Traditional miniaturized fluorescence microscopes are critical tools for modern biology. Invariably, they struggle to simultaneously image with a high spatial resolution and a large field of view (FOV). Lensless microscopes offer a solution to this limitation. However, real-time visualization of samples is not possible with lensless imaging, as image reconstruction can take minutes to complete. This poses a challenge for usability, as real-time visualization is a crucial feature that assists users in identifying and locating the imaging target. The issue is particularly pronounced in lensless microscopes that operate at close imaging distances. Imaging at close distances requires shift-varying deconvolution to account for the variation of the point spread function (PSF) across the FOV. Here, we present a lensless microscope that achieves real-time image reconstruction by eliminating the use of an iterative reconstruction algorithm. The neural network-based reconstruction method we show here, achieves more than 10000 times increase in reconstruction speed compared to iterative reconstruction. The increased reconstruction speed allows us to visualize the results of our lensless microscope at more than 25 frames per second (fps), while achieving better than 7 µm resolution over a FOV of 10 mm2. This ability to reconstruct and visualize samples in real-time empowers a more user-friendly interaction with lensless microscopes. The users are able to use these microscopes much like they currently do with conventional microscopes.
Mesoscopic calcium imaging enables studies of cell-type specific neural activity over large areas. A growing body of literature suggests that neural activity can be different when animals are free to move compared to when they are restrained. Unfortunately, existing systems for imaging calcium dynamics over large areas in non-human primates (NHPs) are table-top devices that require restraint of the animal’s head. Here, we demonstrate an imaging device capable of imaging mesoscale calcium activity in a head-unrestrained male non-human primate. We successfully miniaturize our system by replacing lenses with an optical mask and computational algorithms. The resulting lensless microscope can fit comfortably on an NHP, allowing its head to move freely while imaging. We are able to measure orientation columns maps over a 20 mm2field-of-view in a head-unrestrained macaque. Our work establishes mesoscopic imaging using a lensless microscope as a powerful approach for studying neural activity under more naturalistic conditions.
more » « less- Award ID(s):
- 1730574
- NSF-PAR ID:
- 10490297
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 15
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The simple and compact optics of lensless microscopes and the associated computational algorithms allow for large fields of view and the refocusing of the captured images. However, existing lensless techniques cannot accurately reconstruct the typical low-contrast images of optically dense biological tissue. Here we show that lensless imaging of tissue in vivo can be achieved via an optical phase mask designed to create a point spread function consisting of high-contrast contours with a broad spectrum of spatial frequencies. We built a prototype lensless microscope incorporating the ‘contour’ phase mask and used it to image calcium dynamics in the cortex of live mice (over a field of view of about 16 mm 2 ) and in freely moving Hydra vulgaris , as well as microvasculature in the oral mucosa of volunteers. The low cost, small form factor and computational refocusing capability of in vivo lensless microscopy may open it up to clinical uses, especially for imaging difficult-to-reach areas of the body.more » « less
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Ultra-large mesoscopic imaging advances in the cortex open new pathways to develop neuroprosthetics to restore foveal vision in blind patients. Using targeted optogenetic activation, an optical prosthetic can focally stimulate spatially localized lateral geniculate nucleus (LGN) synaptic boutons within the primary visual cortex (V1). If we localize a cluster within a specific hypercolumn’s input layer, we will find that activation of a subset of these boutons is perceptually fungible with the activation of a different subset of boutons from the same hypercolumn input module. By transducing these LGN neurons with light-sensitive proteins, they are now sensitive to light and we can optogenetically stimulate them in a pattern mimicking naturalistic visual input. Optogenetic targeting of these purely glutamatergic inputs is free from unwanted co-activation of inhibitory neurons (a common problem in electrode-based prosthetic devices, which result in diminished contrast perception). We must prosthetically account for rapidly changing cortical activity and gain control, so our system integrates a real-time cortical read-out mechanism to continually assess and provide feedback to modify stimulation levels, just as the natural visual system does. We accomplish this by readingout a multi-colored array of genetically-encoded and transduced bioluminescent calcium responses in V1 neurons. This hyperspectral array of colors can achieve single-cell resolution. By tracking eye movements in the blind patients, we will account for oculomotor effects by adjusting the contemporaneous stimulation of the LGN boutons to mimic the effects of natural vision, including those from eye movements. This system, called the Optogenetic Brain System (OBServ), is designed to function by optimally activating visual responses in V1 from a fully-implantable coplanar emitter array coupled with a video camera and a bioluminescent read-out system. It follows that if we stimulate the LGN input modules in the same pattern as natural vision, the recipient should perceive naturalistic prosthetic vision. As such, the system holds the promise of restoring vision in the blind at the highest attainable acuity, with maximal contrast sensitivity, using an integrated nanophotonic implantable device that receives eye-tracked video input from a head-mounted video camera, using relatively non-invasive prosthetic technology that does not cross the pia mater of the brain.more » « less
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Abstract Objective . Intracortical microstimulation (ICMS) can be an effective method for restoring sensory perception in contemporary brain–machine interfaces. However, the mechanisms underlying better control of neuronal responses remain poorly understood, as well as the relationship between neuronal activity and other concomitant phenomena occurring around the stimulation site.Approach . Different microstimulation frequencies were investigatedin vivo on Thy1-GCaMP6s mice using widefield and two-photon imaging to evaluate the evoked excitatory neural responses across multiple spatial scales as well as the induced hemodynamic responses. Specifically, we quantified stimulation-induced neuronal activation and depression in the mouse visual cortex and measured hemodynamic oxyhemoglobin and deoxyhemoglobin signals using mesoscopic-scale widefield imaging.Main results . Our calcium imaging findings revealed a preference for lower-frequency stimulation in driving stronger neuronal activation. A depressive response following the neural activation preferred a slightly higher frequency stimulation compared to the activation. Hemodynamic signals exhibited a comparable spatial spread to neural calcium signals. Oxyhemoglobin concentration around the stimulation site remained elevated during the post-activation (depression) period. Somatic and neuropil calcium responses measured by two-photon microscopy showed similar dependence on stimulation parameters, although the magnitudes measured in soma was greater than in neuropil. Furthermore, higher-frequency stimulation induced a more pronounced activation in soma compared to neuropil, while depression was predominantly induced in soma irrespective of stimulation frequencies.Significance . These results suggest that the mechanism underlying depression differs from activation, requiring ample oxygen supply, and affecting neurons. Our findings provide a novel understanding of evoked excitatory neuronal activity induced by ICMS and offer insights into neuro-devices that utilize both activation and depression phenomena to achieve desired neural responses. -
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