Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Simultaneous multiplane imaging with reverberation two-photon microscopy.

Multiphoton microscopy has gained enormous popularity because of its unique capacity to provide high-resolution images from deep within scattering tissue. Here, we demonstrate video-rate multiplane imaging with two-photon microscopy by performing near-instantaneous axial scanning while maintaining three-dimensional micrometer-scale resolution. Our technique, termed reverberation microscopy, enables the monitoring of neuronal populations over large depth ranges and can be implemented as a simple add-on to a conventional design.

Interferometric speckle visibility spectroscopy (iSVS) for measuring decorrelation time and dynamics of moving samples with enhanced signal-to-noise ratio and relaxed reference requirements.

Diffusing wave spectroscopy (DWS) is a group of techniques used to measure the dynamics of a scattering medium in a non-invasive manner. DWS methods rely on detecting the speckle light field from the moving scattering medium and measuring the speckle decorrelation time to quantify the scattering medium's dynamics. For DWS, the signal-to-noise (SNR) is determined by the ratio between measured decorrelation time to the standard error of the measurement. This SNR is often low in certain applications because of high noise variances and low signal intensity, especially in biological applications with restricted exposure and emission levels. To address this photon-limited signal-to-noise ratio problem, we investigated, theoretically and experimentally, the SNR of an interferometric speckle visibility spectroscopy (iSVS) compared to more traditional DWS methods. We found that iSVS can provide excellent SNR performance through its ability to overcome camera noise. We also proved an iSVS system has more relaxed constraints on the reference beam properties. For an iSVS system to function properly, we only require the reference beam to exhibit local temporal stability, while incident angle, reference phase and intensity uniformity do not need to be constrained. This flexibility can potentially enable more unconventional iSVS implementation schemes.

Correcting sampling bias in speckle contrast imaging.

When performing spatial or temporal laser speckle contrast imaging (LSCI), contrast is generally estimated from localized windows containing limited numbers of independent speckle grains NS. This leads to a systematic bias in the estimated speckle contrast. We describe an approach to determine NS and largely correct for this bias, enabling a more accurate estimation of the speckle decorrelation time without recourse to numerical fitting of data. Validation experiments are presented where measurements are ergodic or non-ergodic, including in vivo imaging of mouse brain.

Flatfield ultrafast imaging with single-shot non-synchronous array photography.

We present a method for acquiring a sequence of time-resolved images in a single shot, called single-shot non-synchronous array photography (SNAP). In SNAP, a pulsed laser beam is split by a diffractive optical element into an array of angled beamlets whose illumination fronts remain perpendicular to the optical axis. Different time delays are imparted to each beamlet by an echelon, enabling them to probe ultrafast dynamics in rapid succession. The beamlets are imaged onto different regions of a camera by a lenslet array. Because the illumination fronts remain flat (head-on) independently of beamlet angle, the exposure time in SNAP is fundamentally limited only by the laser pulse duration, akin to a "global shutter" in conventional imaging. We demonstrate SNAP by capturing the evolution of a laser induced plasma filament over 20 frames at an average rate of 4.2 trillion frames per second (Tfps) and a peak rate of 5.7 Tfps.

Depth resolution in multifocus laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) can be used to evaluate blood flow based on spatial or temporal speckle statistics, but its accuracy is undermined by out-of-focus image blur. In this Letter, we show how the fraction of dynamic versus static light scattering is dependent on focus, and describe a deconvolution strategy to correct for out-of-focus blur. With the aid of a z-splitter, which enables instantaneous multifocus imaging, we demonstrate depth-resolved LSCI that can robustly extract multi-plane structural and flow-speed information simultaneously. This method is applied to in vivo imaging of blood vessels in a mouse cortex and provides improved estimates of blood flow speed throughout a depth range of 300µm.

Development of a beam propagation method to simulate the point spread function degradation in scattering media.

Scattering is one of the main issues that limit the imaging depth in deep tissue optical imaging. To characterize the role of scattering, we have developed a forward model based on the beam propagation method and established the link between the macroscopic optical properties of the media and the statistical parameters of the phase masks applied to the wavefront. Using this model, we have analyzed the degradation of the point-spread function of the illumination beam in the transition regime from ballistic to diffusive light transport. Our method provides a wave-optic simulation toolkit to analyze the effects of scattering on image quality degradation in scanning microscopy. Our open-source implementation is available at https://github.com/BUNPC/Beam-Propagation-Method.

Fast hyperspectral phase and amplitude imaging in scattering tissue.

Hyperspectral imaging in scattering tissue generally suffers from low light collection efficiency. In this Letter, we propose a microscope based on Fourier transform spectroscopy and oblique back-illumination microscopy that provides hyperspectral phase and amplitude images of thick, scattering samples with high throughput. Images can be acquired at >0.1 Hz rates with spectral resolution better than 200 cm-1, over a wide spectral range of 450-1700 nm. Proof-of-principle demonstrations are presented with chorioallantoic membrane of a chick embryo, illustrating the possibility of high-resolution hemodynamics imaging in thick tissue, based on transmission contrast.

Unified description of three-dimensional optical diffraction microscopy: from transmission microscopy to optical coherence tomography: tutorial.

In this tutorial, we present a general model linking the data provided by any optical diffraction microscope to the sample permittivity. Our analysis is applicable to essentially all microscope configurations, in transmission or reflection mode, using scanning or full-field illumination, with or without interferometric measurements. We include also a generalization of our analysis to vector fields.

Extended depth-of-field microscopy with a high-speed deformable mirror.

We present a wide-field fluorescence microscopy add-on that provides a fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror with an update rate of 20 kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.

Dual fluorescence-absorption deconvolution applied to extended-depth-of-field microscopy.

Fast imaging over large volumes can be obtained in a simple manner with extended-depth-of-field (EDOF) microscopy. A standard technique of Wiener deconvolution can correct for the blurring inherent in EDOF images. We compare Wiener deconvolution with an alternative, parameter-free technique based on the dual reconstruction of fluorescence and absorption layers in a sample. This alternative technique provides significantly enhanced reconstruction contrast owing to a quadratic positivity constraint that intrinsically favors sparse solutions. We demonstrate the advantages of this technique with mouse neuronal images acquired in vivo.

Phase-gradient microscopy in thick tissue with oblique back-illumination.

Phase-contrast techniques, such as differential interference contrast microscopy, are widely used to obtain morphological images of unstained biological samples. The transillumination geometry required for these techniques restricts their application to thin samples. We introduce oblique back-illumination microscopy, a method of collecting en face phase-gradient images of thick scattering samples, enabling near-video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.

Controlled light propagation through complex media introduction.

A sampling of papers is assembled to represent the active field of controlled light propagation through complex media.

Axial range of conjugate adaptive optics in two-photon microscopy: erratum.

We correct an omission from the Acknowledgments section of our manuscript.

Axial range of conjugate adaptive optics in two-photon microscopy.

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

High-resolution 3D phase imaging using a partitioned detection aperture: a wave-optic analysis.

Quantitative phase imaging has become a topic of considerable interest in the microscopy community. We have recently described one such technique based on the use of a partitioned detection aperture, which can be operated in a single shot with an extended source [Opt. Lett.37, 4062 (2012)OPLEDP0146-959210.1364/OL.37.004062]. We follow up on this work by providing a rigorous theory of our technique using paraxial wave optics, where we derive fully 3D spread functions for both phase and intensity. Using these functions, we discuss methods of phase reconstruction for in- and out-of-focus samples, insensitive to weak attenuations of light. Our approach provides a strategy for detection-limited lateral resolution with an extended depth of field and is applicable to imaging smooth and rough samples.

Field of view advantage of conjugate adaptive optics in microscopy applications.

The imaging performance of an optical microscope can be degraded by sample-induced aberrations. A general strategy to undo the effect of these aberrations is to apply wavefront correction with a deformable mirror (DM). In most cases the DM is placed conjugate to the microscope pupil, called pupil adaptive optics (AO). When the aberrations are spatially variant an alternative configuration involves placing the DM conjugate to the main source of aberrations, called conjugate AO. We provide a theoretical and experimental comparison of both configurations for the simplified case where spatially variant aberrations are produced by a well-defined phase screen. We pay particular attention to the resulting correction field of view (FOV). Conjugate AO is found to provide a significant FOV advantage. While this result is well known in the astronomical community, our goal here is to recast it specifically for the optical microscopy community.

Optical sectioning microscopy with planar or structured illumination.

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

Fast volumetric phase-gradient imaging in thick samples.

Oblique back-illumination microscopy (OBM) provides high resolution, sub-surface phase-gradient images from arbitrarily thick samples. We present an image formation theory for OBM and demonstrate that OBM lends itself to volumetric imaging because of its capacity for optical sectioning. In particular, OBM can provide extended depth of field (EDOF) images from single exposures, by rapidly scanning the focal plane with an electrically tunable lens. These EDOF images can be further enhanced by deconvolution. We corroborate our theory with experimental volumetric images obtained from transparent bead samples and mouse cortical brain slices.

HiLo microscopy with caustic illumination.

HiLo microscopy is an optical sectioning structured illumination microscopy technique based on computationally combining two images: one with uniform illumination and the other with structured illumination. The most widely used structured illumination in HiLo microscopy is random speckle patterns, due to their simplicity and resilience to tissue scattering. Here, we present a novel HiLo microscopy strategy based on random caustic patterns. Building on an off-the-shelf diffuser and a low-coherence LED source, we demonstrate that caustic HiLo can achieve 4.5 µm optical sectioning capability with a 20× 0.75 NA objective. In addition, with the distinct intensity statistical properties of caustic patterns, we show that our caustic HiLo outperforms speckle HiLo, achieving enhanced optical sectioning capability and preservation of fine features by imaging scattering fixed brain sections of 100 µm, 300 µm, and 500 µm thicknesses. We anticipate that this new structured illumination technique may find various biomedical imaging applications.