NON-LINEAR OPTICAL IMAGING OF OBESITY-RELATED HEALTH RISKS:REVIEW

This review highlights the recent applications of non-linear optical(NLO)microscopy to study obesity-related health risks.A strong emphasis is given to the applications of coherent anti-Stokes Raman scattering(CARS)microscopy where multiple non-linear optical imaging modalities including CARS,sum-frequency generation(SFG),and two-photon fluorescence are employed simultaneously on a single microscope platform.Specific examples on applications of NLO microscopy to study lipid-droplet biology,obesity-cancer relationship,atherosclerosis,and lipidrich biological structures are discussed.

FOREWORD

We are pleased to publish the third issue(Vol.2,No.1)of Journal of Innovative Optical Health Sciences(JIOHS)which focuses on the developments and biomedical applications of nonlinear optical(NLO)microscopy.NLO microscopy is becoming a powerful tool for bioimaging due to several unique advantages over traditional methods.Nonlinear dependence on excitation intensity gives NLO microscopy inherent three-dimensional(3D)imaging capability without the need for a confocal pinhole.This is particularly advantageous in the case of tissue imaging where significant scattering can reduce the signal collection efficiency by confocal detection.Laser scanning facilitates real-time NLO imaging of live tissues and animals.NLO microscopy utilizes near IR excitation which provides both superior optical penetration into tissues as well as reduced photodamage due to reduced interaction with endogenous molecules.This issue includes seven original papers and five review articles.

Multimodal Metabolic Imaging Reveals Pigment Reduction and Lipid Accumulation in Metastatic Melanoma

Objective and Impact Statement.Molecular signatures are needed for early diagnosis and improved treatment of metastatic melanoma.By high-resolution multimodal chemical imaging of human melanoma samples,we identify a metabolic reprogramming from pigmentation to lipid droplet(LD)accumulation in metastatic melanoma.Introduction.Metabolic plasticity promotes cancer survival and metastasis,which promises to serve as a prognostic marker and/or therapeutic target.However,identifying metabolic alterations has been challenged by difficulties in mapping localized metabolites with high spatial resolution.Methods.We developed a multimodal stimulated Raman scattering and pump-probe imaging platform.By time-domain measurement and phasor analysis,our platform allows simultaneous mapping of lipids and pigments at a subcellular level.Furthermore,we identify the sources of these metabolic signatures by tracking deuterium metabolites at a subcellular level.By validation with mass spectrometry,a specific fatty acid desaturase pathway was identified.Results.We identified metabolic reprogramming from a pigment-containing phenotype in low-grade melanoma to an LD-rich phenotype in metastatic melanoma.The LDs contain high levels of cholesteryl ester and unsaturated fatty acids.Elevated fatty acid uptake,but not de novo lipogenesis,contributes to the LD-rich phenotype.Monounsaturated sapienate,mediated by FADS2,is identified as an essential fatty acid that promotes cancer migration.Blocking such metabolic signatures effectively suppresses the migration capacity both in vitro and in vivo.Conclusion.By multimodal spectroscopic imaging and lipidomic analysis,the current study reveals lipid accumulation,mediated by fatty acid uptake,as a metabolic signature that can be harnessed for early diagnosis and improved treatment of metastatic melanoma.

Far-field super-resolution chemical microscopy

Far-feld chemical microscopy providing molecular electronic or vibrational fingerprint information opens a new window for the study of three-dimensional biological,material,and chemical systems.Chemical microscopy provides a nondestructive way of chemical identification without exterior labels.However,the diffraction limit of optics hindered it from discovering more details under the resolution limit.Recent development of super-resolution techniques gives enlightenment to open this door behind far-field chemical microscopy.Here,we review recent advances that have pushed the boundary of far-field chemical microscopy in terms of spatial resolution.We further highlight applications in biomedical research,material characterization,environmental study,cultural heritage conservation,and integrated chip inspection.

Mid-infrared chemical imaging of intracellular tau fibrils using fluorescence-guided computational photothermal microscopy

Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases.However,it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment.To address this challenge,we developed a computational chemical microscope integrating 3D midinfrared photothermal imaging with fluorescence imaging,termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography(FBS-IDT).Based on a low-cost and simple optical design,FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fbrils,an important type of amyloid protein aggregates,in their intracellular environment.Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation.Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils.3D visualization of theβ-sheet for tau fibril structure is achieved.

Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy

Real-time vibrational spectroscopic imaging is desired for monitoring cellular states and cellular processes in a label-free manner.Raman spectroscopic imaging of highly dynamic systems is inhibited by relatively slow spectral acquisition on millisecond to second scale.Here,we report microsecond scale vibrational spectroscopic imaging by lock-in free parallel detection of spectrally dispersed stimulated Raman scattering signal.Using a homebuilt tuned amplifier array,our method enables Raman spectral acquisition,within the window defined by the broadband pulse,at the speed of 32 μs and with close to shot-noise limited detection sensitivity.Incorporated with multivariate curve resolution analysis,our platform allows compositional mapping of lipid droplets in single live cells,observation of intracellular retinoid metabolism,discrimination of fat droplets from protein-rich organelles in Caenorhabditis elegans,spectral detection of fast flowing tumor cells and monitoring drug diffusion through skin tissue in vivo.The reported technique opens new opportunities for compositional analysis of cellular compartment in a microscope setting and high-throughput spectral profiling of single cells in a flow cytometer setting.

Non-genetic photoacoustic stimulation of single neurons by a tapered fiber optoacoustic emitter

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments.Here,we developed a tapered fiber optoacoustic emitter(TFOE)generating an ultrasound field with a high spatial precision of 39.6 pm,enabling optoacoustic activation of single neurons or subcellular structures,such as axons and dendrites.Temporally,a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation.The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons.Direct measurements of the electrical response of single neurons to acoustic stimulation,which is difficult for conventional ultrasound stimulation,have been demonstrated.By coupling TFOE with ex vivo brain slice electrophysiology,we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation.These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology,which could shed new insights into the mechanism of ultrasound neurostimulation.

A fiber optoacoustic guide with augmented reality for precision breastconserving surgery

Lumpectomy,also called breast-conserving surgery,has become the standard surgical treatment for early-stage breast cancer.However,accurately locating the tumor during a lumpectomy,especially when the lesion is small and nonpalpable,is a challenge.Such difficulty can lead to either incomplete tumor removal or prolonged surgical time,which result in high re-operation rates(~25%)and increased surgical costs.Here,we report a fiber optoacoustic guide(FOG)with augmented reality(AR)for sub-millimeter tumor localization and intuitive surgical guidance with minimal interference.The FOG is preoperatively implanted in the tumor.Under external pulsed light excitation,the FOG omnidirectionally broadcasts acoustic waves through the optoacoustic effect by a specially designed nano-composite layer at its tip.By capturing the acoustic wave,three ultrasound sensors on the breast skin triangulate the FOG tip’s position with 0.25-mm accuracy.An AR system with a tablet measures the coordinates of the ultrasound sensors and transforms the FOG tip’s position into visual feedback with<1-mm accuracy,thus aiding surgeons in directly visualizing the tumor location and performing fast and accurate tumor removal.We further show the use of a head-mounted display to visualize the same information in the surgeons’first-person view and achieve hands-free guidance.Towards clinical application,a surgeon successfully deployed the FOG to excise a“pseudo tumor”in a female human cadaver.With the high-accuracy tumor localization by FOG and the intuitive surgical guidance by AR,the surgeon performed accurate and fast tumor removal,which will significantly reduce re-operation rates and shorten the surgery time.

Stimulated Raman scattering signal generation in a scattering medium using self-reconstructing Bessel beams

Scattering is a huge challenge for microscopic imaging.Indeed,it is difficult to observe target chemicals in scattering media by means of the current Gaussian beam-based stimulated Raman scattering(SRS)microscopy,since the tight focus of the Gaussian beam is destroyed after propagating through a certain distance.Bessel beams,featuring self-reconstructing property,may bring a solution to this problem.By combining Bessel beams with SRS microscopy,we can probe the SRS signal from a scattering medium.In this paper,using the beam propagation method,we first simulate the propagation of the Bessel beam as well as the generation and self-reconstruction of SRS signals.By adding glass beads along the beam propagation path in order to simulate scattering,the propagation of the Bessel beams and the generation of the SRS signals will change.Then,we design a series of simulations to investigate the influence of the size,position,number,and distribution of the added glass beads on the generation of the SRS signals.A preliminary experiment is also carried out to confirm the simulation predictions.Results demonstrate that the SRS signals can be generated or be recovered at a certain depth in scattering media,and that such signals are greatly affected by the parameters of the scatters.

Bond-selective transient phase imaging via sensing of the infrared photothermal effect

Phase-contrast microscopy converts the phase shift of light passing through a transparent specimen,e.g.,a biological cell,into brightness variations in an image.This ability to observe structures without destructive fixation or staining has been widely utilized for applications in materials and life sciences.Despite these advantages,phase-contrast microscopy lacks the ability to reveal molecular information.To address this gap,we developed a bond-selective transient phase(BSTP)imaging technique that excites molecular vibrations by infrared light,resulting in a transient change in phase shift that can be detected by a diffraction phase microscope.By developing a time-gated pump-probe camera system,we demonstrate BSTP imaging of live cells at a 50 Hz frame rate with high spectral fidelity,sub-microsecond temporal resolution,and sub-micron spatial resolution.Our approach paves a new way for spectroscopic imaging investigation in biology and materials science.

Optically-generated focused ultrasound for noninvasive brain stimulation with ultrahigh precision

High precision neuromodulation is a powerful tool to decipher neurocircuits and treat neurological diseases.Current non-invasive neuromodulation methods offer limited precision at the milimeter level.Here,we report opticallygenerated focused ultrasound(OFUS)for non-invasive brain stimulation with ultrahigh precision.OFUS is generated by a soft optoacoustic pad(SOAP)fabricated through embedding candle soot nanoparticles in a curved polydimethylsiloxane film.SOAP generates a transcranial ultrasound focus at 15 MHz with an ultrahigh lateral resolution of 83μm,which is two orders of magnitude smaller than that of conventional transcranial-focused ultrasound(tFUS).Here,we show effective OFUS neurostimulation in vitro with a single ultrasound cycle.We demonstrate submillimeter transcranial stimulation of the mouse motor cortex in vivo.An acoustic energy of 0.6 mJ/cm?,four orders of magnitude less than that of tFUS,is suffcient for successful OFUS neurostimulation.OFUS offers new capabilities for neuroscience studies and disease treatments by delivering a focus with ultrahigh precision noninvasively.

Two-Photon Luminescence Imaging of Bacillus Spores Using Peptide-Functionalized Gold Nanorods

Bacillus subtilis spores(a simulant of Bacillus anthracis)have been imaged by two-photon luminescence(TPL)microscopy,using gold nanorods(GNRs)functionalized with a cysteine-terminated homing peptide.Control experiments using a peptide with a scrambled amino acid sequence confi rmed that the GNR targeting was highly selective for the spore surfaces.The high sensitivity of TPL combined with the high affi nity of the peptide labels enables spores to be detected with high fi delity using GNRs at femtomolar concentrations.It was also determined that GNRs are capable of signifi cant TPL output even when irradiated at near infrared(NIR)wavelengths far from their longitudinal plasmon resonance(LPR),permitting considerable fl exibility in the choice of GNR aspect ratio or excitation wavelength for TPL imaging.

Spectroscopic stimulated Raman scattering imaging of highly dynamic specimens through matrix completion

Spectroscopic stimulated Raman scattering(SRS)imaging generates chemical maps of intrinsic molecules,with no need for prior knowledge.Despite great advances in instrumentation,the acquisition speed for a spectroscopic SRS image stack is fundamentally bounded by the pixel integration time.In this work,we report three-dimensional sparsely sampled spectroscopic SRS imaging that measures~20%of pixels throughout the stack.In conjunction with related work in low-rank matrix completion(e.g.,the Netflix Prize),we develop a regularized non-negative matrix factorization algorithm to decompose the sub-sampled image stack into spectral signatures and concentration maps.This design enables an acquisition speed of 0.8 s per image stack,with 50 frames in the spectral domain and 40,000 pixels in the spatial domain,which is faster than the conventional raster laser-scanning scheme by one order of magnitude.Such speed allows real-time metabolic imaging of living fungi suspended in a growth medium while effectively maintaining the spatial and spectral resolutions.This work is expected to promote broad application of matrix completion in spectroscopic laser-scanning imaging.

Ultraefficient thermoacoustic conversion through a split ring resonator

Microwaves,which have a∼10-cm wavelength,can penetrate deeper into tissue than photons,heralding exciting deep tissue applications such as modulation or imaging via the thermoacoustic effect.Thermoacoustic conversion efficiency is however very low,even with an exogenous contrast agent.We break this low-conversion limit,using a split ring resonator to effectively collect and confine the microwaves into a submillimeter hot spot for ultrasound emission and achieve a conversion efficiency over 2000 times higher than other reported thermoacoustic contrast agents.Importantly,the frequency of emitted ultrasound can be precisely tuned and multiplexed by modulation of the microwave pulses.Such performance is inaccessible by a piezoelectric-based transducer or a photoacoustic emitter and,therefore,split ring resonators open up new opportunities to study the frequency response of cells in ultrasonic biomodulation.For applications in deep tissue localization,a split ring resonator can be used as a wireless,battery-free ultrasound beacon placed under a breast phantom.

New “HOPE” laser for photoacoustic imaging of water

A hybrid optical parametrically-oscillating laser at 1930 nm enables photoacoustic mapping of water content in deep tissue with good sensitivity and high spatial resolution.

Computational coherent Raman scattering imaging:breaking physical barriers by fusion of advanced instrumentation and data science

Coherent Raman scattering(CRS)microscopy is a chemical imaging modality that provides contrast based on intrinsic biomolecular vibrations.To date,endeavors on instrumentation have advanced CRS into a powerful analytical tool for studies of cell functions and in situ clinical diagnosis.Nevertheless,the small cross-section of Raman scattering sets up a physical boundary for the design space of a CRS system,which trades off speed,signal fidelity and spectral bandwidth.The synergistic combination of instrumentation and computational approaches offers a way to break the trade-off.In this review,we first introduce coherent Raman scattering and recent instrumentation developments,then discuss current computational CRS imaging methods,including compressive micro-spectroscopy,computational volumetric imaging,as well as machine learning algorithms that improve system performance and decipher chemical information.We foresee a constant permeation of computational concepts and algorithms to push the capability boundary of CRS microscopy.