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 subcellular level, near-diffraction-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 spatiotemporal levels. Fig. 13. Imaging Golgi-derived membrane vesicles in living COS-7 cells at a volume acquisition time of 5 ms using HR-LFM. (a) Reconstructed 3D images of vesicles in living COS-7 cells labeled with mEmerald-Golgi-7 acquired at a volume acquisition time of 5 ms. The left and right are reconstructed 3D images at t = 0.005s and 5.000s of a 1000-time-point series, respectively. The depth-information is color-coded according to the color scale bar. (b) Zoomedin images of the corresponding boxed region in (a) at t = 0.005s, 3.000s, 3.500s, 4.000s, 4.500s and 5.000s, respectively. White arrows indicate vesicles moving during time-points. (c) Zoomed-in image (leftmost) of the boxed region in (b) at t = 0.005s and its z-stack images from z = 0.2 µm to +1.3 µm at an axial step size of 100 nm, resolving several nearby vesicles. (d) Zoomed-in images of the corresponding boxed region in (a) at t = 0.005s, 0.500s, 1.000s, 1.500s, 2.000s, 2.500s, 3.000s and 3.500s, respectively. Scale bars: 10 µm (a), 1 µm (b-d).

3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

“…Both the lateral and axial resolutions reach the diffraction limit around the native object plane (250 nm and 320 nm, respectively) and maintain similar performance over a large axial distance (~10 μm). The slight super-resolution capability in the axial domain close to the focal plane is mainly due to the higher angular sensitivity and deconvolution process, which is identical to the previous study with even smaller angular resolution 38 . In addition, we tested the influence of different overlap ratios in the spatial domain and found that a similar performance can be achieved with only 67% spatial overlap, corresponding to 3×3 lateral shifts (Extended Data Fig.…”

Comparing synthetic refocusing to deconvolution for the extraction of neuronal calcium transients from light-fields

“…Methods to increase speed without the need to use high performance computing are desirable. Reconstruction speed has been improved by a number of groups [14,18,37,38].…”

Capturing volumetric dynamics at high speed in the brain by confocal light field microscopy

“…Light field microscopy (LFM) completely parallelizes the fluorescence collection and could capture information over an entire volume simultaneously in a single camera frame. Therefore, it is potentially the most favorable scheme for high speed three-dimensional (3D) imaging of fast dynamics in large biological tissues [23][24][25][26] . However, conventional LFMs lack optical sectioning capability and could not provide optimal spatial resolution and signal to noise ratio (SNR) when imaging into large brains.…”