We describe a simple and robust approach for characterizing the spatially varying pupil aberrations of microscopy systems. In our demonstration with a standard microscope, we derive the location-dependent pupil transfer functions by first capturing multiple intensity images at different defocus settings. Next, a generalized pattern search algorithm is applied to recover the complex pupil functions at ~350 different spatial locations over the entire field-of-view. Parameter fitting transforms these pupil functions into accurate 2D aberration maps. We further demonstrate how these aberration maps can be applied in a phase-retrieval based microscopy setup to compensate for spatially varying aberrations and to achieve diffraction-limited performance over the entire field-of-view. We believe that this easy-to-use spatially-varying pupil characterization method may facilitate new optical imaging strategies for a variety of wide field-of-view imaging platforms.
(170.0180) Microscopy; (100.0100) Image processing
Flow cytometry is a well-established and powerful high-throughput fluorescence measurement tool that also allows for the sorting and enrichment of subpopulations of cells expressing unique fluorescence signatures. Owing to the reliance on intensity-only signals, flow cytometry sorters cannot easily discriminate between fluorophores that spectrally overlap. In this paper we demonstrate a new method of cell sorting using a fluorescence lifetime-dependent methodology. This approach, referred to herein as phase-filtered cell sorting (PFCS), permits sorting based on the average fluorescence lifetime of a fluorophore by separating fluorescence signals from species that emit differing average fluorescence lifetimes. Using lifetime-dependent hardware, cells and microspheres labeled with fluorophores were sorted with purities up to 90%. PFCS is a practical approach for separating populations of cells that are stained with spectrally overlapping fluorophores or that have interfering autofluorescence signals.
(170.1420) Biology; (170.3650) Lifetime-based sensing; (170.6280) Spectroscopy, fluorescence and luminescence; (300.2530) Fluorescence, laser-induced
A technique on high frame rate(28fps), high frequency co-registered ultrasound and photoacoustic imaging for visualizing zebrafish heart blood flow was demonstrated. This approach was achieved with a 40MHz light weight(0.38g) ring-type transducer, serving as the ultrasound transmitter and receiver, to allow an optic fiber, coupled with a 532nm laser, to be inserted into the hole. From the wire target study, axial resolutions of 38µm and 42µm were obtained for ultrasound and photoacoustic imaging, respectively. Carbon nanotubes were utilized as contrast agents to increase the flow signal level by 20dB in phantom studies, and zebrafish heart blood flow was successfully observed.
(110.0110) Imaging systems; (110.5120) Photoacoustic imaging
The capability to perform multicolor, wide field-of-view (FOV) fluorescence microscopy imaging is important in screening and pathology applications. We developed a microscopic slide-imaging system that can achieve multicolor, wide FOV, fluorescence imaging based on the Talbot effect. In this system, a light-spot grid generated by the Talbot effect illuminates the sample. By tilting the excitation beam, the Talbot-focused spot scans across the sample. The images are reconstructed by collecting the fluorescence emissions that correspond to each focused spot with a relay optics arrangement. The prototype system achieved an FOV of 12 × 10 mm2 at an acquisition time as fast as 23 s for one fluorescence channel. The resolution is fundamentally limited by spot size, with a demonstrated full-width at half-maximum spot diameter of 1.2 μm. The prototype was used to image green fluorescent beads, double-stained human breast cancer SK-BR-3 cells, Giardia lamblia cysts, and the Cryptosporidium parvum oocysts. This imaging method is scalable and simple for implementation of high-speed wide FOV fluorescence microscopy.
(180.2520) Fluorescence microscopy; (110.6760) Talbot and self-imaging effects
We introduce an interferometric technique for eliminating the nonresonant background of broadband coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy has been used for imaging a number of biological samples and processes, but the studies are mostly limited to detecting lipids in biological systems by probing the C-H stretch. Nonresonant background and incoherent noise sources can easily overwhelm less intense signals from other molecular vibrations. In this study, we demonstrate a one-laser broadband interferometric technique that separates the spontaneous Raman scattering-related component of the CARS signal from the nonresonant background using liquid benzonitrile as a model system.
A snapshot 3-Dimensional Optical Coherence Tomography system was developed using Image MappingSpectrometry. This system can give depth information (Z) at different spatial positions (XY) withinone camera integration time to potentially reduce motion artifact and enhance throughput. Thecurrent (x,y,λ) datacube of (85×356×117) provides a 3Dvisualization of sample with 400 μm depth and 13.4μm in transverse resolution. Axial resolution of 16.0μm can also be achieved in this proof-of-concept system. We present ananalysis of the theoretical constraints which will guide development of future systems withincreased imaging depth and improved axial and lateral resolutions.
(110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging
The stimulated Raman scattering signal is often accompanied by unwanted background arising from other pump-probe modalities. We demonstrate an approach to overcome this challenge based on spectral domain modulation, enabled by a compact, cost-effective angle-to-wavelength pulse shaper. The pulse shaper switches between two spectrally narrow windows, which are cut out of a broadband femtosecond pulse and selected for on- and off- Raman resonance excitation, at 2.1 MHz frequency for detection of stimulated Raman scattering signal. Such spectral modulation reduced the unwanted pump-probe signals by up to 20 times and enabled stimulated Raman scattering imaging of molecules in a pigmented environment.
(180.4315) Nonlinear microscopy; (290.5910) Scattering, stimulated Raman
X-ray diffraction patterns may be obtained from individual submicron protein nanocrystals using a femtosecond pulse from a free-electron X-ray laser. Many “single-shot” patterns are read out every second from a stream of nanocrystals lying in random orientations. The short pulse terminates before significant atomic (or electronic) motion commences, minimizing radiation damage. Simulated patterns for Photosystem I nanocrystals are used to develop a method for recovering structure factors from tens of thousands of snapshot patterns from nanocrystals varying in size, shape and orientation. We determine the number of shots needed for a required accuracy in structure factor measurement and resolution, and investigate the convergence of our Monte-Carlo integration method.
The intrinsic weak birefringence in all-normal dispersion highly nonlinear fiber, particularly ultra-high-numerical-aperture fiber, generates supercontinuum with long term polarization instabilities, even for seed pulses launched along the perceived slow axis of the fiber. Highly co/anti-correlated fluctuations in energy between regions of power spectral density mask the extent of the spectral noise in total integrated power measurements. The instability exhibits a seed pulse power threshold above which the output polarization state of the supercontinuum seeds from noise. Eliminating this instability through the utilization of nonlinear fiber with a large designed birefringence, encourages the exploration of compression schemes and seed sources. Here, we include an analysis of the difficulties for seeding supercontinuum with the highly attractive ANDi-type lasers. Lastly, we introduce an intuitive approach for understanding supercontinuum development and evolution. By modifying the traditional characteristic dispersion and nonlinear lengths to track pulse properties within the nonlinear fiber, we find simple, descriptive handles for supercontinuum evolution.
(060.2420) Fibers, polarization-maintaining; (190.4370) Nonlinear optics, fibers; (320.5520) Pulse compression; (320.6629) Supercontinuum generation
Temporal focusing allows for optically sectioned wide-field microscopy. The optical sectioning arises because this method takes a pulsed input beam, stretches the pulses by diffracting off a grating, and focuses the stretched pulses such that only at the focal plane are the pulses re-compressed. This approach generates nonlinear optical processes at the focal plane and results in depth discrimination. Prior theoretical models of temporal focusing processes approximate the contributions of the different spectral components by their mean. This is valid for longer pulses that have narrower spectral bandwidth but results in a systematic deviation when broad spectrum, femtosecond pulses are used. Further, prior model takes the paraxial approximation but since these pulses are focused with high numerical aperture (NA) objectives, the effects of the vectorial nature of light should be considered. In this paper we present a paraxial and a vector theory of temporal focusing that takes into account the finite spread of the spectrum. Using paraxial theory we arrive at an analytical solution to the electric field at the focus for temporally focused wide-field two-photon (TF2p) microscopy as well as in the case of a spectrally chirped input beam. We find that using paraxial theory while accounting for the broad spectral spread gives results almost twice vector theory. Experiment results agree with predictions of the vector theory giving an axial full-width half maximum (FWHM) of
μmrespectively as long as spectral spread is taken into account. Using our system parameters, the optical sectioning of the TF2p microscope is found to be
μm. The optical transfer function (OTF) of a TF2p microscope is also derived and is found to pass a significantly more limited band of axial frequencies than a point scanning two-photon (2p) microscope or a single photon (1p) confocal microscope.
(170.0180) Microscopy; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy; (180.6900) Three-dimensional microscopy; (190.7110) Ultrafast nonlinear optics
We present a method to dynamically image structures at nanometer spatial resolution with far-field instruments. We propose the use of engineered nanoprobes with distinguishable spectral responses and the measurement of coherent scattering, rather than fluorescence. Approaches such as PALM/STORM have relied on the rarity of emission events in time to distinguish signals from distinct probes. By distinguishing signals in the spectral domain, we enable the acquisition of data in a multiplex fashion and thus circumvent the fundamental problem of slow data acquisition of current techniques. The described method has the potential to image dynamic systems with a spatial resolution only limited to the size of the scattering probes.
(100.6640) Superresolution; (110.4234) Multispectral and hyperspectral imaging
Lens-free holographic on-chip imaging is an emerging approach that offers both wide field-of-view (FOV) and high spatial resolution in a cost-effective and compact design using source shifting based pixel super-resolution. However, color imaging has remained relatively immature for lens-free on-chip imaging, since a ‘rainbow’ like color artifact appears in reconstructed holographic images. To provide a solution for pixel super-resolved color imaging on a chip, here we introduce and compare the performances of two computational methods based on (1) YUV color space averaging, and (2) Dijkstra’s shortest path, both of which eliminate color artifacts in reconstructed images, without compromising the spatial resolution or the wide FOV of lens-free on-chip microscopes. To demonstrate the potential of this lens-free color microscope we imaged stained Papanicolaou (Pap) smears over a wide FOV of ~14 mm2 with sub-micron spatial resolution.
(110.0180) Microscopy; (170.3880) Medical and biological imaging
The enhanced generation of a spontaneous Raman signal by way of elastic scattering is demonstrated. Using Monte Carlo simulations, we show that elastic scattering, by increasing the path length of light through the medium, enhances the generation of a Raman signal. This is investigated over a large parameter space, demonstrating that this effect is robust, and providing additional physical insight into the dynamics of light propagation in a turbid medium. Both the temporal and spatial profiles of the Raman signal are shown to depend heavily on the amount of scattering present.
(170.3660) Light propagation in tissues; (170.5280) Photon migration; (170.5660) Raman spectroscopy; (170.7050) Turbid media
Recently, mechanobiology has received increased attention. For investigation of biofilm and cellular tissue, measurements of the surface topography and deformation in real-time are a pre-requisite for understanding the growth mechanisms. In this paper, a novel three-dimensional (3D) fluorescent microscopic method for surface profilometry and deformation measurements is developed. In this technique a pair of cameras are connected to a binocular fluorescent microscope to acquire micrographs from two different viewing angles of a sample surface doped or sprayed with fluorescent microparticles. Digital image correlation technique is used to search for matching points in the pairing fluorescence micrographs. After calibration of the system, the 3D surface topography is reconstructed from the pair of planar images. When the deformed surface topography is compared with undeformed topography using fluorescent microparticles for movement tracking of individual material points, the full field deformation of the surface is determined. The technique is demonstrated on topography measurement of a biofilm, and also on surface deformation measurement of the biofilm during growth. The use of 3D imaging of the fluorescent microparticles eliminates the formation of bright parts in an image caused by specular reflections. The technique is appropriate for non-contact, full-field and real-time 3D surface profilometry and deformation measurements of materials and structures at the microscale.
(150.6910) Three-dimensional sensing; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (160.1435) Biomaterials
Pump-probe microscopy is an imaging technique that delivers molecular contrast of pigmented samples. Here, we introduce pump-probe nonlinear phase dispersion spectroscopy (PP-NLDS), a method that leverages pump-probe microscopy and spectral-domain interferometry to ascertain information from dispersive and resonant nonlinear effects. PP-NLDS extends the information content to four dimensions (phase, amplitude, wavelength, and pump-probe time-delay) that yield unique insight into a wider range of nonlinear interactions compared to conventional methods. This results in the ability to provide highly specific molecular contrast of pigmented and non-pigmented samples. A theoretical framework is described, and experimental results and simulations illustrate the potential of this method. Implications for biomedical imaging are discussed.
(300.6420) Spectroscopy, nonlinear; (120.3180) Interferometry; (180.4315) Nonlinear microscopy; (170.3880) Medical and biological imaging; (190.3270) Kerr effect
Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Mateŕn functional family. By measuring the wavelength-dependent backscattering coefficient μb(λ) and the scattering coefficient μs, we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these sub-diffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The sub-diffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.
(170.4500) Optical coherence tomography; (290.0290) Scattering; (170.3660) Light propagation in tissues
Isotropic optical focusing – the focusing of light with axial confinement that matches its lateral confinement, is important for a broad range of applications. Conventionally, such focusing is achieved by overlapping the focused beams from a pair of opposite-facing microscope objective lenses. However the exacting requirements for the alignment of the objective lenses and the method’s relative intolerance to sample turbidity have significantly limited its utility. In this paper, we present an optical phase conjugation (OPC)-assisted isotropic focusing method that can address both challenges. We exploit the time-reversal nature of OPC playback to naturally guarantee the overlap of the two focused beams even when the objective lenses are significantly misaligned (up to 140 microns transversely and 80 microns axially demonstrated). The scattering correction capability of OPC also enabled us to accomplish isotropic focusing through thick scattering samples (demonstrated with samples of ~7 scattering mean free paths). This method can potentially improve 4Pi microscopy and 3D microstructure patterning.
(070.5040) Phase conjugation; (090.1995) Digital holography; (090.2880) Holographic interferometry; (110.7050) Turbid media; (220.1000) Aberration compensation; (220.1080) Active or adaptive optics
Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 μM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.
Vibrational spectroscopy has been widely applied in different fields due to its label-free chemical-sensing capability. Coherent anti-Stokes Raman scattering (CARS) provides stronger signal and faster acquisition than spontaneous Raman scattering, making it especially suitable for molecular imaging. Coherently-controlled single-beam CARS simplifies the conventional multi-beam setup, but the vibrational bandwidth and non-trivial spectrum retrieval have been limiting factors. In this work, a coherent supercontinuum generated in an all-normal-dispersion nonlinear fiber is phase-shaped within a narrow bandwidth for broadband vibrational spectroscopy. The Raman spectra can be directly retrieved from the CARS measurements, covering the fingerprint regime up to 1750 cm−1. The retrieved spectra of several chemical species agree with their spontaneous Raman data. The compact fiber supercontinuum source offers broad vibrational bandwidth with high stability and sufficient power, showing the potential for spectroscopic imaging in a wide range of applications.
(190.4370) Nonlinear optics, fibers; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering; (320.6629) Supercontinuum generation; (320.5540) Pulse shaping
Three-dimensional optical tomographic imaging plays an important role in biomedical research and clinical applications. We introduce spectral tomographic imaging (STI) via spectral encoding of spatial frequency principle that not only has the capability for visualizing the three-dimensional object at sub-micron resolution but also providing spatially-resolved quantitative characterization of its structure with nanoscale accuracy for any volume of interest within the object. The theoretical basis and the proof-of-concept numerical simulations are presented to demonstrate the feasibility of spectral tomographic imaging.
(170.6960) Tomography; (110.6955) Tomographic imaging; (110.1758) Computational imaging; (300.6300) Spectroscopy, Fourier transforms
In single particle spectroscopy, the degree of observed fluorescence anti-bunching in a second-order cross correlation experiment is indicative of its bi-exciton quantum yield and whether or not a particle is well isolated. Advances in quantum dot synthesis have produced single particles with bi-exciton quantum yields approaching unity. Consequently, this creates uncertainty as to whether a particle has a high bi-exciton quantum yield or if it exists as a cluster. We report on a time-gated anti-bunching technique capable of determining the relative contributions of both multi-exciton emission and clustering effects. In this way, we can now unambiguously determine if a particle is single. Additionally, this time-gated anti-bunching approach provides an accurate way for the determination of bi-exciton lifetime with minimal contribution from higher order multi-exciton states.
(300.0300) Spectroscopy; (300.2530) Fluorescence, laser-induced; (300.6500) Spectroscopy, time-resolved
Magnetomotive optical coherence tomography (MM-OCT) is a functional extension of OCT which utilizes magnetically responsive materials that are modulated by an external magnetic field for contrast enhancement and for elastography to assess the structural and viscoelastic properties of the surrounding tissues. Traditionally, magnetomotive contrast relies on the interaction between the displacement of magnetic particles induced by an external magnetic field and the micro-environmental restoring (elastic) force acting on the particles. When the restoring force from a sample containing magnetic particles is weak or non-existent, the MM-OCT signal-to-noise ratio (SNR) can degrade significantly. We have developed a novel solenoid configuration to enable MM-OCT imaging in samples that do not have an elastic restoring force, such as liquids. This coil configuration may potentially enable real-time MM-OCT imaging.
(110.4500) Optical coherence tomography; (170.4090) Modulation techniques; (160.4236) Nanomaterials; (230.3810) Magneto-optic systems
The unique superiority of transformation optics devices designed from coordinate transformation is their capability of recovering both ray trajectory and optical path length in light manipulation. However, very few experiments have been done so far to verify this dual-recovery property from viewpoints of both ray trajectory and optical path length simultaneously. The experimental difficulties arise from the fact that most previous optical transformation optics devices only work at the nano-scale; the lack of intercomparison between data from both optical path length and ray trajectory measurement in these experiments obscured the fact that the ray path was subject to a subwavelength lateral shift that was otherwise not easily perceivable and, instead, was pointed out theoretically [B. Zhang et al. Phys. Rev. Lett. 104, 233903, (2010)]. Here, we use a simple macroscopic transformation optics device of phase-preserved optical elevator, which is a typical birefringent optical phenomenon that can virtually lift an optical image by a macroscopic distance, to demonstrate decisively the unique optical path length preservation property of transformation optics. The recovery of ray trajectory is first determined with no lateral shift in the reflected ray. The phase preservation is then verified with incoherent white-light interferometry without ambiguity and phase unwrapping.
(160.2710) Inhomogeneous optical media; (260.2110) Electromagnetic optics; (290.5839) Scattering, invisibility; (120.3180) Interferometry
This paper presents a two-frequency binary phase-shifting technique to measure three-dimensional (3D) absolute shape of beating rabbit hearts. Due to the low contrast of the cardiac surface, the projector and the camera must remain focused, which poses challenges for any existing binary method where the measurement accuracy is low. To conquer this challenge, this paper proposes to utilize the optimal pulse width modulation (OPWM) technique to generate high-frequency fringe patterns, and the error-diffusion dithering technique to produce low-frequency fringe patterns. Furthermore, this paper will show that fringe patterns produced with blue light provide the best quality measurements compared to fringe patterns generated with red or green light; and the minimum data acquisition speed for high quality measurements is around 800 Hz for a rabbit heart beating at 180 beats per minute.
(120.0120) Instrumentation, measurement, and metrology; (110.6880) Three-dimensional image acquisition; (320.7100) Ultrafast measurements; (120.5050) Phase measurement
Confocal microscopy is an oft-used technique in biology. Deconvolution of 3D images reduces blurring from out-of-focus light and enables quantitative analyses, but existing software for deconvolution is slow and expensive. We present a parallelized software method that runs within ImageJ and deconvolves 3D images ~100 times faster than conventional software (few seconds per image) by running on a low-cost graphics processor board (GPU). We demonstrate the utility of this software by analyzing microclusters of T cell receptors in the immunological synapse of a CD4 + T cell and dendritic cell. This software provides a low-cost and rapid way to improve the accuracy of 3D microscopic images obtained by any method.
(180.2520) Fluorescence microscopy; (100.1830) Deconvolution