Comparison of dermal vs internal light administration in human lungs using the TDLAS‐GASMAS technique—Phantom studies

Oxygen and water vapor content, in the lungs of a 3D‐printed phantom model based on CT‐images of a preterm infant, is evaluated using Tunable Diode Laser Absorption Spectroscopy (TDLAS) in Gas in Scattering Media Absorption Spectroscopy (GASMAS), that is, the TDLAS‐GASMAS technique. Oxygen gas is detected through an absorption line near 764 nm and water vapor through an absorption line near 820 nm. A model with a lung containing interior structure is compared to a model with a hollow lung. Compared to the model with the hollow lung, both the mean absorption path length and the transmitted intensity are found to be lower for the model with the structured lung. A new approach, where laser light is delivered internally into the model through an optical fiber, is compared to dermal light administration, that is, illumination onto the skin, for the model with structure inside the lung. The internal light administration generally resulted in larger gas absorption, and higher signal‐to‐noise ratios, compared to the dermal light administration. The results from the phantom measurements show great promise for the internal illumination approach and a natural next step would be to investigate it further in clinical studies.     

Inside Back Cover: Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations (J. Biophotonics 3/2018)

The picture depicts the different 3d‐printed organs, thorax, lungs, heart and bone. Assembled it is used as an optical phantom of a preterm infant for performing percutaneous optical measurements of the gas content in the lungs. In order to simulate the optical properties of the tissue, the heart and thorax can be filled with liquid phantoms, a mixture of Intralipid and Indian Ink. Further details can be found in the article by Jim Larsson et al. ( e201700097 ).

     

Multi‐modal acousto‐optic/ultrasound imaging of ex vivo liver tumors at 790 nm using a Sn2P2S6 wavefront adaptive holographic setup

Biological tissues are very strong light‐scattering media. As a consequence, current medical imaging devices do not allow deep optical imaging unless invasive techniques are used. Acousto‐optic imaging is a light‐ultrasound coupling technique that takes advantage of the ballistic propagation of ultrasound in biological tissues to access optical contrast with a millimeter resolution. We have developed a photorefractive‐crystal‐based system that performs self‐adaptive wavefront holography and works within the optical therapeutic window. As it works at an appropriate wavelength range for biological tissues imaging, it was tested on ex vivo liver samples containing tumors as a pre‐clinical study. Optical contrast was obtained even if acoustical one was not significant.

Ultrasound image (left) and acousto‐optic image (right) of a liver biopsy with tumors. Acousto‐optic imaging exhibits tumors that are not detected through ultrasound.     

Optofluidic single‐cell absorption flow analyzer for point‐of‐care diagnosis of malaria

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point‐of‐care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off‐the‐shelf components like laser diode, photo‐detector and a micro‐controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

     

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

Back Cover: Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods (J. Biophotonics 2/2018)

Gold nanoparticles serve as imaging contrast agents useful for two‐photon nonlinear microscopy of biological cells and tissues. In this study, 100‐nm‐sized gold particles with a multitude of nanopores embedded inside have been physically synthesized and investigated for the plasmonic enhancement in two‐photon luminescence. Exhibiting remarkable potential for two‐photon imaging, the porous gold nanoparticles boost near‐infrared light absorption substantially and allow emission signals 20 times brighter than gold nanorods being currently used as typical imaging agents. Further details can be found in the article by Joo H. Park et al. ( e201700174 )

     

SPR‐based plastic optical fibre biosensor for the detection of C‐reactive protein in serum

A plastic optical fibre biosensor based on surface plasmon resonance for the detection of C‐reactive protein (CRP) in serum is proposed. The biosensor was integrated into a home‐made thermo‐stabilized microfluidic system that allows avoiding any thermal and/or mechanical fluctuation and maintaining the best stable conditions during the measurements. A working range of 0.006–70 mg L^–1 and a limit of detection of 0.009 mg L^–1 were achieved. These results are among the best compared to other SPR‐based biosensors for CRP detection, especially considering that they were achieved in a real and complex medium, i.e. serum. In addition, since the sensor performances satisfy those requested in physiologically‐relevant clinical applications, the whole biosensing platform could well address high sensitive, easy to realize, real‐time, label‐free, portable and low cost diagnosis of CRP for future lab‐on‐a‐chip applications.

3D sketch (left) of the thermo‐stabilized home‐made flow cell developed to house the SPR‐based plastic optical fibre biosensor. Exemplary response curve (shift of the SPR wavelength versus time) of the proposed biosensor (right) for the detection of C‐reactive protein in serum.     

Rapid and label‐free optical detection of individual carbon air pollutant nanoparticulates in biomedical samples

Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label‐free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump‐probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump‐probe approach allows label‐free optical detection and unambiguous localization of CB particles in (bio)fluids and 3D cellular environments. In combination with fluorescence microscopy, this method allows for simultaneous colocalization of CB with different cellular components using fluorophores as shown here for human lung fibroblasts. We further demonstrate the versatility of pump‐probe detection in a flow cell.      

Label‐free and non‐invasive discrimination of HaCaT and melanoma cells in a co‐culture model by hyperspectral confocal reflectance microscopy

A novel hyperspectral confocal microscopy method to separate different cell populations in a co‐culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non‐invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

Set of hyperspectral images of melanoma‐keratinocytes co‐culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label‐free spectral identification of cell populations (right).     

Quantitative subsurface spatial frequency‐domain fluorescence imaging for enhanced glioma resection

The rate of complete resection of glioma has improved with the introduction of 5‐aminolevulinic acid‐induced protoporphyrin IX (PpIX) fluorescence image guidance. Surgical outcomes are further enhanced when the fluorescence signal is decoupled from the intrinsic tissue optical absorption and scattering obtained from diffuse reflectance measurements, yielding the absolute PpIX concentration, [PpIX]. Spatial frequency domain imaging was used previously to measure [PpIX] in near‐surface tumors under blue fluorescence excitation. Here, we extend this to subsurface [PpIX] fluorescence under red‐light excitation. The decay rate of the modulation amplitude of the fluorescence signal was used to calculate the PpIX depth, which was then applied in a forward diffusion model to estimate [PpIX] at depth. For brain‐like optical properties in phantoms with PpIX fluorescent inclusions, the depth can be recovered up to depths of 9.5 mm ± 0.4 mm, with [PpIX] ranging from 5 to 15 μg/mL within an average deviation of 15% from the true [PpIX] value.      

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     

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.      

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Label‐free,zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

We experimentally demonstrate a label‐free biosensor for the ERBB2 cancer gene DNA target based on the distance‐dependent detection of surface‐enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non‐porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays.

With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label‐free biosensor that makes use of distance‐dependent detection of surface‐enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.     

Non‐Invasive Multi‐Dimensional Two‐Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non‐invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two‐photon microscopy to non‐invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence‐based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients.

Two photon based autofluorescence features of immune cells enables non‐invasive differentiation.     

Multi‐wavelength light emitting diode‐based disposable optrode array for in vivo optogenetic modulation

We present a light emitting diode (LED)‐based optical waveguide array that can optogenetically modulate genetically targeted neurons in the brain. The reusable part of the system consists of control electronics and conventional multi‐wavelength LED. The disposable part comprises optical fibers assembled with microlens array fabricated on a silicon die. Both parts can be easily assembled and separated by snap fit structure. Measured light intensity is 3.35 mW/mm² at 469 nm and 0.29 mW/mm² at 590 nm when the applied current is 80 mA. In all the tested conditions, the light‐induced temperature rise is under 0.5°C and over 90% of the relative light intensity is maintained at 2 mm‐distance from the fiber tips. We further tested the efficiency of the optical array in vivo at 469 nm. When the optical array delivers light stimulation on to the visual cortex of a mouse expressing channelrhodopsin‐2, the neural activity is significantly increased. The light‐driven neural activity is successfully transformed into a percept of the mouse, showing significant learning of the task detecting the cortical stimulation. Our results demonstrate that the proposed optical array interfaces well with the neural circuits in vivo and the system is applicable to guide animal behaviors.      

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.      

Towards the use of bioresorbable fibers in time‐domain diffuse optics

In the last years bioresorbable materials are gaining increasing interest for building implantable optical components for medical devices. In this work we show the fabrication of bioresorbable optical fibers designed for diffuse optics applications, featuring large core diameter (up to 200 μm) and numerical aperture (0.17) to maximize the collection efficiency of diffused light. We demonstrate the suitability of bioresorbable fibers for time‐domain diffuse optical spectroscopy firstly checking the intrinsic performances of the setup by acquiring the instrument response function. We then validate on phantoms the use of bioresorbable fibers by applying the MEDPHOT protocol to assess the performance of the system in measuring optical properties (namely, absorption and scattering coefficients) of homogeneous media. Further, we show an ex‐vivo validation on a chicken breast by measuring the absorption and scattering spectra in the 500–1100 nm range using interstitially inserted bioresorbable fibers. This work represents a step toward a new way to look inside the body using optical fibers that can be implanted in patients. These fibers could be useful either for diagnostic (e. g. for monitoring the evolution after surgical interventions) or treatment (e. g. photodynamic therapy) purposes. Picture : Microscopy image of the 100 μm core bioresorbable fiber.

     

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.