Fast, volumetric live-cell imaging using high-resolution light-field microscopy

Abstract

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, neardiffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators

“…For this study, we used the original LFM design, which has since been improved upon significantly by multiple groups. [57][58][59][60][61] These advances variously improve the spatial resolution and decrease the computational cost of deconvolution; however, the basic image formation concept remains the same in all the new modalities. The results in this study are likely applicable to voltage imaging with these newer light-field modalities as the reconstruction approaches are similar.…”

“…We expect this effect to be small for the somatic ROIs we used, as the pixels external to the ROIs typically still contained scattered or dendritic voltage signals, and so the increased granularity of the wide-field ROIs would not provide a large SNR advantage. Furthermore, this disadvantage is greatly ameliorated in more recent LFM implementations, 57,59,61 which typically have better lateral resolution, especially at the LFM focal plane.…”

“…Newer light-field designs have significantly improved LFM performance and circumvented the degenerate sampling at the native focal plane. High-resolution LFM displaces the degenerate sampling away from the native focal plane, enabling higher resolution object reconstruction 57 . Cohen et al 58 removed the sampling degeneracy and improved the reconstruction via placing a phase mask in the objective back focal plane.…”

Artifact-free deconvolution in light field microscopy

“…Similar to us, in [2] the authors compute the wavefront at the MLA in a defocused LFM setup. They first model the PSF at the NIP as in [3,22] and then propagate the wavefront for a distance of ∆ MLA (the signed distance between the tube lens and the MLA plane) via Fresnel diffraction integral.…”

“…Since it was first proposed by Levoy et al in 2006 [1], light field microscopy has proven very useful in biological applications involving fast dynamics, due to its high speed 3D imaging capability. The potential of the modality has been show-cased in various applications, including live cell imaging [2], fish larvae neuro-dynamics recording [3,4], heart imaging and blood flow monitoring [5].…”