Computational illumination for high-speed in vitro Fourier ptychographic microscopy

Abstract

We demonstrate a new computational illumination technique that achieves large space-bandwidth-time product, for quantitative phase imaging of unstained live samples in vitro. Microscope lenses can have either large field of view (FOV) or high resolution, not both. Fourier ptychographic microscopy (FPM) is a new computational imaging technique that circumvents this limit by fusing information from multiple images taken with different illumination angles. The result is a gigapixelscale image having both wide FOV and high resolution, i.e. large space-bandwidth product (SBP). FPM has enormous potential for revolutionizing microscopy and has already found application in digital pathology. However, it suffers from long acquisition times (on the order of minutes), limiting throughput. Faster capture times would not only improve imaging speed, but also allow studies of live samples, where motion artifacts degrade results. In contrast to fixed (e.g. pathology) slides, live samples are continuously evolving at various spatial and temporal scales. Here, we present a new source coding scheme, along with real-time hardware control, to achieve 0.8 NA resolution across a 4× FOV with sub-second capture times. We propose an improved algorithm and new initialization scheme, which allow robust phase reconstruction over long time-lapse experiments. We present the first FPM results for both growing and confluent in vitro cell cultures, capturing videos of subcellular dynamical phenomena in popular cell lines undergoing division and migration. Our method opens up FPM to applications with live samples, for observing rare events in both space and time.

Incubator embedded cell culture imaging system (EmSight) based on Fourier ptychographic microscopy

“…We note that we captured wells sequentially in our neuron imaging experiment because the time between each imaging sequence (1 hr) was long enough to capture each well one at a time. For a simultaneous imaging from 6 wells, we can upgrade imaging cameras to cameras with high dynamic range and apply the multiple LED illumination method from [29,39] in order to boost capturing speed for a frame data set.…”

“…Recently, live cell imaging via FPM has been reported [29]. In that paper, the authors implemented a high speed FPM imaging system (greater than 1 frame per second) that is capable of tracking fast dynamic movement of cells during in-vitro culture.…”

“…This work is distinct in that the EmSight system is designed to be sufficiently compact to be housed in an incubator, cost-effective and is capable of imaging several wells in a culture well plate over a long timeline (a few weeks). Both the Emsight system and the system in [29] are designed for cell culture experiments. However, they are each designed to fit the needs of different cell culture experimental formats.…”

“…However, they are each designed to fit the needs of different cell culture experimental formats. EmSight is designed for streaming a lot of culture FPM data associated with multiple cultures from an incubator; while the system in [29] is focused on very fast imaging of a single cell culture.…”

Ptychographic microscopy via wavelength scanning

“…Using this concept, Fourier Ptychography Microscopy (FPM) has been developed recently. 21,22 Despite the advantage of large field of view, high spatial bandwidth product, FPM is also limited to the assumption of fully coherent illumination, which is not practical for LED array. And the algorithm of FPM assumes that the imaging system has same optical transfer function for different incident angles, so the inaccurate determination of phase slope of LED light could affect the final resolution.…”