One of the major challenges in large scale optical imaging of neuronal activity is to simultaneously achieve sufficient temporal and spatial resolution across a large volume. Here, we introduce sparse decomposition light-field microscopy (SDLFM), a computational imaging technique based on light-field microscopy (LFM) that takes algorithmic advantage of the high temporal resolution of LFM and the inherent temporal sparsity of spikes to improve effective spatial resolution and signal-to-noise ratios (SNRs). With increased effective spatial resolution and SNRs, neuronal activity at the single-cell level can be recovered over a large volume. We demonstrate the single-cell imaging capability of SDLFM with
Light-field fluorescence microscopy can record large-scale population activity of neurons expressing genetically-encoded fluorescent indicators within volumes of tissue. Conventional light-field microscopy (LFM) suffers from poor lateral resolution when using wide-field illumination. Here, we demonstrate a structured-illumination light-field microscopy (SI-LFM) modality that enhances spatial resolution over the imaging volume. This modality increases resolution by illuminating sample volume with grating patterns that are invariant over the axial direction. The size of the SI-LFM point-spread-function (PSF) was approximately half the size of the conventional LFM PSF when imaging fluorescent beads. SI-LFM also resolved fine spatial features in lens tissue samples and fixed mouse retina samples. Finally, SI-LFM reported neural activity with approximately three times the signal-to-noise ratio of conventional LFM when imaging live zebrafish expressing a genetically encoded calcium sensor.
more » « less- Award ID(s):
- 1847540
- NSF-PAR ID:
- 10247487
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 12
- Issue:
- 7
- ISSN:
- 2156-7085
- Page Range / eLocation ID:
- Article No. 3887
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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in vivo imaging of neuronal activity of whole brains of larval zebrafish with estimated lateral and axial resolutions ofand , respectively, acquired at volumetric imaging rates up to 50 Hz. We also show that SDLFM increases the quality of neural imaging in adult fruit flies. -
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Summary Since its invention 29 years ago, two‐photon laser‐scanning microscopy has evolved from a promising imaging technique, to an established widely available imaging modality used throughout the biomedical research community. The establishment of two‐photon microscopy as the preferred method for imaging fluorescently labelled cells and structures in living animals can be attributed to the biophysical mechanism by which the generation of fluorescence is accomplished. The use of powerful lasers capable of delivering infrared light pulses within femtosecond intervals, facilitates the nonlinear excitation of fluorescent molecules only at the focal plane and determines by objective lens position. This offers numerous benefits for studies of biological samples at high spatial and temporal resolutions with limited photo‐damage and superior tissue penetration. Indeed, these attributes have established two‐photon microscopy as the ideal method for live‐animal imaging in several areas of biology and have led to a whole new field of study dedicated to imaging biological phenomena in intact tissues and living organisms. However, despite its appealing features, two‐photon intravital microscopy is inherently limited by tissue motion from heartbeat, respiratory cycles, peristalsis, muscle/vascular tone and physiological functions that change tissue geometry. Because these movements impede temporal and spatial resolution, they must be properly addressed to harness the full potential of two‐photon intravital microscopy and enable accurate data analysis and interpretation. In addition, the sources and features of these motion artefacts are varied, sometimes unpredictable and unique to specific organs and multiple complex strategies have previously been devised to address them. This review will discuss these motion artefacts requirement and technical solutions for their correction and after intravital two‐photon microscopy.
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Abstract Implantable image sensors have the potential to revolutionize neuroscience. Due to their small form factor requirements; however, conventional filters and optics cannot be implemented. These limitations obstruct high-resolution imaging of large neural densities. Recent advances in angle-sensitive image sensors and single-photon avalanche diodes have provided a path toward ultrathin lens-less fluorescence imaging, enabling plenoptic sensing by extending sensing capabilities to include photon arrival time and incident angle, thereby providing the opportunity for separability of fluorescence point sources within the context of light-field microscopy (LFM). However, the addition of spectral sensitivity to angle-sensitive LFM reduces imager resolution because each wavelength requires a separate pixel subset. Here, we present a 1024-pixel, 50 µm thick implantable shank-based neural imager with color-filter-grating-based angle-sensitive pixels. This angular-spectral sensitive front end combines a metal–insulator–metal (MIM) Fabry–Perot color filter and diffractive optics to produce the measurement of orthogonal light-field information from two distinct colors within a single photodetector. The result is the ability to add independent color sensing to LFM while doubling the effective pixel density. The implantable imager combines angular-spectral and temporal information to demix and localize multispectral fluorescent targets. In this initial prototype, this is demonstrated with 45 μm diameter fluorescently labeled beads in scattering medium. Fluorescent lifetime imaging is exploited to further aid source separation, in addition to detecting pH through lifetime changes in fluorescent dyes. While these initial fluorescent targets are considerably brighter than fluorescently labeled neurons, further improvements will allow the application of these techniques to in-vivo multifluorescent structural and functional neural imaging.more » « less
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Background Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.more » « less
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