Jerome Mertz - US grants

DESCRIPTION (provided by applicant): The goal of this project is to develop a new type of nonlinear-optical microscope, and to establish its potential for thick-tissue imaging. The microscope uses a femtosecond-pulsed infrared laser beam to trans-illuminate the tissue sample. As opposed to other nonlinear-optical techniques such as two-photon excited fluorescence (TPEF) microscopy, or second-harmonic generation (SHG) microscopy, the laser beam does not generate signal within the sample. Instead, the laser beam traverses the sample completely and is then focused onto a nonlinear crystal. The detected signal is the SHG produced by the crystal. The contrast mechanism is as follows: the laser beam incurs spatially dependent phase fluctuations as it traverses the sample. These phase fluctuations lead to a defocusing of the beam on the crystal, which is monitored as a reduction in the SHG signal. Because the SHG is sensitive to defocusing, the crystal acts as a virtual self-aligned confocal pinhole, thereby leading to phase sensitivity and intrinsic 3d resolution. This technique, called auto-confocal microscopy (ACM), requires no labeling, is technically simple, and may be readily combined with existing nonlinear imaging modalities such as TPEF microscopy. The R21 Phase (1 yr) will consist in building an ACM, and quantifying its performance in terms of resolution, depth-penetration, etc., using simple samples such as latex beads (or cultured cells) in agarose (or collagen) gels. The ACM is expected to reveal local index-of-refraction fluctuations throughout a sample with high temporal resolution (microsecs), and as yet uncharacterized spatial resolution depending on the sample thickness. The R33 Phase (3 yrs) will involve vertebrate-animal tissue imaging. For thin enough samples (to be determined), an ACM should rapidly assess sample structure from local (micron) to global (millimeter) length scales. Our proposed project will apply such wide dynamic-range measurements to a variety of topics including brain-slice imaging, diseased tissue screening, and the monitoring of epithelial tissue dynamics, using simultaneous ACM and TPEF contrasts.

DESCRIPTION (provided by applicant): The aim of this project is to extend optical microscopy into the deep ultraviolet (DUV) wavelength range, and characterize DUV endogenous signals obtained from unlabeled biological tissue. We define DUV wavelengths as ranging from 230 nm to 350nm. This range is rarely investigated in microscopy because DUV light does not transmit through ordinary glass, and hence cannot be observed with standard microscope optics. Our basic hypothesis is that DUV optical microscopy can reveal clinically relevant information on cells and tissue structures that is normally unobservable. Our proposed technique will be based on the use of a nonlinear microscope to produce DUV three-photon excited autofluorescence (3PEF) and third-harmonic generation (3HG). These signals are intrinsic and do not require sample labeling. We will exploit the advantages of nonlinear microscopy over standard (linear) microscopy for accessing the DUV spectral window, and will develop a novel light collection technique specifically designed for detection in the backward direction, thereby allowing DUV imaging in thick tissues. In addition, we will integrate our nonlinear microscope with a custom designed spectrograph to allow both high-resolution imaging and spectral analysis. As far as we know, DUV imaging (as defined here) in a thick tissue with a nonlinear microscope has never been performed before. Our goal in this project is to demonstrate sub-cellular resolution DUV imaging in unlabelled brain and cancer tissue, and to demonstrate the unambiguous identification of molecular species in the DUV spectral range using recently developed techniques of spectral decomposition. Ultimately, we hope to confirm our hypothesis that intrinsic optical signals in the DUV spectral window, which has largely been overlooked so far, can provide useful and clinically relevant information.

[unreadable] DESCRIPTION (provided by applicant): Our aim is to develop of a new type of imaging "macroscope" that performs ultra-deep (several millimeters) fluorescence imaging in thick tissue while providing out-of-focus background rejection. Our proposed method for out-of-focus blur rejection is based on a novel technique called Dynamic Speckle Illumination (DSI) microscopy that was recently invented in our lab. Our goal is to build a portable DSI macroscope and establish its potential for in-vivo molecular-imaging applications such as cancer research and diagnosis, as well as small animal imaging and surgery. DSI microscopy consists of a simple modification to a standard widefield fluorescence microscope. Sample illumination is performed with random laser speckle patterns rather than with a lamp. The main advantage of DSI microscopy is that it provides depth discrimination and out-of-focus blur reduction in thick tissues without the use of a complicated scanning mechanism. We have developed the full theory of DSI microscopy and already demonstrated sub-micron-resolution imaging with confocal-like background rejection of GFP-labeled neurons, down to about 100[unreadable]m in mice brain. We propose to develop a new instrument that performs much deeper imaging but with lower resolution. Our targeted depth is several millimeters, down to perhaps a centimeter. To attain this goal we propose to significantly re-design of our DSI microscope to incorporate a long working-distance telecentric objective, near-infrared laser illumination, and a more sensitive CCD camera. We also plan to combine DSI with structured illumination contrast and multipsectral imaging (for the latter, we will work with Cambridge Research & Instrumentation). The defining advantage of our DSI macroscope compared to conventional commercially available macroscopes will be that DSI provides out-of-focus background rejection, enabling significantly better localization and visualization of labeled structures within thick tissue. Initial testing of our DSI prototype will be performed on tissue phantoms provided by a radiology lab. Widefield fluorescence macroscopy with out-of-focus blur rejection. Public health: We propose to develop a simple and inexpensive device that performs ultra-deep fluorescence imaging with out-of-focus blur rejection, for small animal cancer-model imaging. Our goal is to build a new tool for cancer research that can eventually be implemented in the clinic. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by the applicant): Novel Techniques in Microscopy (NTM) is a new conference that will be launched on April 25-29 under the sponsorship of the Optical Society of America (OSA). The reason for this conference stems from a need, perceived by both the OSA and the biomedical optics community, for a conference that specifically focuses on the development of optical imaging techniques as opposed to applications. Particular emphasis will be placed on techniques designed to ultimately provide novel or improved modalities for biological or biomedical imaging. As such, this conference will be in an area of direct relevance to the National Institutes of Health, specifically NIBIB. The goal of this conference will be to facilitate interactions between inventors in the field, researchers and students, and industrial participants. Since this conference is new, the targeted number of participants will be modest, less than a hundred. If all goes well, it is hoped that it will henceforth be offered biennially. The main aims of this conference are: Aim 1: To identify and bring together the most promising technologies and scientists in microscopic imaging. Aim 2: To enable presentation and understanding of the key state-of-the-art methods and technologies. Aim 3: To promote interaction between the invited scientists and participating students, and to enable collaborations. Aim 4: To establish and strengthen relationships with industry participants for the development of new technologies. The purpose of this proposal to request funds to support young investigators and graduate students and postdoctoral fellows to attend this meeting.

ABSTRACT The goal of this project is to bring a new optical imaging technology to the biomedical community, and establish it as a simple, versatile and robust alternative to confocal microscopy. Our technique is called HiLo imaging, and was invented by our lab in the summer of 2008. HiLo imaging allows confocal-like out-of-focus fluorescence background rejection without the use of a confocal scanning mechanism. In brief, it requires the acquisition of two images, one with structured illumination and another with standard uniform illumination. HiLo imaging works with both fluorescent and non-fluorescent samples. Since it is based on conventional widefield imaging, it can readily be adapted to standard microscopes. Moreover, since it requires only two images, it can be operated at very high acquisition rates that easily surpass video rate (to be demonstrated). This project is focused mostly on instrumentation development. Having already established the basic principles of HiLo microscopy, we plan to experimentally quantify key performance parameters, including axial resolution, signal to noise ratio and depth penetration. To demonstrate the versatility of HiLo imaging and its broad range of applicability, we plan to confirm the effectiveness of HiLo imaging in situations where confocal microscopy is impractical or impossible. Specifically, we plan to build both a HiLo endomicroscope and a HiLo macroscope. The former will be used to perform intravital confocal-like imaging in rat colons, in collaboration with the Dr. Satish Singh group at the Boston University School of Medicine Gastroenterology Department, with the goal of demonstrating the effectiveness of HiLo endomicroscopy for optical biopsy and cancer diagnosis. Our second device, a HiLo macroscope, will exploit the versatility of HiLo imaging by providing what confocal microscopy cannot, namely video-rate, out-of-focus background rejection over a very wide field of view (about 1cm) and with a long working distance (several cms). The applications of this macroscope will be in molecular imaging of small animals, and we propose to demonstrate its effectiveness at characterizing abnormal vasculature in mouse tumor models, in collaboration with the Dr. Rakesh Jain group at the Massachusetts General Hospital Department of Radiation Oncology. HiLo imaging is a new technology. Our goal is to firmly establish the advantages of this technology so that it will become routine instrumentation in biomedical imaging.

DESCRIPTION (provided by applicant): The imaging of absorbing (i.e. nonfluorescent) chromophores in thick tissue poses a challenge for microscopists. Recently, a new technique called photothermal microscopy (PM) has been gaining attention, which involves the focusing two laser beams into a sample. One beam (the heating beam) is tuned to a chromophore absorption line while the other is set at wavelength outside any absorption bands (the probe beam). When the heating beam is absorbed, energy is deposited into the tissue, producing a local density fluctuation. This density change results in a small transient change in the refractive index about the chromophore, which is then monitored by the probe beam. In early applications, PM has been shown to track gold nanoparticles in cell cultures with unprecedented sensitivity. More recently, PM has also been shown to be remarkably effective at imaging endogenous chromophores in live cells. Examples include imaging of mitochondria and erythrocytes with 3D spatial resolution comparable to confocal microscopy. As promising as PM is, several open questions still remain. Specifically: is it possible to perform PM in thick tissue, and what exactly are the chromophore species responsible for PM contrast? To date, PM has only been performed with thin samples in a transmitted light configuration. Moreover, the chromophore species responsible for PM contrast are either unknown (in the case of mitochondrial imaging) or speculated (in the case of erythrocyte imaging). We propose to 1) develop a novel scanning PM that can perform photothermal imaging in thick tissue, for the first time, using on one- or two-photon absorption, and 2) unambiguously identify and characterize both existing and new endogenous contrast agents using a photothermal spectroscopy screening platform equipped with an ultra-wide bandwidth (UV to THz) laser. We will initially concentrate on the study of cytochrome in mitochondria, heme protein in blood cells, and channel rhodopsin. A completion of the above aims will be indispensible for PM to gain widespread acceptance in the biomedical imaging community, and will lay the groundwork for a new technology that provides high sensitivity, high resolution absorption contrast with molecular specificity. PUBLIC HEALTH RELEVANCE: We propose to develop an optical microscopy technique that provides ultrahigh sensitivity 3D imaging of absorbing (i.e. non-fluorescent) proteins or molecules in tissue. This technology will be useful for in- vivo imaging research applications and rapid tissue diagnosis in the clinic.

DESCRIPTION (provided by applicant): Fluorescence resonance energy transfer (FRET) can serve as a nanoscale molecular ruler. When used in imaging applications, it is a highly sensitive reporter of donor-acceptor molecular configuration. In most cases, FRET is utilized with standard one-photon excitation. Extensions of FRET to two-photon excitation are generally hampered by the problem of donor and acceptor bleed through, which imposes the requirement of technically complicated spectral unmixing algorithms or fluorescence lifetime measurements. We propose to considerably simplify the detection of FRET with two-photon excitation. Our goal is to establish the feasibility of modulating FRET signal using ultrasonic waves. Since FRET is sensitive to distance between donor-acceptor pairs at the nanoscale, mechanical compression and tension produced by sound waves are likely to modulate the FRET signal, serving as a useful signature to distinguish FRET from donor and acceptor bleed-through, which is a particular problem in multiphoton FRET. In particular, we propose to conduct preliminary experiments to establish the premise that ultrasound modulates FRET. We will investigate two types of samples: genetically encoded FRET probes in tissue (or tissue-like environments), and labeled microbubbles in solution. In the former case, we will investigate the application of our technique to in-vivo imaging in C.Elegans. FRET is a powerful tool for functional imaging. We believe that our hybrid ultrasound-optical technique will significantly improve the isolation of FRET signals and therefore be of benefit to any FRET-based functional imaging application. Moreover, when coupled with two-photon excitation, as proposed here, our technique should be well adapted to thick tissue imaging, thereby benefiting the in-vivo imaging community. PUBLIC HEALTH RELEVANCE: Fluorescence resonance energy transfer (FRET) is a powerful research technique to image cellular function in tissue. We propose to improve FRET signal generation with the use of ultrasound. This will benefit any FRET-based imaging applications, and will be specifically adapted to in-vivo microscopy in thick tissue.

DESCRIPTION (provided by applicant): Phase contrast microscopy is one of the most widely used techniques for biological imaging because it provides exquisite high-resolution images of sample morphology, without the use of sample labeling. However standard phase contrast techniques, like differential interference contrast (DIC), only work in the transmission direction and thus cannot be used when imaging thick samples. For this reason, standard phase contrast techniques have not made a great impact in in-vivo biomedical or clinical imaging. We have developed a new phase contrast technique, called Oblique Back-illumination Microscopy (OBM), that works in a reflected light configuration, and is thus amenable to in-vivo endomicroscopy applications. OBM requires no labeling and provides high resolution DIC-like images of sub-surface sample morphology. As far as we know, DIC-like imaging in an epi-detection configuration has never been demonstrated before. Our goal in this project is to demonstrate that OBM can enable clinicians to perform histopathology and tissue diagnosis in situ, without the need for a physical biopsy. We will limit our study to the evaluation of OBM endomicroscopy for preclinical cancer diagnosis. In particular, we will concentrate on colorectal imaging in rodents, in-vivo and ex-vivo. Small animal imaging with endomicroscopes presents technical challenges related to probe miniaturization that are more restrictive than human imaging. Nevertheless, we believe such imaging is a necessary first step that must be taken prior to clinical evaluations. Accordingly, our specific aims are to 1) construct a miniaturized OBM endomicroscope probe suitable for in-vivo colorectal imaging in rats, 2) perform longitudinal in-vivo imaging of a chemical rat cancer model, and 3) perform ex-vivo imaging of a genetic mouse cancer model to compare with standard H&E histopathology. A difficulty with the development of a new technique is that, while we have already established that OBM produces high resolution images in thick tissue, we do not yet have a roadmap for what role these images can play in clinical applications (precisely because no-one has been able to perform such imaging before). Our goal for this project is to establish this role by correlating OBM imaging results with the known chronology of cancer in well characterized animal models, thus enabling us to define classification criteria associated with this new technology. Ultimately, we hope this preclinical study will lead toward the development of a simple, safe, versatile, low-cost endomicroscopy technique that can be operated in conjunction with standard endoscopy for in-situ colorectal cancer diagnosis in the clinic.

? DESCRIPTION (provided by applicant): With the continuing expansion of optogenetic tools, the central importance of optical measurement of neural activity has and will continue to grow. However, large scale optical approaches, such as pursued by the BRAIN Initiative, must overcome high light scattering in brain tissue. Moreover, optical approaches should be feasible in behaving animals, including freely moving mice. We will develop an ultra-miniature microendoscope, termed a needle optrode, made with a bare fiber bundle beveled to a fine tip. The smaller size and beveling improves tissue penetration and lowers tissue damage compared to existing technology, and arranges the field of view for imaging across layered structures such as neocortex. The core project contribution is achieving miniaturization through a lensless design, using an innovative coupling of array detector to enable fluorescence background rejection and remote focusing. We will demonstrate the power of our approach for collecting large scale, multiregion data by recording simultaneously from aligned cortical and thalamic regions of the mouse somatosensory system, in anesthetized mice. Completion of these aims will prepare us to record simultaneously throughout an entire thalamocortical whisker processing network, including both feedforward and feedback projections, and be a first step towards performing these recordings in an awake animal actively engaging objects of interest in a tactile task. Moreover, our approach --- providing multiregion, cellular resolution recording of genetically identified cell types, with possible extension to behaving animals --- will support similar experiments across brain regions and systems, as one of the high priority components of the BRAIN initiative.

? DESCRIPTION (provided by applicant): Microscope techniques to image inside brain tissue are generally limited by poor depth penetration. Micro-endoscopy, wherein a probe is physically inserted into the tissue, can overcome this limitation in depth penetration, but at the expense of invasiveness and tissue damage due to the size of the probe. Our goal here is to palliate these problems by developing an ultra-miniature microendoscope probe based on a single, lensless optical fiber. The direct transmission of an image through an optical ?ber is di?cult because spatial information becomes scrambled upon propagation. We have recently demonstrated an image transmission strategy where spatial information is ?rst converted to spectral information. Our strategy is based on a principle of spread-spectrum encoding, borrowed from wireless communications, wherein object pixels are converted into distinct spectral codes that span the full bandwidth of the object spectrum. Image recovery is performed by numerical inversion of the detected spectrum at the ?ber output. We have provided a simple demonstration of spread-spectrum encoding using macroscopic Fabry-Perot etalons. Our technique enables the 2D imaging of luminous (i.e. fluorescent or bioluminescent) objects with high throughput independent of pixel number. Moreover, it is insensitive to ?ber bending, contains no moving parts, and opens the attractive possibility of extreme miniaturization down to the size of a single optical fiber. Our goal here is to develop, characterize, and establish the versatility of a new class of ultra-miniature fiber probes that can provide functional 2D brain imaging at arbitrary depths and with minimal tissue damage. Our strategy will involve probe development, machine-learning algorithm development, and the actual demonstration of microendoscopic imaging in freely moving behaving animals.

PI: Mertz, Jerome
Proposal: 1508988

The objective of this proposal is to build an ultraminiaturized imaging device that can be used to image at arbitrary depths within tissue, and to reach confined regions in samples that are difficult to access.

A key feature of the device is that it will be able to focus to variable depths, even though it is lensless in design and contains no moving parts. The device can be used with any type of luminous object, such as fluorescence, bioluminescence or even white-light illuminated scenes. The device is based on an invention the PI recently patented that enables high-throughput imaging through a single optical fiber. This uses the principle of a spread-spectrum encoder (SSE), wherein light-ray directions entering a fiber are converted into unique, broadband, fingerprint spectral codes that then propagate through the fiber to be detected and decoded at the other end. Because image information (i.e. ray directions) is converted into spectral information, this information becomes insensitive to fiber bending or motion, so that the technique can readily be used for microendoscopy applications.

ABSTRACT Multiphoton microscopy is one of the preferred techniques for high-resolution functional brain imaging because of its remarkable depth penetration in thick tissue. In standard configurations, such imaging involves scanning a femtosecond laser focus in 3D throughout a sample. The laser power is fixed during the scan and image information is contained in the time dependence of the detected fluorescence signal. Several problems can occur with this technique. First, in common cases where the sample contains extreme variations in brightness, for example between large somas and much finer dendritic processes, it is often impossible to capture the full range of signals without either saturating the detector when scanning over bright regions, or losing signal when scanning over dim regions. Second, when imaging time-varying signals from functional reporters such as GCaMP, large brightness variations occur that cannot be predicted in advance, forcing the user to use a low illumination to minimize the possibility of detector saturation, thus potentially compromising SNR. Third, when performing volumetric scans through an extended range of depths, a single laser power becomes either too weak at large depths or too strong at shallow depths. We propose a simple solution to solve all these problems. The solution involves actively regulating the laser power pixel-by-pixel using feedback electronics. We have demonstrated that our technique can improve the dynamic range of two-photon microscopes by several orders of magnitude for moderately fast pixel times of 20?s, achieving an unprecedentedly high dynamic range (HDR) of 1011:1. Our goals for this project are the: 1) Development of ultrafast feedback electronics for video-rate HDR imaging. 2) Development of switched multiplexing technique for large-scale multi-region HDR imaging. 3) Application of multiphoton HDR imaging to anatomical and functional mouse brain imaging. Our goal is to enable comprehensive large-scale multiphoton imaging with unprecedented dynamic range in a simple manner that can be readily implementable by many labs at reasonable cost and with minimal hardware modifications.

ABSTRACT Human retinal imaging is conventionally performed with a fundus camera, which sends light into the subject?s eye and records images of the light reflected from the retina. Our goal is to develop an alternative imaging strategy based on light delivered transcranially through the subject?s temple. The light diffuses through the bone and illuminates the retina not from the front, as in a standard fundus camera, but rather mostly from the back. As such, we image light transmitted through the retina rather than reflected from the retina. In addition to being glare-free and allowing deeper image penetration well into the choroid, we hypothesize that transmitted-light imaging reveals qualitatively different tissue structure than reflected-light imaging. Our strategy will be compatible with existing fundus cameras, enabling transmission and reflection modalities to be operated quasi-simultaneously, allowing a direct comparison of the two modalities and a potential fusion of the information they provide. Specifically, we propose to equip our device with eight different LED illumination wavelengths, any two of which can be operated simultaneously, from which we propose to identify a variety of chromophore distributions such as oxy- and deoxy-hemoglobin and melanin. We hypothesize that such chromophore mapping over extended depths will provide valuable information useful for the potential diagnosis and study of macular degeneration, and for the real-time monitoring of hemodynamics. To gauge the performance of our device and its potential utility in the clinic, we have enlisted the help of Dr. Elias Reichel at the Tufts Medical Center, who has clinical experience with both fundus and OCT imaging.

The purpose of flow cytometers is to enable the classification of cells or organisms at high throughput. Label-free optical flow cytometers not based on fluorescence are generally based on scattering. The most common of these compares the amount of forward (FS) versus side (SS) scattering. Such two-parameter information permits rudimentary classification based on size or granularity, but it misses more subtle features that can be critical in defining organism identity. Nevertheless, FS/SS flow cytometry remains popular, largely because of its simplicity and capacity for high throughput. We propose to develop a label-free computational flow cytometer that preserves much of the simplicity and high-throughput capacity of FS/SS flow cytometry, but provides significantly enhanced information. Instead of characterizing organisms based on scattering direction (as does FS/SS flow cytometry), we will characterize based on scattering patterns. We will insert a reconfigurable diffractive element in the imaging optics of a flow cytometer to route user-defined basis patterns to independent detectors. The basis patterns will be optimally matched to specific sample features. The respective weights of these basis patterns will serve as signatures to identify organisms of interest. The basis patterns themselves will be determined by machine learning algorithms. Both the device and the learning algorithms will be developed from scratch. We anticipate that our flow cytometer will be able to operate at flow rates on the order of meters per second, commensurate with state-of-the-art FS/SS flow cytometers, while providing significantly more information for improved classification capacity. While our technique should be advantageous for any label-free flow cytometry application requiring high throughput, we will test it here by demonstrating high-throughput classification of microbial communities.

This is a proposal from twelve investigators from the College of Engineering, the College of Arts and Sciences and Sargent College at Boston University requesting purchase of a replacement laser scanning confocal microscope. The instrument they?ve been using for the last 12 years has reached its end of service lifetime. Acquisition of the new system will allow users to continue their NIH funded research programs that depend on confocal imaging based experiments. With the addition of motorized XY position control, high speed and high precision scanning with hybrid/resonance scanning system and sensitive GaAsP photodetectors, data throughput will increase and interval time between samples will decrease allowing experiments not possible with the previous instrument. The users for the proposed instrument have a diversity of research foci. Their general aspiration in requesting the new instrument is to localize molecules, cells and tissues in 3 dimensions using fluorescence molecules and backscattered light. Specifically, we have users imaging: lab created artificial tissues, cell and tissue level mechanics and response to mechanical stimulation, novel tools to study brain function, unique imaging methods and probes, and intracellular signal transduction cascades. These approaches have been productive for these investigators in the past and the continuing availability of these techniques are necessary for continued progress on their NIH funded work. In this proposal, we describe the instrument we aim to acquire, the benefits of this particular instrument over others, provide a management plan for instrument use and cost recovery, and illustrate the institutions commitment to maintaining this resource. Overall the acquisition of this laser scanning confocal microscope will support projects focused on improving diverse aspect of human health and the treatment of disease.

ABSTRACT Ultrasound (US) imaging is one of the most common methods of medical imaging, and has had tremendous impact in the practice and delivery of healthcare. Advantages of ultrasound imaging are that it is non-invasive, cost-effective, and provides images with penetration depths commensurate with human organ imaging. In this last regard, US imaging has a considerable advantage over optical imaging techniques, which are hampered by very poor depth penetration in comparison. This proposal rests on the fact that there is a close analogy between optical imaging and US imaging. We have recently developed a new optical microscopy technique called Oblique Back-illumination Microscopy (OBM) that provides DIC-like phase contrast in arbitrarily thick tissue. While OBM is a remarkably simple method to obtain fast, high resolution, label-free imaging of tissue structure, it is limited in depth penetration to about 100µm. Such limited depth penetration restricts the applicability of OBM to superficial imaging of epithelial tissue only. Motivated by the close analogy between optical and US imaging, we propose to extend the concept of OBM directly to acoustics, enabling the possibility of what we believe to be an entirely new modality of US imaging, called Oblique Backscattering Ultrasound (OBUS) imaging. Specifically, OBUS imaging is unusual in that it is based on the detection of transmitted rather than reflected sound, even though it is configured in a reflection geometry. As such, it can operate in arbitrarily thick tissue, thus differing from previous transmission US imaging techniques. Because it is based on phase contrast, OBUS imaging reveals fundamentally different sample features than standard echography. Moreover, OBUS imaging is speckle-free, which has been a long- standing challenge in US imaging. We propose to improve OBUS by combining it with a technique called Differential Aberration Imaging, enabling it to work with the same samples and with the same hardware as a conventional echographic US imaging. Information from both contrast modalities will be obtained simultaneously, and combined to enable augmented sample reconstruction. Our goal will be to lay the necessary groundwork for the future development of this new technology, which we believe represents a paradigm shift in US imaging that may have significant clinical impact.

ABSTRACT Neuronal signals can vary on millisecond timescales, with communicating neurons often separated by hundreds of microns. Imaging such fast dynamics over extended volumes presents a challenge for standard fluorescence microscopes. For example, a new generation of genetically encoded voltage indicators are becoming available whose response times are on the order of milliseconds. To address this challenge, we propose to develop a new type of microscope that can perform near- 1kHz-rate high resolution volumetric imaging over 1mm x 1mm x 0.2mm scales. Our proposed solution, called Multi-Z confocal microscopy, is based on two key ideas. First, it combines high-NA detection with low-NA illumination. The former leads to high signal collection efficiency; the latter leads to axially extended illumination over an extended range of Z depths. Second, it detects multiple signals from this extended depth range using multiple confocal pinholes that are axially distributed. The pinholes are reflecting, so that signal rejected by one pinhole is sent to the next pinhole, and so forth. In this manner, no signal is lost, and signal collection efficiency remains high. Two versions of our microscope will be developed, based on line-scan and sheet-scan illumination. The former provides better optical sectioning and will be designed for calcium imaging. The latter provides much higher speed (near kHz-rate) and will be designed for voltage imaging. In contrast to conventional line-scan or light-sheet microscopes, our lines and sheets are oriented parallel to the optical axis rather than perpendicular to the axis. The versatility of both versions of our microscope will be augmented with the addition of optogenetic stimulation and combined confocal reflectance contrast. We have enlisted the help of Drs. Alberto Cruz-Martin (BU, Biology) and Xue Han (BU, BME), who both specialize in in-vivo mouse imaging and have expertise in the genetic or viral delivery of novel probes, animal preparation, head fixation, behavior protocols, etc.. For voltage imaging, we will test a state-of-the-art indicator called SomArchon1 (provided by the Dr. Ed Boyden lab). The ability to image large sample volumes at 1kHz rates is of general applicability and can be broadly impactful. Our goal will be to demonstrate the effectiveness of our Multi-Z microscopy technique by performing calcium and voltage imaging over entire populations of neurons in behaving mice.

ABSTRACT Many brain areas, such as neocortex and olfactory bulb, are vertically organized into layers containing distinct cell types that show different activity profiles and project to different downstream targets. Fast, volumetric imaging is thus indispensable to capture the dynamics of such neuronal populations within their stratified environments. While multiphoton microscopy (MPM) has become the gold standard for high resolution imaging from deep within brain tissue, it is generally restricted to 2D planar imaging. We propose to develop a technique to perform volumetric MPM where a long-range z-stack is acquired by near-instantaneous axial scanning, while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, enables the monitoring of neuronal populations over large scales, including the depth scale, with no speed penalty compared to conventional MPM. Reverberation MPM is a new technique which we have demonstrated only recently with proof of principle two-photon experiments. Much of our proposal will be focused on further developing this tool and characterizing its performance. Moreover, we propose to extend our technique to three-photon microscopy, for increased depth penetration. Our goal is to perform comprehensive 3D-resolved imaging of neuronal populations within volumes up to 1×1×1mm3, spanning the entire thickness of the mouse cortex. A key advantage of reverberation MPM is its extreme simplicity. It requires only the addition of a reverberation loop to a conventional MPM equipped with fast detection electronics. Moreover, it allows the acquisition of an arbitrary number of planes without increasing setup complexity. Other advantages are that our system is light efficient and easily compatible with video-rate scanning, making it ideal for volumetric calcium imaging using genetically encoded calcium indicators. These advantages make reverberation MPM particularly attractive as a general tool for fast, high resolution, large-scale volumetric imaging in brain tissue.