Third harmonic generation (THG) provides a valuable, label-free approach to imaging biological systems. To date, THG microscopy has been performed using point scanning methods that rely on intensity measurements lacking phase information of the complex field. We report the first demonstration of THG holographic microscopy and the reconstruction of the complex THG signal field with spatial synthetic aperture imaging. Phase distortions arising from measurement-to-measurement fluctuations and imaging components cause optical aberrations in the reconstructed THG field. We have developed an aberration-correction algorithm that estimates and corrects for these phase distortions to reconstruct the spatial synthetic aperture THG field without optical aberrations.
We investigate the angular field of view (AFOV) of a holographic image reconstructed from the digital Fresnel hologram in holographic display. The theoretical analysis reveals that the AFOV of a holographic image is fundamentally determined by the hologram numerical aperture (HNA) other than a diffraction angle of pixel pitch of a pixelated modulator. This property is proved for various types of the digital holograms by using a numerical simulation and optical experiments. The high-HNA hologram reconstructs the image with a high viewing-angle, although the image contraction is inevitable due t…
The finite-difference time-domain (FDTD) method is employed to solve the three dimensional Maxwell equation for the situation of near-field microscopy using a sub-wavelength aperture. Experimental result on unexpected high spatial resolution is reproduced by our computer simulation.
We propose a three-dimensional holographic reconstruction procedure applicable with no a priori knowledge about the recording conditions enabling distortion-free three-dimensional object reconstruction.
Usually, off-axis digital holographic microscopy requires a coherent light source in order to record a full-field hologram. Nevertheless, a LASER-based illumination leads to a non-negligible coherent noise, decreasing then the imaging quality. We hereby report a simple method to reduce the coherent noise contribution using a low-temporal-coherence illumination while maintaining a large interference area. A diffraction grating is hence introduced in the reference arm of the interferometer, allowing the coherence plane of the reference beam to be tilted following angular dispersion. The phase pl…
The theoretical approach to describe the defocusing microscopy technique by U. Agero et al. [Phys. Rev. E {\bf 67}, 051904 (2003)] assumes that the size of the objective lens aperture is infinite. This treatment gives that the intensity at the image plane depends on the laplacian of the phase introduced in the field by a pure phase object. In the present paper, we consider an arbitrary size for the aperture of the objective lens and we conclude that the intensity at the image plane depends also on the gradient of the phase introduced by the object and the phase itself. In this case, even an ob…
Holographic microscopy has developed into a powerful tool for 3D particle tracking, yielding nanometer-scale precision at high frame rates. However, current particle tracking algorithms ignore the effect of the microscope objective on the formation of the recorded hologram. As a result, particle tracking in holographic microscopy is currently limited to particles well above the microscope focus. Here, we show that modeling the effect of an aberration-free lens allows tracking of particles above, near, and below the focal plane in holographic microscopy, doubling the depth of field. Finally, we…
We report a novel microscopy platform, termed Multi-Aperture Fourier ptychographic microscopy (MA-FPM), capable of realizing gigapixel complex field images with large data acquisition bandwidths - in gigapixels per second. MA-FPM is a synthetic aperture technique- an array of objectives together with tilt-shift illumination are used to synthesize high-resolution, wide field-of-view images. Here, the phase is recovered using Fourier ptychography (FP) algorithms, unlike conventional optical synthetic aperture techniques where holographic measurements are used. The parallel data-acquisition capab…
An holographic microscopy reconstruction method compatible with high numerical aperture microscope objective (MO) up to NA=1.4 is proposed. After off axis and reference field curvature corrections, and after selection of the +1 grating order holographic image, a phase mask that transforms the optical elements of the holographic setup into an afocal device is applied in the camera plane. The reconstruction is then made by the angular spectrum method. The field is first propagated in the image half space from the camera to the afocal image of the MO optimal plane (plane for which MO has been des…
We theoretically and experimentally demonstrate a multifrequency excitation and detection scheme in apertureless near field optical microscopy, that exceeds current state of the art sensitivity and background suppression. By exciting the AFM tip at its two first flexural modes, and demodulating the detected signal at the harmonics of their sum, we extract a near field signal with a twofold improved sensitivity and deep sub-wavelength resolution, reaching $λ/230$. Furthermore, the method offers rich control over experimental degrees of freedom, expanding the parameter space for achieving comple…
We have extended Laser Doppler holographic microscopy to transmission geometry. The technique is validated with living fish embryos imaged by a modified upright bio-microcope. By varying the frequency of the holographic reference beam, and the combination of frames used to calculate the hologram, multimodal imaging has been performed. Doppler images of the blood vessels for different Doppler shifts, images where the flow direction is coded in RGB colors or movies showing blood cells individual motion have been obtained as well. The ability to select the Fourier space zone that is used to calcu…
We use digital holographic microscopy and Mie scattering theory to simultaneously characterize and track individual colloidal particles. Each holographic snapshot provides enough information to measure a colloidal sphere's radius and refractive index to within 1%, and simultaneously to measure its three-dimensional position with nanometer in-plane precision and 10 nanometer axial resolution.
This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environment. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire sample's scattered field, with a lateral…
We have developed a new on-axis digital holographic technique, heterodyne holography. The resolution of this technique is limited mainly by the amount of data recorded on two-dimensional photodetectors, i.e., the number of pixels and their size. We demonstrate that it is possible to increase the resolution linearly with the amount of recorded data by aperture synthesis as done in the radar technique but with an optical holographic field.
Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately $T^{-1/2}$, where T is the transmissivity of the mask. Numerical aperture based differentiation of background from scattered light represents a general approach to increasin…
We report experimental results on heterodyne holographic microscopy of subwavelength-sized gold particles. The apparatus uses continuous green laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled device array detector enables 3D localization of brownian particles in water
The viewing-angle enlargement of a holographic image is a crucial factor for realizing the holographic display. The numerical aperture (NA) of digital hologram other than a pixel specification has been known to determine the angular field extent of image. Here, we provide a valid foundation for the dependence of viewing angle on the hologram numerical aperture by investigating mathematically the internal structure of the sampled point spread function showing a self-similarity of its modulating curves and especially, analyzing this scheme on the basis of quantum mechanical framework. The enhanc…
Fiber imaging bundles are widely used as thin, passive image conduits for miniaturised and endoscopic microscopy, particularly for confocal fluorescence imaging. Holographic microscopy through fiber bundles is more challenging; phase conjugation approaches are complex and require extensive calibration. This articles describes how simple inline holographic microscopy can be performed through an imaging bundle using a partially coherent illumination source from a multimode fiber. The sample is imaged in transmission, with the intensity hologram sampled by the bundle and transmitted to a remote c…
Using conventional refraction-based optical lens, it is challenging to achieve both high-resolution imaging and long-working-distance condition. To increase the numerical aperture of a lens, the working distance should be compensated, and vice versa. Here we propose and demonstrate a new concept in optical microscopy that can achieve both high-resolution imaging and long-working-distance conditions by utilising a scattering layer instead of refractive optics. When light passes through a scattering layer, it creates a unique interference pattern. To retrieve the complex amplitude image from the…
We present a new technique that combines off-axis Digital Holography and Dark Field Microscopy to track 100nm gold particles diffusing in water. We show that a single hologram is sufficient to localize several particles in a thick sample with a localization accuracy independent of the particle position. From our measurements we reconstruct the trajectories of the particles and derive their 3D diffusion coefficient. Our results pave the way for quantitative studies of the motion of single nanoparticle in complex media.
We propose a real-time digital holographic microscopy, that enables simultaneous multiple reconstructed images with arbitrary resolution, depth and positions, using Shifted-Fresnel diffraction instead of Fresnel diffraction. In this system, we used four graphics processing units (GPU) for multiple reconstructions in real-time. We show the demonstration of four reconstruction images from a hologram with arbitrary depths, positions, and resolutions.
Light diffraction through a subwavelength aperture located at the apex of a metallic screen with conical geometry is investigated theoretically. A method based on a multipole field expansion is developed to solve Maxwell's equations analytically using boundary conditions adapted both for the conical geometry and for the finite conductivity of a real metal. The topological properties of the diffracted field are discussed in detail and compared to those of the field diffracted through a small aperture in a flat screen, i. e. the Bethe problem. The model is applied to coated, conically tapered op…
We present a cost-effective, simple and fast digital holographic microscopy method based upon Rayleigh-Sommerfeld back propagation for identification of the geometrical shape of a cell. The method was tested using synthetic hologram images generated by ray-tracing software and from experimental images of semi-transparent spherical beads and living red blood cells. Our results show that by only using the real part of the back-reconstructed amplitude the proposed method can provide information of the geometrical shape of the object and at the same time accurately determine the axial position of …
We introduce a new modality for dynamic phase imaging in confocal microscopy based on synthetic optical holography. By temporal demultiplexing of the detector signal into a series of holograms, we record time-resolved phase images directly in the time domain at a bandwidth as determined by the photo detector and digitizer. We demonstrate our method by optical imaging of transient vibrations in an atomic force microscope cantilever with 100 ns time resolution, and observe the dynamic deformation of the cantilever surface after excitation with broadband mechanical pulses. Temporal Fourier transf…
Lensless microscopy with coherent or partially coherent light sources is a well known imaging technique, commonly referred as digital in-line holographic microscopy. In the established methods, both the spatial and temporal coherence of light play a crucial role in determining the resolution of reconstructed object. We report lensless microscopy with a spatially extended white LED, a light source of low spatial and very low temporal coherence. The wave-field propagation between two parallel planes can be obtained using a convolution operation, where the convolution kernel depends on the object…
We demonstrate a gigapixel inline digital holographic microscopy using a consumer scanner. The consumer scanner can maximally scan an A4 size image (297mm x 210mm) with 4800 dpi (= 5.29 um), theoretically achieving a resolution of 56,144 x 39,698 = 2.22 gigapixels. The system using a consumer scanner has a simple structure, compared with synthetic aperture digital holography using a camera mounted on a two-dimensional moving stage. In this demonstration, we captured an inline hologram with 23,602 x 18,023 pixels (= 0.43 gigapixels). In addition, to accelerate the reconstruction time of the gig…
In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This cha…
We discuss theoretically the concept of spatial resolution in near-field scanning optical microscopy (NSOM) in light of a recent work [Opt. Express 17 (2009) 19969] which reported on the achievement of active tips made of a single ultrasmall fluorescent nanodiamond grafted onto the apex of a substrate tip and on their validation in NSOM imaging. Since fluorescent nanodiamonds tend to decrease steadily in size, we assimilate a nanodiamond-based tip to a point-like single photon source and compare its ultimate resolution with that offered by standard metal-coated aperture NSOM tips. We demonstra…
Phase retrieval and imaging phase measurements are fields of intense research. It has recently been shown that phase retrieval from self-referencing interferograms (SRI) can be reformulated leading to a stable, linear equation provided the amplitude of the field is known from prior measurement steps (HOLOCAM). Consequently, the numerical solution thereof is straightforward. This is a big achievement since convergence is otherwise not always guaranteed. Applications are expected in X-ray microscopy, general phase retrieval, holography, tomography and optical imaging.
Aperture based scanning near field optical microscopes are important instruments to study light at the nanoscale and to understand the optical functionality of photonic nanostructures. In general, a detected image is affected by both, the transverse electric and magnetic field components of light. The discrimination of the individual field components is challenging, as these four field components are contained within two signals in the case of a polarization-resolved measurement. Here, we develop a methodology to solve the inverse imaging problem and to retrieve the vectorial field components …
Laboratoire Kastler Brossel（卡斯特勒·布罗塞尔实验室）；Ecole Normale Supérieure（巴黎高等师范学院）；UPMC（巴黎第六大学（皮埃尔和玛丽·居里大学））；Laboratoire Charles Coulomb（查尔斯·库仑实验室）；Université Montpellier II（蒙彼利埃第二大学）
In this paper we present a digital holographic microscope to track gold colloids in three dimensions. We report observations of 100nm gold particles in motion in water. The expected signal and the chosen method of reconstruction are described. We also discuss about how to implement the numerical calculation to reach real-time 3D tracking.
This study developed handheld and low-cost digital holographic microscopy (DHM) by adopting an in-line type hologram, a webcam, a high power RGB light emitting diode (LED), and a pinhole. It cost less than 20,000 yen (approximately 250 US dollars at 80 yen/dollar), and was approximately 120 mm x 80 mm x 55 mm in size. In addition, by adjusting the recording-distance of a hologram, the lateral resolution power at the most suitable distance was 17.5 um. Furthermore, this DHM was developed for use in open source libraries, and is therefore low-cost and can be easily developed by anyone. In this r…
Spatial effects of interference and interaction of light modes in the subwavelength part of the near-field optical microscopy probe have been theoretically studied. It was found that the mode interference can lead to higher spatial compression of light (lambda = 500 nm in free space) within the transverse size of 25 nm inside the probe output aperture of 100 nm in diameter. The results principally demonstrate the possibility of increasing spatial resolution of the near-field optical microscopy technique.
This paper presents an application of digital Fourier holography for selective imaging of scatterers with different sizes in turbid media such as biological tissues. A combination of Fourier holography and high-resolution digital recording, digital Fourier microscopy (DFM) permits crucial flexibility in applying filtering to highlight scatterers of interest in the tissue. The high-resolution digital hologram is a result of the collation of Fourier holographic frames to form a large-size composite hologram. It is expected that DFM has an improved signal-to-noise ratio as compared to conventiona…
A holographic microscope captures interference patterns, or holograms, that encode three-dimensional (3D) information about the object being viewed. Computation is essential to extracting that 3D information. By wrapping low-level scattering codes and taking advantage of Python's data analysis ecosystem, HoloPy makes it easy for experimentalists to use modern, sophisticated inference methods to analyze holograms. The resulting data can be used to understand how small particles or microorganisms move and interact. The project illustrates how computational tools can enable experimental methods a…
University of California, Los Angeles（加州大学洛杉矶分校）；California NanoSystems Institute（加州纳米系统研究所）；David Geffen School of Medicine（大卫·格芬医学院）
Polarized light microscopy provides high contrast to birefringent specimen and is widely used as a diagnostic tool in pathology. However, polarization microscopy systems typically operate by analyzing images collected from two or more light paths in different states of polarization, which lead to relatively complex optical designs, high system costs or experienced technicians being required. Here, we present a deep learning-based holographic polarization microscope that is capable of obtaining quantitative birefringence retardance and orientation information of specimen from a phase recovered …
We propose to use high numerical aperture single mode optical fibers like photonic crystal fiber for lensless in-line holographic microscopy. Highly divergent beam helps to overcome the spatial sampling limitation of the image sensor. In this paper, a submicron lateral resolution has been demonstrated, with an imaging sensor of pixel pitch 1.12 micrometer and a photonic crystal fiber of mode field diameter 1.8 micrometer. In earlier methods of single-shot lensless imaging, submicron resolution has been obtained at very small working distance and field of view. The proposed method improves the …
Recent progress in phase modulation using nanofabricated electron holograms has demonstrated how the phase of an electron beam can be controlled. In this paper, we apply this concept to the correction of spherical aberration in a scanning transmission electron microscope and demonstrate an improvement in spatial resolution. Such a holographic approach to spherical aberration correction is advantageous for its simplicity and cost-effectiveness.
This Letter reports a demonstration of off-axis compressed holography in low-light level imaging conditions. An acquisition protocol relying on a single exposure of a randomly undersampled diffraction map of the optical field, recorded in the high heterodyne gain regime, is proposed. The image acquisition scheme is based on compressed sensing, a theory establishing that near-exact recovery of an unknown sparse signal is possible from a small number of nonstructured measurements. Image reconstruction is further enhanced by introducing an off-axis spatial support constraint to the image estimati…
We propose an holographic microscopy reconstruction method, which propagates the hologram, in the object half space, in the vicinity of the object. The calibration yields reconstructions with an undistorted reconstruction grid i.e. with orthogonal $x$, $y$ and $z$ axis and constant pixels pitch. The method is validated with an USAF target imaged by a $\times$60 microscope objective, whose holograms are recorded and reconstructed for different USAF locations along the longitudinal axis: -75 to +75 $μ$m. Since the reconstruction numerical phase mask, the reference phase curvature and MO form an …
We present a computational method for full-range interferometric synthetic aperture microscopy (ISAM) under dispersion encoding. With this, one can effectively double the depth range of optical coherence tomography (OCT), whilst dramatically enhancing the spatial resolution away from the focal plane. To this end, we propose a model-based iterative reconstruction (MBIR) method, where ISAM is directly considered in an optimization approach, and we make the discovery that sparsity promoting regularization effectively recovers the full-range signal. Within this work, we adopt an optimal nonuniform…
Quantitative phase imaging (QPI) is a valuable label-free modality that has gained significant interest due to its wide potentials, from basic biology to clinical applications. Most existing QPI systems measure microscopic objects via interferometry or nonlinear iterative phase reconstructions from intensity measurements. However, all imaging systems compromise spatial resolution for field of view and vice versa, i.e., suffer from a limited space bandwidth product. Current solutions to this problem involve computational phase retrieval algorithms, which are time-consuming and often suffer from…
High Sensitivity Transient Holographic Full-Polarization Microscopy
Institute for Physical Chemistry, Ruprecht-Karls-University Heidelberg（海德堡大学物理化学研究所）；Ruprecht-Karls-University Heidelberg（海德堡大学）；Karlsruhe Institute of Technology（卡尔斯鲁厄理工学院）
Changes in morphological characteristics of cells from two cultured cancer cell lines, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer have been monitored using digital holographic microscopy. The observed dose-dependent post-treatment dynamics of phase shift variations demonstrated several scenarios of cell death. In particular the phase shift increase at low doses can be associated with apoptosis while its decrease at high doses can be associated with necrosis. Two cell types were shown to be differently responsive to treatment at the same doses. Although th…
We investigate - both experimentally and theoretically - the inelastic interaction between fast electrons and the electromagnetic field scattered by metallic apertures and nanostructures on dielectric membranes using photon induced near-field electron microscopy. The experiments - performed in a high brightness ultrafast transmission electron microscope - on gold apertures on silicon nitride membranes reveal strong modulations of the electron-light coupling strength. We demonstrates that this effect results from the combined action of the electric field scattered by the aperture edges and the …
The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization (IVF), and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI). In this paper, we have studied whether Digital Holographic (DH) microscopy may be useful to obtain quantitative data on human sperm head structure and compared this technique to high power digitally enhanced Nomarski microscope. The main advantage of DH is that a high resolution 3-D quanti…
The 3D structure of light scattering from dark-field illuminated live 3T3 cells marked with 40 nm gold nanomarkers is explored. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. Images are obtained using a novel 3D reconstruction method providing longitudinally undistorted 3D images. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cellular structures, well defined bright scattering patte…
