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, 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 spatio-temporal levels.
Highlights
Light-field microscopy (LFM) simultaneously captures both the 2D spatial and 2D angular information of the incident light, allowing computational reconstruction of the full 3D volume of a specimen from a single camera frame [1,2,3,4]
HeLa cells were obtained from the American Type Culture Collection (ATCC), maintained in 1 × Minimum Essential Medium (MEM) (Corning CellGro) with 10% fetal bovine serum (FBS) (Atlanta Biologicals) and 50 μg/ml gentamycin (Amresco), and incubated at 37°C with 5% CO2
The cells were plated on a 35 mm2 MatTek glass-bottom dishes (MatTek), incubated at 37°C for 16 hours, and fixed with 4% formaldehyde (15735, Electron Microscopy Sciences) prepared in phosphate buffered saline (PBS) for 10 mins at 37°C
Summary
Light-field microscopy (LFM) simultaneously captures both the 2D spatial and 2D angular information of the incident light, allowing computational reconstruction of the full 3D volume of a specimen from a single camera frame [1,2,3,4]. Fluorescent imaging techniques acquire 3D spatial information in a sequential or scanning fashion [5,6,7,8,9,10,11,12], inevitably compromising temporal resolution and increasing photodamage for live imaging. The 4D imaging scheme of LFM effectively liberates volume acquisition time (limited primarily by the camera’s frame rate) from the spatial parameters (e.g. the field of view (FOV) and spatial resolution), making LFM a promising tool for high-speed, volumetric imaging of living biological systems with low photodamage across many spatial levels. The challenge remains for current LFM to achieve subcellular level, near-diffraction-limited 3D spatial resolution
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