3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.     

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.      

Recent advances in optical fiber devices for microfluidics integration

This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab‐on‐chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi‐sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.

Detection and trapping of molecules can be achieved with optical fibers directly located within the fluidic microchannel.     

Two‐photon imaging of fiber‐coupled neurons

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two‐photon imaging is shown to enable a quantitative in situ analysis of such fiber–single‐neuron coupling in the live brain of transgenic mice. Fiber‐optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two‐photon imaging. These results pave the way for fiber‐optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber‐optic single‐neuron stimulation into the optical imaging framework.

     

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization‐resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line‐to‐line switching of polarization using an electro‐optical modulator and works in epi‐detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.      

Reconnectable fiberscopes for chronic in vivo deep‐brain imaging

Reconnectable bundles consisting of thousands of optical fibers are shown to enable high‐quality image transmission, offering a platform for the creation of implantable fiberscopes for minimally invasive in vivo brain imaging. Experiments on various lines of transgenic mice verify the performance of this fiberscope as a powerful tool for chronic in vivo neuroimaging using genetically encoded calcium indicators, neuronal activity markers as well as axon growth regulators and brain‐specific protein drivers in deep regions of live brain.      

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.

     

Uniform light delivery in volumetric optoacoustic tomography

Accurate image reconstruction in volumetric optoacoustic tomography implies the efficient generation and collection of ultrasound signals around the imaged object. Non‐uniform delivery of the excitation light is a common problem in optoacoustic imaging often leading to a diminished field of view, limited dynamic range and penetration, as well as impaired quantification abilities. Presented here is an optimized illumination concept for volumetric tomography that utilizes additive manufacturing via 3D printing in combination with custom‐made optical fiber illumination. The custom‐designed sample chamber ensures convenient access to the imaged object along with accurate positioning of the sample and a matrix array ultrasound transducer used for collection of the volumetric image data. Ray tracing is employed to optimize the positioning of the individual fibers in the chamber. Homogeneity of the generated light excitation field was confirmed in tissue‐mimicking agar spheres. Applicability of the system to image entire mouse organs ex vivo has been showcased. The new approach showed a clear advantage over conventional, single‐sided illumination strategies by eliminating the need to correct for illumination variances and resulting in enhancement of the effective field of view, greater penetration depth and significant improvements in the overall image quality.      

Diaphragm‐based optical fiber sensor for pulse wave monitoring and cardiovascular diseases diagnosis

Arterial pulse wave has been considered as a vital sign in assessment of cardiovascular diseases. Noninvasive pulse sensor with compact structure, immunity to electro‐magnetic interference and high sensitivity is the research focus in recent years. While, optical fiber biosensor is a competitive option to meet these needs. Here, a diaphragm‐based optical fiber pulse sensor was proposed to achieve high‐precision radial pulse wave monitoring. A wearable device was developed, composed of a sports wristband and an aluminum diaphragm‐based optical fiber sensor tip of only 1 cm in diameter, which was highly sensitive to the weak acoustic signal. In particular, coherent phase detection was adopted to improve detection signal‐to‐noise ratio, so as to recover the high‐fidelity pulse waveforms. A clinical experiment was carried out to detect and morphological analyze the pulse waveforms of four subjects, the results of which preliminarily demonstrated the feasibility of pulse diagnosis method. The proposed pulse fiber sensor provides a comfortable way for pulse diagnosis, which is promising in early cardiovascular diseases indicating.     

Contact‐free endoscopic photoacoustic sensing using speckle analysis

Photoacoustic endoscopy (PAE) is an emerging imaging modality, which offers a high imaging penetration and a high optical contrast in soft tissue. Most of the developed endoscopic photoacoustic sensing systems use miniaturized contact ultrasound transducers or complex optical approaches. In this work, a new fiber‐based detection technique using speckle analysis for contact‐free signal detection is presented. Phantom and ex vivo experiments are performed in transmission and reflection mode for proof of concept. In summary, the potential of the technique for endoscopic photoacoustic signal detection is demonstrated. The new technique might help in future to broaden the applications of PAE in imaging or guiding minimally invasive laser procedures.      

Remission spectrometry for blood vessel detection during stereotactic biopsy of brain tumors

Stereotactic biopsy is used to enable diagnostic confirmation of brain tumors and treatment planning. Despite being a well‐established technique, it is related to significant morbidity and mortality rates mostly caused by hemorrhages due to blood vessel ruptures. This paper presents a method of vessel detection during stereotactic biopsy that can be easily implemented by integrating two side‐view fibers into a conventional side‐cutting biopsy needle. Tissue within the needle window is illuminated through the first fiber; the second fiber detects the remitted light. By taking the ratio of the intensities at two wavelengths with strongly differing hemoglobin absorption, blood vessels can be recognized immediately before biopsy sampling. Via ray tracing simulations and phantom experiments, the dependency of the remission ratio R = I₅₇₈/I₆₅₀ on various parameters (blood oxygenation, fiber‐to‐vessel and inter‐fiber distance, vessel diameter and orientation) was investigated for a bare‐fiber probe. Up to 800–1200 µm away from the probe, a vessel can be recognized by a considerable reduction of the remission ratio from the background level. The technique was also successfully tested with a real biopsy needle probe on both optical phantoms and ex‐vivo porcine brain tissue, thus showing potential to improve the safety of stereotactic biopsy.

Dual‐wavelength remission measurement for the detection of blood vessels during stereotactic biopsy.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Development of hyperthermia measurable fiber radiometric thermometer for thermotherapy

Temperature monitoring is extremely important during thermotherapy. Fiber‐optic temperature sensors are preferred because of their flexibility and immunity to electromagnetic interference. Although many types of fiber‐optic sensors have been developed, clinically adopting them remains challenging. Here, we report a silica fiber‐based radiometric thermometer using a low‐cost extended InGaAs detector to detect black body radiation between 1.7 and 2.4 μm. For the first time, this silica fiber‐based thermometer is capable of measuring temperatures down to 35°C, making it suitable for monitoring hyperthermia during surgery. In particular, the thermometer has potential for seamless integration with current silica fiber catheters, which are widely used in laser interstitial thermotherapy. The feasibility, capability and sensitivity of tracking tissue temperature variation were proved through ex vivo tissue studies. After further improvement, the technology has the potential to be translated into clinics for monitoring tissue temperature.     

Bioluminescence imaging of hollow fibers in living animals: its application in monitoring molecular pathways

We have applied noninvasive optical imaging technology to the in vivo hollow fiber assay, using tumor cell lines in which optical reporters are expressed in response to activation/inhibition of a specific molecular pathway. In vivo noninvasive imaging of molecular pathways in cells within hollow fibers enables a rapid and accurate evaluation of drug targets and provides useful insights to guide novel drug discovery. In this protocol we show, as an example, that a luciferase reporter, driven by the responsive element of nuclear factor NF-kappaB, was induced in cells within hollow fibers implanted in living mice, and a detailed procedure for in vivo bioluminescence imaging of hollow fibers is described. This approach can, in principle, be applied to image any molecular pathways of interest when appropriate reporter cells are generated. Hollow fiber encapsulation and implantation takes 2 d, and in vivo validation of reporter takes 1-2 weeks.     

Microfocusing sapphire capillary needle for laser surgery and therapy: Fabrication and characterization

A sapphire shaped capillary needle designed for collimating and focusing of laser radiation was proposed and fabricated by the edge‐defined film‐fed growth technique. It features an as‐grown surface quality, high transparency for visible and near‐infrared radiation, high thermal and chemical resistance and the complex shape of the tip, which protects silica fibers. The needle's geometrical parameters can be adjusted for use in various situations, such as type of tissue, modality of therapy and treatment protocol. The focusing effect was demonstrated numerically and observed experimentally during coagulation of the ex vivo porcine liver samples. This needle in combination with 0.22NA optical fiber allows intensive and uniform coagulation of 150 mm³ volume interstitially and 30 mm³ superficially by laser exposure with 280 J without tissue carbonization and fiber damaging along with delicate treatment of small areas. The demonstrated results reveal the perspectives of the proposed sapphire microfocusing needle for laser surgery and therapy.     

Dual‐wavelength reflectance spectroscopy of the superior vena cava: A method for placing central venous catheters at the cavoatrial junction

There are a limited number of methods to guide and confirm the placement of a peripherally inserted central catheter (PICC) at the cavoatrial junction. The aim of this study was to design, test and validate a dual‐wavelength, diode laser‐based, single optical fiber instrument that would accurately confirm PICC tip location at the cavoatrial junction of an animal heart, in vivo. This was accomplished by inserting the optical fiber into a PICC and ratiometrically comparing simultaneous visible and near‐infrared reflection intensities of venous and atrial tissues found near the cavoatrial junction. The system was successful in placing the PICC line tip within 5 mm of the cavoatrial junction.      

Conduction velocities and fiber diameters in a cutaneous nerve of the pigeon

Summary The characteristics of fibers of a cutaneous nerve supplying the wing skin of the pigeon have been investigated with electrophysiological and electron microscopic techniques.Recordings of the compound action potential showed four distinct peaks with conduction velocities of about 30 m/s, 12 m/s, 4 m/s and 0.5 m/s.From electron micrographs both fiber diameters and thickness of myelin sheath were assessed and used as criteria for segregating various fiber populations. Altogether four groups could be discerned: large thickly myelinated fibers, small thickly myelinated fibers, small thinly myelinated fibers, and unmyelinated or C-fibers. The subdivision of the thickly myelinated fibers into two populations is evidenced mainly by corresponding peaks in the compound action potential. The thinly myelinated fibers with a mean diameter of 2 m contributed about 90% of all myelinated fibers in this nerve.When comparing fiber dimensions and conduction velocities of this avian nerve with those of mammalian cutaneous nerves, the lower CV's of avian nerve fibers can be explained by smaller diameters and thinner myelin sheaths.The results of this investigation are a prerequisite for latency considerations in central somatosensory pathways in birds.Abbreviations CAP compound action potential - CV conduction velocity - D fiber diameter - d axon diameter - g ratio d/D - m thickness of myelin sheath     

Air‐core fiber or photonic‐crystal rod,which is more suitable for energetic femtosecond pulse generation and three‐photon microscopy at the 1700‐nm window?

Energetic femtosecond pulses at the 1700‐nm window are a prerequisite for deep‐tissue three‐photon microscopy (3PM). Soliton self‐frequency shift (SSFS) in photonic‐crystal (PC) rod has been the only technique to generate such pulses suitable for 3PM. Here we demonstrate through SSFS in an air‐core fiber, we can generate most energetic femtosecond soliton pulses at the 1700‐nm window, 5.2 times higher than that from PC rod. However, the air‐core soliton pulse width is 5.9 times longer than that of PC rod soliton. Based on comparative 3PM excited with both air‐core and PC rod solitons, we propose the more suitable source for 3PM. We further elucidate the challenge of generating shorter soliton pulses from air‐core fibers through numerical simulation.     

Correcting the limited view in optical‐resolution photoacoustic microscopy

Optical‐resolution photoacoustic microscopy (OR‐PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection‐mode detection in OR‐PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited‐view phenomenon in OR‐PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof‐of‐concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection‐mode OR‐PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture‐cross phantom and in a mouse brain in vivo.      

Endoluminal application of glass‐capped diffuser for ex vivo endovenous photocoagulation

Endovenous laser ablation (EVLA) has frequently been used to treat varicose veins for 20 years. In spite of 90?95% occlusion rates, clinical complications such as burn and ecchymosis still occur due to excessive thermal injury to perivenous tissue. In the current study, a glass‐capped diffusing applicator is designed to validate the feasibility of EVLA as an effective therapeutic device by applying circumferential light distribution. The proposed device is evaluated with a flat fiber as a reference in terms of temperature elevation, fiber degradation, and degree of coagulative necrosis after 532 nm‐assisted EVLA at 100 J/cm. The diffusing fiber generates a 40% lower maximum temperature with a 90% lower transient temperature change in blood, compared to the flat fiber. Due to low irradiance (13.5 kW/cm²) and wide light distribution, the diffuser tip experiences no significant thermal degradation while severe carbonization occurs at the flat fiber tip. Ex vivo tissue tests verify that the diffusing fiber induces circumferential and consistent tissue denaturation to the vein wall (107.8 ± 7.8 µm) along with 19% vessel shrinkage. The proposed glass‐capped diffusing applicator can be a feasible therapeutic device for EVLA with minimal complications by entailing low maximum temperatures and uniform tissue denaturation in the venous tissue.