific analysis is proposed to evaluate the potential of thermally induced side effects.

     

Low‐coherent optical diffraction tomography by angle‐scanning illumination

Temporally low‐coherent optical diffraction tomography (ODT) is proposed and demonstrated based on angle‐scanning Mach‐Zehnder interferometry. Using a digital micromirror device based on diffractive tilting, the full‐field interference of incoherent light is successfully maintained during every angle‐scanning sequences. Further, current ODT reconstruction principles for temporally incoherent illuminations are thoroughly reviewed and developed. Several limitations of incoherent illumination are also discussed, such as the nondispersive assumption, optical sectioning capacity and illumination angle limitation. Using the proposed setup and reconstruction algorithms, low‐coherent ODT imaging of plastic microspheres, human red blood cells and rat pheochromocytoma cells is experimentally demonstrated.      

High sensitivity non‐invasive detection of calcifications deep inside biological tissue using Transmission Raman Spectroscopy

The aim of this research was to develop a novel approach to probe non‐invasively the composition of inorganic chemicals buried deep in large volume biological samples. The method is based on advanced Transmission Raman Spectroscopy (TRS) permitting chemical specific detection within a large sampling volume. The approach could be beneficial to chemical identification of the breast calcifications detected during mammographic X‐ray procedures. The chemical composition of a breast calcification reflects the pathology of the surrounding tissue, malignant or benign and potentially the grade of malignancy. However, this information is not available from mammography, leading to excisional biopsy and histopathological assessment for a definitive diagnosis. Here we present, for the first time, a design of a new high performance deep Raman instrument and demonstrate its capability to detect type II calcifications (calcium hydroxyapatite) at clinically relevant concentrations and depths of around 40 mm in phantom tissue. This is around double the penetration depth achieved with our previous instrument design and around two orders of magnitude higher than that possible when using conventional Raman spectroscopy.

     

Three‐dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo

Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three‐dimensional detection of pathophysiological parameters within skin. It was recently shown that single‐wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three‐dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15–125 MHz. We employ a per‐pulse tunable laser to inherently co‐register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label‐free visualization of physiological biomarkers in skin in vivo.

Cross‐sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50–90%. All scale bars: 250 µm     

Which wavelength is optimal for transcranial low‐level laser stimulation?

One of the challenges in transcranial low‐level laser therapy (LLLT) is to optimally choose illumination parameters, such as wavelength. However, there is sparse study on the wavelengths comparison especially on human transcranial LLLT. Here, we employed Monte Carlo modeling and visible human phantom to compute the penetrated photon fluence distribution within cerebral cortex. By comparing the fluence distribution, penetration depth and the intensity of laser‐tissue‐interaction within brain among all candidate wavelengths, we found that 660, 810 nm performed much better than 980, 1064 nm with much stronger, deeper and wider photon penetration into cerebral tissue; 660 nm was shown to be the best and slightly better than 810 nm. Our computational finding was in a surprising accordance with previous LLLT‐neurobehavioral studies on mice. This study not only offered quantitative comparison among wavelengths in the effect of LLLT light penetration effectiveness but also anticipated a delightful possibility of online, precise and visible optimization of LLLT illumination parameters.      

Unassisted Water Splitting Using a GaSbxP(1−x) Photoanode

Here, unbiased water splitting with 2% solar‐to‐hydrogen efficiency under AM 1.5 G illumination using new materials based on GaSb_0.03P_0.97 alloy is reported. Freestanding GaSb_xP_1?x is grown using halide vapor phase epitaxy. The native conductivity type of the alloy is modified by silicon doping, resulting in an open‐circuit potential (OCP) of 750 mV, photocurrents of 7 mA cm^?2 at 10 sun illumination, and corrosion resistance in an aqueous acidic environment. Alloying GaP with Sb at 3 at% improves the absorption of high‐energy photons above 2.68 eV compared to pure GaP material. Electrochemical Impedance Spectroscopy and illuminated OCP measurements show that the conduction band of GaSb_xP_1?x is at ?0.55 V versus RHE irrespective of the Sb concentration, while photocurrent spectroscopy indicates that only radiation with photon energies greater than 2.68 eV generate mobile and extractable charges, thus suggesting that the higher‐laying conduction bands in the Γ 1 valley of the alloys are responsible for exciton generation.     

The role of dermal photoreceptors during the sea lamprey (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>) spawning migration

Light avoidance in larval lampreys is mediated by dermal photoreceptors located in the tail. These photoreceptors continue to function in adults, but they seem redundant because post-metamorphic lampreys possess well-developed eyes. This study examined the role of dermal photoreceptors in adult sea lampreys by testing whether temperature-induced changes in refuge-seeking behavior are mediated by a reduction in dermal photosensitivity. In a lighted arena containing a single shaded refuge platform, lampreys at 22°C displayed five times less search activity and were less likely to attach beneath the refuge platform than lampreys at either 7 or 15°C. A behavioral assay for tail photosensitivity (locomotor response to tail illumination) revealed a corresponding reduction in dermal photosensitivity at 22°C. Moreover, the responses to head illumination (eyes and pineal) did not correspond with the observed light avoidance behaviors. The head was less responsive to light than the tail and was not influenced by temperature. These results provide strong evidence that the dermal photoreceptors continue to mediate light avoidance in adult lampreys, even though adults possess fully functional eyes. The fact that the eyes apparently do not take on this role suggests that there is functional specialization between these two light sensing systems.     

Real‐time functional optical‐resolution photoacoustic microscopy using high‐speed alternating illumination at 532 and 1064 nm

Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps.      

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Large‐depth‐of‐field full‐field optical angiography

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF) using a contrast pyramid fusion algorithm (CPFA). The absorption intensity fluctuation modulation effect is utilized to obtain full‐field optical angiography (FFOA) images at different focus positions. The CPFA is used to process these FFOA images with different focuses. By selecting high‐contrast areas, the CPFA can highlight the characteristics and details of blood vessels to obtain LD‐FFOA images. In the optimal case of the proposed method, the DOF for FFOA is more than tripled using 10 differently focused FFOA images. Both the phantom and animal experimental results show that the LD‐FFOA resolves FFOA defocusing issues induced by surface and thickness inhomogeneities in biological samples. The proposed method can be potentially applied to practical biological experiments.