High sampling rate single-pixel digital holography system employing a DMD and phase-encoded patterns.

A single-pixel digital holography system with phase-encoded illumination using a digital micromirror device (DMD) as a spatial light modulator (SLM) is presented. The enhanced switching rate of DMDs, far exceeding the stringent frame-rate of liquid crystal SLMs, allows recording and reconstruction of complex amplitude distributions in just a few seconds. A single amplitude binary modulation device is used for concurrently displaying the phase-encoded sampling patterns, compensating the distortion of the wavefront, and applying phase-shifting, by means of computer generated holograms. Our detection system consists of a simple photodiode that sequentially records the irradiance fluctuations corresponding to the interference between object and reference beams. The system recovers phase and amplitude information even when a diffuser is placed in front of the photodiode.

Image transmission through dynamic scattering media by single-pixel photodetection.

Smart control of light propagation through highly scattering media is a much desired goal with major technological implications. Since interaction of light with highly scattering media results in partial or complete depletion of ballistic photons, it is in principle impossible to transmit images through distances longer than the extinction length. Nevertheless, different methods for image transmission, focusing, and imaging through scattering media by means of wavefront control have been published over the past few years. In this paper we show that single-pixel optical systems, based on compressive detection, can also overcome the fundamental limitation imposed by multiple scattering to successfully transmit information. But, in contrast with the recently introduced schemes that use the transmission matrix technique, our approach does not require any a-priori calibration process that ultimately makes the present method suitable to use with dynamic scattering media. This represents an advantage over previous methods that rely on optical feedback wavefront control, especially for short speckle decorrelation times.

Speckle-enabled in vivo demixing of neural activity in the mouse brain.

Functional imaging of neuronal activity in awake animals, using a combination of fluorescent reporters of neuronal activity and various types of microscopy modalities, has become an indispensable tool in neuroscience. While various imaging modalities based on one-photon (1P) excitation and parallel (camera-based) acquisition have been successfully used for imaging more transparent samples, when imaging mammalian brain tissue, due to their scattering properties, two-photon (2P) microscopy systems are necessary. In 2P microscopy, the longer excitation wavelengths reduce the amount of scattering while the diffraction-limited 3D localization of excitation largely eliminates out-of-focus fluorescence. However, this comes at the cost of time-consuming serial scanning of the excitation spot and more complex and expensive instrumentation. Thus, functional 1P imaging modalities that can be used beyond the most transparent specimen are highly desirable. Here, we transform light scattering from an obstacle into a tool. We use speckles with their unique patterns and contrast, formed when fluorescence from individual neurons propagates through rodent cortical tissue, to encode neuronal activity. Spatiotemporal demixing of these patterns then enables functional recording of neuronal activity from a group of discriminable sources. For the first time, we provide an experimental, in vivo characterization of speckle generation, speckle imaging and speckle-assisted demixing of neuronal activity signals in the scattering mammalian brain tissue. We found that despite an initial fast speckle decorrelation, substantial correlation was maintained over minute-long timescales that contributed to our ability to demix temporal activity traces in the mouse brain in vivo. Informed by in vivo quantifications of speckle patterns from single and multiple neurons excited using 2P scanning excitation, we recorded and demixed activity from several sources excited using 1P oblique illumination. In our proof-of-principle experiments, we demonstrate in vivo speckle-assisted demixing of functional signals from groups of sources in a depth range of 220-320 µm in mouse cortex, limited by available speckle contrast. Our results serve as a basis for designing an in vivo functional speckle imaging modality and for maximizing the key resource in any such modality, the speckle contrast. We anticipate that our results will provide critical quantitative guidance to the community for designing techniques that overcome light scattering as a fundamental limitation in bioimaging.

Demixing fluorescence time traces transmitted by multimode fibers.

Optical methods based on thin multimode fibers (MMFs) are promising tools for measuring neuronal activity in deep brain regions of freely moving mice thanks to their small diameter. However, current methods are limited: while fiber photometry provides only ensemble activity, imaging techniques using of long multimode fibers are very sensitive to bending and have not been applied to unrestrained rodents yet. Here, we demonstrate the fundamentals of a new approach using a short MMF coupled to a miniscope. In proof-of-principle in vitro experiments, we disentangled spatio-temporal fluorescence signals from multiple fluorescent sources transmitted by a thin (200 µm) and short (8 mm) MMF, using a general unconstrained non-negative matrix factorization algorithm directly on the raw video data. Furthermore, we show that low-cost open-source miniscopes have sufficient sensitivity to image the same fluorescence patterns seen in our proof-of-principle experiment, suggesting a new avenue for novel minimally invasive deep brain studies using multimode fibers in freely behaving mice.

Large field-of-view non-invasive imaging through scattering layers using fluctuating random illumination.

Non-invasive optical imaging techniques are essential diagnostic tools in many fields. Although various recent methods have been proposed to utilize and control light in multiple scattering media, non-invasive optical imaging through and inside scattering layers across a large field of view remains elusive due to the physical limits set by the optical memory effect, especially without wavefront shaping techniques. Here, we demonstrate an approach that enables non-invasive fluorescence imaging behind scattering layers with field-of-views extending well beyond the optical memory effect. The method consists in demixing the speckle patterns emitted by a fluorescent object under variable unknown random illumination, using matrix factorization and a novel fingerprint-based reconstruction. Experimental validation shows the efficiency and robustness of the method with various fluorescent samples, covering a field of view up to three times the optical memory effect range. Our non-invasive imaging technique is simple, neither requires a spatial light modulator nor a guide star, and can be generalized to a wide range of incoherent contrast mechanisms and illumination schemes.