Micron-scale Spatial Resolution Research Articles

Dosimetry of microbeam radiotherapy by flexible hydrogenated amorphous silicon detectors

Objective. Detectors that can provide accurate dosimetry for microbeam radiation therapy (MRT) must possess intrinsic radiation hardness, a high dynamic range, and a micron-scale spatial resolution. In this work we characterize hydrogenated amorphous silicon detectors for MRT dosimetry, presenting a novel combination of flexible, ultra-thin and radiation-hard features. Approach. Two detectors are explored: an n-type/intrinsic/p-type planar diode (NIP) and an NIP with an additional charge selective layer (NIP + CSC). Results. The sensitivity of the NIP + CSC detector was greater than the NIP detector for all measurement conditions. At 1 V and 0 kGy under the 3T Cu–Cu synchrotron broadbeam, the NIP + CSC detector sensitivity of (7.76 ± 0.01) pC cGy−1 outperformed the NIP detector sensitivity of (3.55 ± 0.23) pC cGy−1 by 219%. The energy dependence of both detectors matches closely to the attenuation coefficient ratio of silicon against water. Radiation damage measurements of both detectors out to 40 kGy revealed a higher radiation tolerance in the NIP detector compared to the NIP + CSC (17.2% and 33.5% degradations, respectively). Percentage depth dose profiles matched the PTW microDiamond detector’s performance to within ±6% for all beam filtrations except in 3T Al–Al due to energy dependence. The 3T Cu–Cu microbeam field profile was reconstructed and returned microbeam width and peak-to-peak values of (51 ± 1) μm and (405 ± 5) μm, respectively. The peak-to-valley dose ratio was measured as a function of depth and agrees within error to the values obtained with the PTW microDiamond. X-ray beam induced charge mapping of the detector revealed minimal dose perturbations from extra-cameral materials. Significance. The detectors are comparable to commercially available dosimeters for quality assurance in MRT. With added benefits of being micron-sized and possessing a flexible water-equivalent substrate, these detectors are attractive candidates for quality assurance, in-vivo dosimetry and in-line beam monitoring for MRT and FLASH therapy.

Hyaluronic acid modified indocyanine green nanoparticles: a novel targeted strategy for NIR-II fluorescence lymphatic imaging.

The lymphatic system, alongside blood circulation, is crucial for maintaining bodily equilibrium and immune surveillance. Despite its importance, lymphatic imaging techniques lag behind those for blood circulation. Fluorescence imaging, particularly in the near-infrared-II (NIR-II) region, offers promising capabilities with centimeter-scale tissue penetration and micron-scale spatial resolution, sparking interest in visualizing the lymphatic system. Although indocyanine green (ICG) has been approved by the Food and Drug Administration (FDA) for use as a near-infrared-I (NIR-I) region fluorescent dye, its limitations include shallow penetration depth and low signal-to-noise ratio. Research suggests that ICG's fluorescence emission tail in the second near-infrared window holds potential for high-quality NIR-II imaging. However, challenges like short circulation half-life and concentration-dependent aggregation hinder its wider application. Here we developed HA@ICG nanoparticles (NPs), a superior ICG-based NIR-II fluorescent probe with excellent biocompatibility, prolonging in vivo imaging, and enhancing photostability compared to ICG alone. Leveraging LYVE-1, a prominent lymphatic endothelial cell receptor that binds specifically to hyaluronic acid (HA), our nanoprobes exhibit exceptional performance in targeting lymphatic system imaging. Moreover, our findings demonstrate the capability of HA@ICG NPs for capillary imaging, offering a means to assess local microcirculatory blood supply. These compelling results underscore the promising potential of HA@ICG NPs for achieving high-resolution bioimaging of nanomedicines in the NIR-II window.

Brillouin microscopic imaging of ex-vivo porcine eye using VIPA-CMOS-based spectrometer

The research on non-invasive ophthalmic imaging has become a hot topic in diagnosing ophthalmic diseases, especially assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. The Brillouin microscopy technique based on spontaneous Brillouin scattering has emerged as a unique elastography method due to its advantages of non-contact, label-free, and high-resolution biomechanical imaging of biological tissues. Here, we developed a three-dimensional (3D) optical microscope system for ex-vivo porcine cornea Brillouin imaging. To do this, we designed and experimentally demonstrated a setup with double-stage VIPA etalons and CMOS camera that integrate to a fully parallel dispersive Brillouin spectrometer. The designed spectrometer exhibits a smaller dispersion rate to enhance the spectral sampling rate and downsizes the size of the double-stage-based system. Using the optical microscope system, we measured and mapped the Brillouin frequency shifts of the cornea and crystalline lens in ex-vivo porcine eyes at micron-scale spatial resolution, and a spectral accuracy of < 50 MHz can be achieved without interpolating the spectra. This work is essential for future applications of Brillouin microscopy in clinical diagnosis and treatment of ophthalmic diseases.

Effects of refractive index mismatch between sample and immersion medium in line-field confocal optical coherence tomography

Line-field confocal optical coherence tomography (LC-OCT) is an optical technique based on low-coherence interference microscopy with line illumination, designed for tomographic imaging of semi-transparent samples with micrometer-scale spatial resolution. A theoretical model of the signal acquired in LC-OCT is presented. The model shows that a refractive index mismatch between the sample and the immersion medium causes a dissociation of the coherence plane and the focal plane, leading to a decrease in the signal amplitude and a degradation of the image’s lateral resolution. Measurements are performed to validate and illustrate the theoretical predictions. A mathematical condition linking various experimental parameters is established to ensure that the degradation of image quality is negligible. This condition is tested experimentally by imaging a phantom. It is verified theoretically in the case of skin imaging, using experimental parameters corresponding to those of the commercially available LC-OCT device.

Quantum Sensing in Tweezer Arrays: Optical Magnetometry on an Individual-Atom Sensor Grid

We implement a scalable platform for quantum sensing comprising hundreds of sites capable of holding individual laser-cooled atoms and demonstrate the applicability of this single-quantum-system sensor array to magnetic field mapping on a two-dimensional grid. With each atom being confined in an optical tweezer within an area of 0.5μm2 at mutual separations of 7.0(2)μm, we obtain micrometer-scale spatial resolution and highly parallelized operation. An additional steerable optical tweezer allows rearrangement of atoms within the grid and enables single-atom scanning microscopy with submicron resolution. This individual-atom sensor platform finds an immediate application in mapping an externally applied dc gradient magnetic field. In a Ramsey-type measurement, we obtain a field resolution of 98(29) nT. We estimate the sensitivity to be 25μT/Hz. Published by the American Physical Society 2024

The Progress of Optical Coherence Tomography in Industry Applications

Optical coherence tomography (OCT) is a nonintrusive optical imaging method offering micrometer-scale spatial resolution, enabling the generation of internal structural cross-sectional images. Since the first OCT was invented in the early 1990s, its utilization has become prevalent in the field of ophthalmology and has become one of the golden standards in clinical diagnosis. Beyond the extensive research in biomedical fields, because of the accomplished exceptional spatial resolution and rapid imaging acquisition rate, the OCT has also been explored as an emerging solution for characterizing structure of new materials, detecting defects of industrial products in situ. In this paper, we reviewed and summarized recent progresses of OCT in several industrial applications, including structural analysis of composite materials, quality inspection of printed electronic devices, and analysis of industrial fluids. Meanwhile, the potential challenges and future development of OCT application in these fields are also outlined.

2D Helium Atom Diffraction from a Microscopic Spot.

A method for measuring helium atom diffraction with micron-scale spatial resolution is demonstrated in a scanning helium microscope (SHeM) and applied to study a micron-scale spot on the (100) plane of a lithium fluoride (LiF) crystal. The positions of the observed diffraction peaks provide an accurate measurement of the local lattice spacing, while a combination of close-coupled scattering calculations and MonteCarlo ray-tracing simulations reproduce the main variations in diffracted intensity. Subsequently, the diffraction results are used to enhance image contrast by measuring at different points in reciprocal space. The results open up the possibility for using helium microdiffraction to characterize the morphology of delicate or electron-sensitive materials on small scales. These include many fundamentally and technologically important samples which cannot be studied in conventional atom scattering instruments, such as small grain size exfoliated 2D materials, polycrystalline samples, and other surfaces that do not exhibit long-range order.

In situ visualization of misorientation-dependent hydrogen diffusion at grain boundaries of pure polycrystalline Ni using a hydrogen video imaging system

Hydrogen, the key element for solving the major environmental issues, exhibits rapid diffusion and localization in lattice defects of metals, which leads to embrittlement. However, the microstructure-hydrogen interactions in metallic materials are poorly understood. Therefore, a technique for capturing the microstructure-dependent hydrogen diffusion in real-time has long been targeted by researchers. Here, we successfully visualized the preferential hydrogen diffusion at grain boundaries (GBs) of pure Ni using a hydrogen video imaging system. A thin polyaniline layer was utilized as a hydrogenochromic sensor, and the hydrogen distribution was analyzed based on the color distribution of polyaniline. The system revealed the misorientation-dependent hydrogen flux at GBs of pure polycrystalline Ni with a micrometer-scale spatial resolution. It suggests that the geometrical structure of the GBs is a critical factor for preferential hydrogen diffusion. The use of the hydrogen video imaging system further advances the mechanistic understanding of hydrogen-material atomic interactions in polycrystalline metals and critically facilitates the development of hydrogen-related materials.

Real-time visualization of skeletal muscle necrosis in mice and swine through NIR-II/I fluorescence imaging.

Avulsion often occurs in the limb due to heavy shearing forces which not only damage skeletal muscle but also main vessels, resulting in life-threatening muscle ischemia and necrosis. Defining muscle activity is vital for surgical repair. Currently, the color, capacity of blood, contractibility, and consistency (4C) are the primary principles for evaluating the activities of torn muscles. Based on clinical experiences, this standard turns out to be delayed diagnosis, which is not defined by specific parameters. Recently, near-infrared (NIR) fluorescence probes emitting within the second near-infrared window (NIR-II, 1000-1700 nm) have been widely used for non-invasive optical imaging because the tissue absorption and autofluorescence in the NIR-II region are negligible, thus allowing deeper penetration depths with micrometer-scale spatial resolution in vivo. As pathogenesis and development of muscle necrosis, necrosis-related protein may participate in this procedure. There is promising future for NIR-II to be used in evaluating muscle activity in avulsion. A new approach is developed based on experiments with mice and large animals (swine). Myoblasts were incubated with indocyanine green (ICG) to identify the necrosis muscles. The model of extremity damaged muscle was established for the real-time visualization and detection of developed necrosis muscle field under new equipment, both in balb/c mice (female) and long-haired swines. A visible NIR-II/I imaging system was first used in a large animal injured skeletal muscle-related model. Our NIR-II/I imaging system is suitable for evaluating the normal and injured skeletal muscle ICG cycle and pointing to the necrotic skeletal muscle tissue. NIR-II imaging is superior to NIR-I imaging in estimating skeletal muscle, best with 1100 nm filter. NIR-II fluorescence with 1100 nm filter is suitable for analyzing the progress of necrosis muscle tissue, leading to a new approach for intraoperative evaluation.

Structure-aware guided filtering for a ring artifact correction in synchrotron x-ray microtomography.

Synchrotron-based x-ray microtomography (S-µCT) is a powerful non-invasive three-dimensional (3D) imaging technique used for visualizing the internal structure of objects with micron-scale spatial resolution. However, in practical applications, ring artifacts often occur in S-µCT, which significantly degrades image quality and hinders interpretation. In this study, we propose a ring artifact correction method based on guided image filtering (GIF). The method first extracts structural prior from the input S-µCT images and then uses it as the guidance image to correct the ring artifacts. Finally, GIF with a self-guidance image is employed to further enhance image quality. Extensive comparisons and analyses on simulations and real data experiments demonstrate that the proposed method is capable of effectively correcting ring artifacts, accompanied by low-dose noise suppression and sparse-view artifact reduction. These findings suggest that the proposed method has great potential to promote the wider applications of S-µCT in the future.

(Invited) In-Situ Optical Diagnostics of Plasma Electrochemical Reactors

Many methods for synthesizing nanomaterials are complex and can require high temperature, acids, bases, or reducing agents. The plasma electrochemical reactor (PEC), composed of an atmospheric-pressure low-temperature plasma in contact with an aqueous electrode, may provide unique physico-chemical conditions that sidestep these disadvantages because non-equilibrium electrochemistry and nucleation are initiated in solution without additional heating or strong/toxic reagents. Indeed, PECs have successfully synthesized graphene quantum dots (GQD) [1,2].The precise mechanism of GQD growth is likely to be complex, involving non-equilibrium plasma chemistry and interactions near the plasma-liquid interface. Up to now, investigations of the liquid-phase chemistry have relied principally on ex situ measurements with little or no spatially-resolved information. In addition, conventional experimental techniques can suffer from a lack of selectivity and/or degradation of dyes, chemical probes, or spin traps introduced into the liquid.To extend the scope of diagnostics, we employ an in situ approach using multiple diagnostic techniques to study a wide range of physical and chemical properties at the plasma-water interface. The centrepiece of this platform is in situ spontaneous Raman microspectroscopy, which is advantageous because of its non-intrusiveness, selectivity, versatility, and straightforward calibration. Shaping the laser beam into a light sheet enables probing of the interfacial region with micron-scale spatial resolution. To observe the effect of the plasma on the solvent, we tracked the Raman spectrum of water. Analysis of the –OH stretch band reveals that the plasma weakens the hydrogen bonding network of water. This effect becomes especially pronounced near the interface, at a depth of a few tens of microns. Likewise, at a similar depth, the concentrations of aqueous H2O2 and NO3 - both exceed those of the bulk liquid [3]. Similar interfacial layers have been modeled in past studies for radical species such as OH but not for long-lived species such as NO3 -.Concerning GQDs, we tracked their production via in situ photoluminescence (PL) and UV-VIS absorption spectroscopies. Both the PL and absorption intensities reached a maximum not at the interface but rather at a depth of a few millimeters. This observation corroborates with liquid flow field measurements by particle image velocimetry, which revealed a low-velocity zone at this depth. Under certain conditions, we could observe the consumption of the carbon precursor over the course of plasma treatment using in situ Raman spectroscopy. Finally, we characterized plasma properties near the interface, such as the electron number density, using optical emission spectroscopy. Together, these results provide the fullest description to date of the reaction environment during GQD synthesis. Acknowledgments Financial support: ANR grants ANR-15-CE06-0007-01 and ANR-11-LABX-0017-01, PHC Orchid 40938YL, CNRS-IEA “GRAFMET”, Fédération Plas@Par, EUR PLASMAScience, Poitou-Charentes region (CPER program) References [1] Orrière, T., Kurniawan, D., Chang, Y. C., Pai, D. Z., & Chiang, W. H. (2020). Nanotechnology 31 (485001).[2] Yang, J. S., Pai, D. Z., & Chiang, W. H. (2019). Carbon 153, 315-319.[3] Pai, D. Z. (2021) J. Phys. D. : Appl. Phys. 54, 355201

Optical-scanning evanescent sensor for high-speed broadband photoacoustic microscopy

Photoacoustic microscopy with high spatial resolution and fast imaging acquisition allows observing dynamic processes of optical absorption-based microanatomic structures in three dimensions. An evanescent sensor accesses ultrasonic detection with high sensitivity and broad bandwidth while suffering from limited field of view (FOV), thus compromising the photoacoustic imaging acquisition rate. Here, we develop an optical-scanning evanescent sensor by fast deflection of the interrogation light along the interface of prism and water using a one-dimensional galvanometer, demonstrating excellent detection sensitivity of ∼132 Pa with a broadband frequency response of &amp;gt;140-MHz at an enlarged FOV of ∼2.90 × 0.19 mm2. Incorporating the optical-scanning evanescent sensor in photoacoustic microscopy, a volumetric image (∼3.0 × 0.25 × 1.0 mm3) with micrometer-scale spatial resolution is acquired within ∼2.5 s by synergistically scanning both photoacoustic illumination laser and sensor's interrogation light. High-speed imaging of flowing microparticles within a capillary tube offers the visualizations of the traveling processes in three dimensions. Potentially, the optical-scanning evanescent sensor allows photoacoustic microscopy accommodating to dynamic imaging at cellular level such as in vivo flow cytometry of circulating tumor cells.

Manipulating solid-state spin concentration through charge transport

Solid-state defects are attractive platforms for quantum sensing and simulation, e.g., in exploring many-body physics and quantum hydrodynamics. However, many interesting properties can be revealed only upon changes in the density of defects, which instead is usually fixed in material systems. Increasing the interaction strength by creating denser defect ensembles also brings more decoherence. Ideally one would like to control the spin concentration at will while keeping fixed decoherence effects. Here, we show that by exploiting charge transport, we can take some steps in this direction, while at the same time characterizing charge transport and its capture by defects. By exploiting the cycling process of ionization and recombination of NV centers in diamond, we pump electrons from the valence band to the conduction band. These charges are then transported to modulate the spin concentration by changing the charge state of material defects. By developing a wide-field imaging setup integrated with a fast single photon detector array, we achieve a direct and efficient characterization of the charge redistribution process by measuring the complete spectrum of the spin bath with micrometer-scale spatial resolution. We demonstrate a two-fold concentration increase of the dominant spin defects while keeping the T2 of the NV center relatively unchanged, which also provides a potential experimental demonstration of the suppression of spin flip-flops via hyperfine interactions. Our work paves the way to studying many-body dynamics with temporally and spatially tunable interaction strengths in hybrid charge-spin systems.

Chemical composition changes of kiwifruit petiole cell walls infected by Pseudomonas syringae pv. actinidiae based on confocal Raman imaging combined with chemometrics

Kiwifruit canker caused by Pseudomonas syringae pv. actinidiae (Psa) is devastating to the kiwifruit industry. Because of the complex pathogenesis of kiwifruit canker, there is still a lack of effective control measures. The aim of this study was to characterize the temporal and spatial changes of biopolymers in the petiole cell walls of healthy and cankered kiwifruit using confocal Raman microscopy (CRM) techniques combined with Chemometrics. We proposed a general investigation of the development of kiwifruit canker and chemical distribution imaging was successfully applied to the analysis of disease dynamics. First, Raman spectra (RS) of healthy and diseased kiwifruit petiole cell walls were acquired at a micron-scale spatial resolution, followed by label-free in situ imaging of the cell wall. The results of RS and chemical imaging showed that the changes of cellulose and lignin on the petiole cell wall of susceptible kiwifruit under Psa stress were time-dependent, and the degradation rate of cellulose and lignin reached about 25% after eight days of incubation. The results of principal component analysis (PCA) and support vector machine (SVM) based on RS showed that healthy, early-stage disease and late-stage disease samples exhibited significant clustering effects and the SVM classification accuracy was as high as 100%. The multivariate curve resolved–alternating least squares (MCR–ALS) is used to optimize the Raman imaging in order to eliminate the noise and interference caused by the peak clamping method and provide a meaningful chemical profile. In conclusion, CRM combined with chemical classification algorithms and imaging analysis has certain feasibility and application prospects in the early warning and cellular-level dynamic characterization of kiwifruit canker.

Molecular-Based FRET Nanosensor with Dynamic Ratiometric NIR-IIb Fluorescence for Real-Time In Vivo Imaging and Sensing.

Real-time fluorescence sensing can provide insight into biodynamics. However, few fluorescent tools are available to overcome the tissue scattering and autofluorescence interference for high-contrast in vivo sensing with high spatiotemporal resolution. Here, we develop a molecular-based FRET nanosensor (MFN) capable of producing a dynamic ratiometric NIR-IIb (1500-1700 nm) fluorescence signal under a frequency-modulated dual-wavelength excitation bioimaging system. The MFN provides reliable signals in highly scattering tissues and enables in vivo real-time imaging at micrometer-scale spatial resolution and millisecond-scale temporal resolution. As a proof of concept, a physiological pH-responsive nanosensor (MFNpH) was designed as a nanoreporter for intravital real-time monitoring of the endocytosis dynamics of nanoparticles in the tumor microenvironment. We also show that MFNpH allows the accurate quantification of pH changes in a solid tumor through video-rate ratiometric imaging. Our study offers a powerful approach for noninvasive imaging and sensing of biodynamics with micrometer-scale spatial resolution and millisecond-scale temporal resolution.

High spatial resolution direct conversion amorphous selenium X-ray detectors with monolithically integrated CMOS readout

Recent progress in the field of micron-scale spatial resolution direct conversion X-ray detectors for high-energy synchrotron light sources serve applications ranging from nondestructive and noninvasive microscopy techniques which provide insight into the structure and morphology of crystals, to medical diagnostic measurement devices. Amorphous selenium (a-Se) as a wide-bandgap thermally evaporated photoconductor exhibits ultra-low thermal generation rates for dark carriers and has been extensively used in X-ray medical imaging. Being an amorphous material, it can further be deposited over large areas at room temperatures and at substantially lower costs as compared to crystalline semiconductors. To address the demands for a high-energy and high spatial resolution X-ray detector for synchrotron light source applications, we have thermally evaporated a-Se on a Mixed-Mode Pixel Array Detector (MM-PAD) Application Specific Integrated Circuit (ASIC). The ASIC format consists of 128 × 128 square pixels each 150 μm on a side. A 200 μm a-Se layer was directly deposited on the ASIC followed by a metal top electrode. The completed detector assembly was tested with 45 kV Ag and 23 kV Cu X-ray tube sources. The detector fabrication, performances, Modulation Transfer Function (MTF) measurements, and simulations are reported.

Small animal cerebral microvascular imaging with super-resolution ultrasound: Techniques and applications

Super-resolution ultrasound (SR-US) is an emerging microvascular imaging technology that provides a micron-scale spatial resolution with tens of millimeters of depth of imaging penetration. The unmatched combination of high spatial resolution and deep imaging penetration opened new doors for many brain imaging applications that benefit from cerebrovascular biomarkers. Recently, SR-US has found its niche in small animal brain applications thanks to its unique capabilities of extending the optical-imaging-level spatial resolution to subcortical, deep brain regions. In this presentation, I will first introduce existing SR-US imaging techniques that are tailored to small animal brain imaging applications. Topics include fast SR-US methods, 3D SR-US imaging, and phase aberration correction for intact skull imaging. I will then focus on reviewing brain imaging applications that use SR-US to characterize cerebral mirovasculature in the aging, stroke, and Alzheimer’s Disease brain. Finally, I will introduce the new functional super-resolution imaging technique that combines SR-US with functional ultrasound (fUS) to realize whole-brain neural activity recording at a micron-scale spatial resolution.

Investigation of neutron imaging applications using fine-grained nuclear emulsion

Neutron imaging is a nondestructive inspection technique that has a wide range of applications. One of the important aspects of neutron imaging is achieving a micrometer-scale spatial resolution. The development of a high-resolution neutron detector is a challenging task. As one potential solution to this task, we investigate whether neutron detectors based on fine-grained nuclear emulsions are suitable for high-resolution neutron imaging applications. High track density is necessary to improve the quality of neutron imaging. However, the available track analysis methods are difficult to apply under high track density conditions. Simulated images are used to determine the required track density for neutron imaging. A track density of the order of 104 tracks per 100×100μm2 is sufficient to utilize neutron detectors for imaging applications. Contrast resolution was also investigated for image datasets with various track densities and neutron transmission rates. Moreover, experiments were performed for the neutron imaging of gadolinium-based gratings with known geometries. The grating structures were successfully resolved. The calculated 1σ 10%–90% edge response using the grayscale optical images of the grating slit with a periodic structure of 9 μm was 0.945±0.004 μm.

Super-SIMS at DREAMS: Status of a unique and complex endeavour

At the DREAMS (DREsden AMS) facility we are implementing a so-called Super-SIMS (SIMS = Secondary Ion Mass Spectrometry) device, which combines the micron-scale spatial resolution of a commercial SIMS (CAMECA IMS 7f-auto) with the very high selectivity through molecule suppression by AMS. We have demonstrated high transmission for major element ions including silicon, fluorine and iodine, however the lack of well characterized calibration materials makes a true quantification of trace and ultra-trace elements contents difficult. Measurements of P in Si show the linearity of the instrument’s relative sensitivity factor over more than three orders of magnitude, and measurements of the isotopic ratio of bromine in ZnS document the reliability of our approach. The goal of the DREAMS Super-SIMS project is to provide quantitative concentration data of ultra-trace elements in geological samples in the context of resource technology.

Nondestructive circadian profiling of starch content in fresh intact Arabidopsis leaf with two-photon fluorescence and second-harmonic generation imaging

Plant chloroplasts conduct photosynthesis to convert solar energy into sugars for the carbon source essential for cell living and growth during the day. One fraction of photosynthetic products is stored in chloroplasts by forming starch granules to continue the provision of carbon energy during the night. Currently, profiling the starch temporal pattern requires either: (i) sacrificing the leaves, or (ii) generating transgenic plants at the risk of changing the metabolisms by incorporating a genetically modified granule-bound starch synthase (GBSS). In this paper, we demonstrated a nondestructive method using two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging to quantify starch granules within chloroplasts of fresh intact leaves across a day-night cycle. We did so using two Arabidopsis lines having normal and excess starch contents: wild-type (Columbia-0) and starch excess 1 (sex1). The starch granules were visualized by SHG imaging, while the chloroplasts in mesophyll cells were visualized by TPF imaging. Our results provided micron scale spatial resolution of starch distribution within leaves and showed starch circadian patterns consistent with those profiled by enzymatic assays in previous studies. We demonstrated that TPF-SHG imaging is a potential tool for revealing the real-time heterogeneity of starch circadian rhythm in leaf cells, without the need for destructive sample preparation.