Three-dimensional super-resolution high-throughput imaging by structured illumination STED microscopy

Stimulated emission depletion (STED) microscopy is able to image fluorescence labeled samples with nanometer scale resolution. STED microscopy is typically a point-scanning method, limited by the high intensity requirement of the depletion beam. With the development of high peak power lasers, two dimensional parallel STED microscopy has been developed. Here, we develop the theoretical basis for extending STED microscopy to three dimensional imaging in parallel. This method uses structured illumination (SI) to generates a three dimensional depletion pattern. Compared to the two dimensional parallel STED microscopy, the 3D SI-STED microscopy generates intensity modulation along the light propagation direction without requiring higher laser power. This method not only achieves axial super-resolution of STED microscopy but also greatly reduces photobleaching and photodamage for 3D volumetric imaging.National Institutes of Health (U.S.) (NIH 1-U01-NS090438-01)National Institutes of Health (U.S.) (NIH 5-P41-EB015871)Hamamatsu Corporatio

Improving femtosecond laser pulse delivery through a hollow core photonic crystal fiber for temporally focused two-photon endomicroscopy

In this paper, we present a strategy to improve delivery of femtosecond laser pulses from a regenerative amplifier through a hollow core photonic crystal fiber for temporally focused wide-field two-photon endomicroscopy. For endomicroscope application, wide-field two-photon excitation has the advantage of requiring no scanning in the distal end. However, wide-field two-photon excitation requires peak power that is 10[superscript 4]–10[superscript 5] times higher than the point scanning approach corresponding to femtosecond pulses with energy on the order of 1–10 μJ at the specimen plane. The transmission of these high energy pulses through a single mode fiber into the microendoscope is a significant challenge. Two approaches were pursued to partially overcome this limitation. First, a single high energy pulse is split into a train of pulses with energy below the fiber damage threshold better utilizing the available laser energy. Second, stretching the pulse width in time by introducing negative dispersion was shown to have the dual benefit of reducing fiber damage probability and compensating for the positive group velocity dispersion induced by the fiber. With these strategy applied, 11 fold increase in the two photon excitation signal has been demonstrated.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (5R01EY017656-02)National Institutes of Health (U.S.) (5R01 NS051320)National Institutes of Health (U.S.) (4R44EB012415-02)National Science Foundation (U.S.) (CBET-0939511)Singapore-MIT AllianceSingapore-MIT Alliance for Research and TechnologySkolkovo Institute of Science and TechnologyHamamatsu CorporationDavid H. Koch Institute for Integrative Cancer Research at MIT. Bridge Project Initiativ

High-throughput three-dimensional lithographic microfabrication

A 3D lithographic microfabrication process has been developed that is high throughput, scalable, and capable of producing arbitrary patterns. It offers the possibility for industrial scale manufacturing of 3D microdevices such as photonic crystals, tissue engineering scaffolds, and microfluidics chips. This method is based on depth-resolved wide-field illumination by temporally focusing femtosecond light pulses. We characterized the axial resolution of this technique, and the result is consistent with the theoretical prediction. As proof-of-concept experiments, we demonstrated photobleaching of 3D resolved patterns in a fluorescent medium and fabricating 3D microstructures with SU-8 photoresist.Deshpande Center for Technological Innovation (Massachusetts Institute of Technology. School of Engineering)Singapore-MIT Alliance for Research and Technology (SMART

Quantifying the Dynamics of Bacterial Secondary Metabolites by Spectral Multiphoton Microscopy

Phenazines, a group of fluorescent small molecules produced by the bacterium Pseudomonas aeruginosa, play a role in maintaining cellular redox homeostasis. Phenazines have been challenging to study in vivo due to their redox activity, presence both intra- and extracellularly, and their diverse chemical properties. Here, we describe a noninvasive in vivo optical technique to monitor phenazine concentrations within bacterial cells using time-lapsed spectral multiphoton fluorescence microscopy. This technique enables simultaneous monitoring of multiple weakly fluorescent molecules (phenazines, siderophores, NAD(P)H) expressed by bacteria in culture. This work provides the first in vivo measurements of reduced phenazine concentration as well as the first description of the temporal dynamics of the phenazine-NAD(P)H redox system in Pseudomonas aeruginosa, illuminating an unanticipated role for 1-hydroxyphenazine. Similar approaches could be used to study the abundance and redox dynamics of a wide range of small molecules within bacteria, both as single cells and in communities

Regeneration of injured skin and peripheral nerves requires control of wound contraction, not scar formation

We review the mounting evidence that regeneration is induced in wounds in skin and peripheral nerves by a simple modification of the wound healing process. Here, the process of induced regeneration is compared to the other two well-known processes by which wounds close, i.e., contraction and scar formation. Direct evidence supports the hypothesis that the mechanical force of contraction (planar in skin wounds, circumferential in nerve wounds) is the driver guiding the orientation of assemblies of myofibroblasts (MFB) and collagen fibers during scar formation in untreated wounds. We conclude that scar formation depends critically on wound contraction and is, therefore, a healing process secondary to contraction. Wound contraction and regeneration did not coincide during healing in a number of experimental models of spontaneous (untreated) regeneration described in the literature. Furthermore, in other studies in which an efficient contraction-blocker, a collagen scaffold named dermis regeneration template (DRT), and variants of it, were grafted on skin wounds or peripheral nerve wounds, regeneration was systematically observed in the absence of contraction. We conclude that contraction and regeneration are mutually antagonistic processes. A dramatic change in the phenotype of MFB was observed when the contraction-blocking scaffold DRT was used to treat wounds in skin and peripheral nerves. The phenotype change was directly observed as drastic reduction in MFB density, dispersion of MFB assemblies and loss of alignment of the long MFB axes. These observations were explained by the evidence of a surface-biological interaction of MFB with the scaffold, specifically involving binding of MFB integrins α[subscript 1]β[subscript 1] and α[subscript 2]β[subscript 1] to ligands GFOGER and GLOGER naturally present on the surface of the collagen scaffold. In summary, we show that regeneration of wounded skin and peripheral nerves in the adult mammal can be induced simply by appropriate control of wound contraction, rather than of scar formation.National Institutes of Health (U.S.) (Grant RO1 NS051320)National Institutes of Health (U.S.) (Grant 5‐P41‐EB015871‐28)Horizon 2020 Framework Programme (European Commission) (Grant DLV‐658850)Singapore-MIT Alliance for Research and Technology (SMART) (Grant 5‐P41‐EB015871‐28)Hamamatsu Corporatio

Quantifying the surface chemistry of 3D matrices in situ

Despite the major role of the matrix (the insoluble environment around cells) in physiology and pathology, there are very few and limited methods that can quantify the surface chemistry of a 3D matrix such as a biomaterial or tissue ECM. This study describes a novel optical-based methodology that can quantify the surface chemistry (density of adhesion ligands for particular cell adhesion receptors) of a matrix in situ. The methodology utilizes fluorescent analogs (markers) of the receptor of interest and a series of binding assays, where the amount of bound markers on the matrix is quantified via spectral multi-photon imaging. The study provides preliminary results for the quantification of the ligands for the two major collagen-binding integrins (α[subscript 1]β[subscript 1], α[subscript 2]β[subscript 1]) in porous collagen scaffolds that have been shown to be able to induce maximum regeneration in transected peripheral nerves. The developed methodology opens the way for quantitative descriptions of the insoluble microenvironment of cells in physiology and pathology, and for integrating the matrix in quantitative models of cell signaling.National Institutes of Health (U.S.) (RO1 NS051320)Singapore-MIT Alliance for Research and Technolog

Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy

Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.National Institutes of Health (U.S.) (grant EY007023)National Institutes of Health (U.S.) (grant NS090473)National Institutes of Health (U.S.) (grant 4-P41-EB015871)National Science Foundation (U.S.) (grant EF1451125)Picower Institute for Learning and Memory (Engineering Collaboration Grant)Massachusetts Life Sciences Initiativ

Parallel super-resolution imaging

Massive parallelization of scanning-based super-resolution imaging allows fast imaging of large fields of view