Tunable mode control through myriad-mode fibers.

Multimode fibers are attractive for imaging, communication, computation, and energy delivery. Unfortunately, intermodal and polarization coupling precludes direct control of the delivered mode composition. We present a technique to tailor the mode composition at the output of a multimode fiber with thousands of modes, which we refer to as myriad-mode fiber, using its experimentally measured transmission matrix. While precise mode control has been demonstrated in typical multimode fibers with up to 210 modes, the method proposed here is particularly useful for high mode number fibers, such as when the number of modes is comparable to the number of modes of the wavefront shaping spatial light modulator. To illustrate the technique, we select different subsets of modes to create focal spots at the output of a fiber with 7140 modes. Importantly, we define efficiency and fidelity metrics to evaluate the mode control and demonstrate the relationship between efficiency, fidelity, and the spatial location of the spots across the distal fiber cross-section.

Characterizing and correcting camera noise in back-illuminated sCMOS cameras.

With promising properties of fast imaging speed, large field-of-view, relative low cost and many others, back-illuminated sCMOS cameras have been receiving intensive attention for low light level imaging in the past several years. However, due to the pixel-to-pixel difference of camera noise (called noise non-uniformity) in sCMOS cameras, researchers may hesitate to use them in some application fields, and sometimes wonder whether they should optimize the noise non-uniformity of their sCMOS cameras before using them in a specific application scenario. In this paper, we systematically characterize the impact of different types of sCMOS noise on image quality and perform corrections to these types of sCMOS noise using three representative algorithms (PURE, NCS and MLEsCMOS). We verify that it is possible to use appropriate correction methods to push the non-uniformity of major types of camera noise, including readout noise, offset, and photon response, to a satisfactory level for conventional microscopy and single molecule localization microscopy. We further find out that, after these corrections, global read noise becomes a major concern that limits the imaging performance of back-illuminated sCMOS cameras. We believe this study provides new insights into the understanding of camera noise in back-illuminated sCMOS cameras, and also provides useful information for future development of this promising camera technology.

Azimuthal multiplexing 3D diffractive optics.

Diffractive optics have increasingly caught the attention of the scientific community. Classical diffractive optics are 2D diffractive optical elements (DOEs) and computer-generated holograms (CGHs), which modulate optical waves on a solitary transverse plane. However, potential capabilities are missed by the inherent two-dimensional nature of these devices. Previous work has demonstrated that extending the modulation from planar (2D) to volumetric (3D) enables new functionalities, such as generating space-variant functions, multiplexing in the spatial or spectral domain, or enhancing information capacity. Unfortunately, despite significant progress fueled by recent interest in metasurface diffraction, 3D diffractive optics still remains relatively unexplored. Here, we introduce the concept of azimuthal multiplexing. We propose, design, and demonstrate 3D diffractive optics showing this multiplexing effect. According to this new phenomenon, multiple pages of information are encoded and can be read out across independent channels by rotating one or more diffractive layers with respect to the others. We implement the concept with multilayer diffractive optical elements. An iterative projection optimization algorithm helps solve the inverse design problem. The experimental realization using photolithographically fabricated multilevel phase layers demonstrates the predicted performance. We discuss the limitations and potential of azimuthal multiplexing 3D diffractive optics.

Sub-acoustic resolution optical focusing through scattering using photoacoustic fluctuation guided wavefront shaping.

Focusing light through turbid media using wavefront shaping generally requires a noninvasive guide star to provide feedback on the focusing process. Here we report a photoacoustic guide star mechanism suitable for wavefront shaping through a scattering wall that is based on the fluctuations in the photoacoustic signals generated in a micro-vessel filled with flowing absorbers. The standard deviation of photoacoustic signals generated from random distributions of particles is dependent on the illumination volume and increases nonlinearly as the illumination volume is decreased. We harness this effect to guide wavefront shaping using the standard deviation of the photoacoustic response as the feedback signal. We further demonstrate sub-acoustic resolution optical focusing through a diffuser with a genetic algorithm optimization routine.

Spectral image scanning microscopy.

For decades, the confocal microscope has represented one of the dominant imaging systems in biomedical imaging at sub-cellular lengthscales. Recently, however, it has increasingly been replaced by a related, but more powerful successor technique termed image scanning microscopy (ISM). In this article, we present ISM capable of measuring spectroscopic information such as that contained in fluorescence or Raman images. Compared to established confocal spectroscopic imaging systems, our implementation offers similar spectral resolution, but higher spatial resolution and detection efficiency. Color sensitivity is achieved by a grating placed in the detection path in conjunction with a camera collecting both spatial and spectral information. The multidimensional data is processed using multi-view maximum likelihood image reconstruction. Our findings are supported by numerical simulations and experiments on micro beads and double-stained HeLa cells.

Two-photon PSF-engineered image scanning microscopy.

We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500 nm with an axial resolution of better than 400 nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.

Minimally invasive multimode optical fiber microendoscope for deep brain fluorescence imaging.

A major open challenge in neuroscience is the ability to measure and perturb neural activity in vivo from well defined neural sub-populations at cellular resolution anywhere in the brain. However, limitations posed by scattering and absorption prohibit non-invasive multi-photon approaches for deep (>2mm) structures, while gradient refractive index (GRIN) endoscopes are relatively thick and can cause significant damage upon insertion. Here, we present a novel micro-endoscope design to image neural activity at arbitrary depths via an ultra-thin multi-mode optical fiber (MMF) probe that has 5-10X thinner diameter than commercially available micro-endoscopes. We demonstrate micron-scale resolution, multi-spectral and volumetric imaging. In contrast to previous approaches, we show that this method has an improved acquisition speed that is sufficient to capture rapid neuronal dynamics in-vivo in rodents expressing a genetically encoded calcium indicator (GCaMP). Our results emphasize the potential of this technology in neuroscience applications and open up possibilities for cellular resolution imaging in previously unreachable brain regions.

Material anisotropy as a degree of freedom in optical design.

We present an approach for the design of refractive optical elements using materials degrees of freedom that are accessible via engineered materials. Starting from first principles and an unconstrained general material, we specify homogeneous refractive lenses that focus light with diffraction-limited resolution resulting from a tailored anisotropic refractive index. We analyze the performance, physical feasibility, and advantages over isotropic lenses. Materials degrees of freedom enable new flexibility for imaging system designs with lower complexity expanding the existing aspheric and graded index paradigms.

Thermal expansion feedback for wave-front shaping.

Focusing inside scattering media is a challenging task with a variety of applications in biomedicine. State of the art methods mostly require invasive feedback inside or behind the sample, limiting practical use. We present a technique for dynamic control and focusing inside scattering media that combines two powerful methods: optical coherence tomography (OCT) and wave-front shaping (WFS). We use OCT as a non-invasive feedback for WFS optimization of a separate, penetrating laser. Energy absorbed in the sample, creates thermal expansions that are used for the feedback mechanism. By maximizing thermal deformations within a selected focal region, we demonstrate enhanced focusing of light through scattering media beyond the ballistic regime and within the penetration range of OCT.

Single multimode fiber endoscope.

Multimode fibers can guide thousands of modes capable of delivering spatial information. Unfortunately, mode dispersion and coupling have so far prevented their use in endoscopic applications. To address this long-lasting challenge, we present a robust scanning fluorescence endoscope. A spatial light modulator shapes the input excitation wavefront to focus light on the distal tip of the fiber and to rapidly scan the focus over the region of interest. A detector array collects the fluorescence emission propagated back from the sample to the proximal tip of the fiber. We demonstrate that proper selection of the multimode fiber is critical for a robust calibration and for high signal-to-background ratio performance. We compare different types of multimode fibers and experimentally show that a focus created through a graded-index fiber can withstand a few millimeters of fiber distal tip translation. The resulting scanning endoscopic microscope images fluorescent samples over a field of view of 80µm with a resolution of 2µm.

Lock-in detection of photoacoustic feedback signal for focusing through scattering media using wave-front shaping.

Wave-front shaping techniques enable focusing and imaging through scattering media. Unfortunately, most approaches require invasive feedback inside or behind the sample, or use of spatial correlations (memory effect) limiting the application to specific types of samples. Recent approaches overcome these limitations by taking advantage of acoustic waves via the photoacoustic (PA) effect or via photon tagging. We present a fully analog signal processing lock-in scheme for PA detection to improve focusing through scattering media and to efficiently extract nonlinear photoacoustic signals towards wave-front optimization. Our implementation improves PA feedback performance in terms of SNR, speed, and resolution.

Single molecule image formation, reconstruction and processing: introduction.

The ability to image at the single molecule scale has revolutionized research in molecular biology. This feature issue presents a collection of articles that provides new insights into the fundamental limits of single molecule imaging and reports novel techniques for image formation and analysis.

Deconvolution approach for 3D scanning microscopy with helical phase engineering.

RESCH (refocusing after scanning using helical phase engineering) microscopy is a scanning technique using engineered point spread functions which provides volumetric information. We present a strategy for processing the collected raw data with a multi-view maximum likelihood deconvolution algorithm, which inherently comprises the resolution gain of pixel-reassignment microscopy. The method, which we term MD-RESCH (for multi-view deconvolved RESCH), achieves in our current implementation a 20% resolution advantage along all three axes compared to RESCH and confocal microscopy. Along the axial direction, the resolution is comparable to that of image scanning microscopy. However, because the method inherently reconstructs a volume from a single 2D scan, a significantly higher optical sectioning becomes directly visible to the user, which would otherwise require collecting multiple 2D scans taken at a series of axial positions. Further, we introduce the use of a single-helical detection PSF to obtain an increased post-acquisition refocusing range. We present data from numerical simulations as well as experiments to confirm the validity of our approach.

Real-time adaptive drift correction for super-resolution localization microscopy.

Super-resolution localization microscopy involves acquiring thousands of image frames of sparse collections of single molecules in the sample. The long acquisition time makes the imaging setup prone to drift, affecting accuracy and precision. Localization accuracy is generally improved by a posteriori drift correction. However, localization precision lost due to sample drifting out of focus cannot be recovered as the signal is originally detected at a lower peak signal. Here, we demonstrate a method of stabilizing a super-resolution localization microscope in three dimensions for extended periods of time with nanometer precision. Hence, no localization correction after the experiment is required to obtain super-resolved reconstructions. The method incorporates a closed-loop with a feedback signal generated from camera images and actuation on a 3D nanopositioning stage holding the sample.

Super-resolution photoacoustic imaging through a scattering wall.

The use of wavefront shaping to compensate for scattering has brought a renewed interest as a potential solution to imaging through scattering walls. A key to the practicality of any imaging through scattering technique is the capability to focus light without direct access behind the scattering wall. Here we address this problem using photoacoustic feedback for wavefront optimization. By combining the spatially non-uniform sensitivity of the ultrasound transducer to the generated photoacoustic waves with an evolutionary competition among optical modes, the speckle field develops a single, high intensity focus significantly smaller than the acoustic focus used for feedback. Notably, this method is not limited by the size of the absorber to form a sub-acoustic optical focus. We demonstrate imaging behind a scattering medium using two different imaging modalities with up to ten times improvement in signal-to-noise ratio and five to six times sub-acoustic resolution.

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.

Three-dimensional super-resolution and localization of dense clusters of single molecules.

When a single molecule is detected in a wide-field microscope, the image approximates the point spread function of the system. However, as the distribution of molecules becomes denser and their images begin to overlap, existing solutions to determine the number of molecules present and their precise three-dimensional locations can tolerate little to no overlap. We propose a localization scheme that can identify several overlapping molecule images while maintaining high localization precision. A solution to this problem involving matched optical and digital techniques, as here proposed, can substantially increase the allowable labeling density and accelerate the data collection time of single-molecule localization microscopy by more than one order of magnitude.

High contrast three-dimensional photoacoustic imaging through scattering media by localized optical fluence enhancement.

We demonstrate enhanced three-dimensional photoacoustic imaging behind a scattering material by increasing the fluence in the ultrasound transducer focus. We enhance the optical intensity using wavefront shaping before the scatterer. The photoacoustic signal induced by an object placed behind the scattering medium serves as feedback to optimize the wavefront, enabling one order of magnitude enhancement of the photoacoustic amplitude. Using the enhanced optical intensity, we scan the object in two-dimensions before post-processing of the data to reconstruct the image. The temporal profile of the photoacoustic signal provides the information used to reconstruct the third dimension.

Real-time resilient focusing through a bending multimode fiber.

Multimode optical fibers are attractive for biomedical and sensing applications because they possess a small cross section and can bend over small radii of curvature. However, mode phase-velocity dispersion and random mode coupling change with bending, temperature, and other perturbations, producing scrambling interference among propagating modes; hence preventing its use for focusing or imaging. To tackle this problem we introduce a system capable of re-focusing light through a multimode fiber in 37ms, one order of magnitude faster than demonstrated in previous reports. As a result, the focus spot can be maintained during significant bending of the fiber, opening numerous opportunities for endoscopic imaging and energy delivery applications. We measure the transmission matrix of the fiber by projecting binary-amplitude computer generated holograms using a digital micro-mirror device controlled by a field programmable gate array. The system shows two orders of magnitude enhancements of the focus spot relative to the background.