Demands of multi-well plate readers has been on the rise for drug discovery and cell line development applications, as it can obtain fluorescence, absorbance, and morphology information from cell cultures grown in tens to hundreds of conditions. Existing systems typically only house one camera, requiring slow mechanical actuation to cover a large area on the multi-well plate, or to sacrifice speed for area, forgoing the precious spatial information. We mitigate the time-resolution trade off with the Fourier ptychographic microscopy (FPM) technology by simultaneously capturing 96 high-resolution phase images (>20,000 cells per plate) with CMOS-based cameras with custom-designed microscope objectives. By illuminating the samples with a permutation of lighting conditions, we achieve synthetic numerical aperture (NA) of 0.3 at an extended depth-of-field of 20 micrometer for at most 96 conditions at one time. Unlike our previous invention of the 6-well plate reader (EmSight), the same illumination condition can be shared among adjacent cameras. Therefore, image acquisition and data transfer can be performed in a massively parallel manner. Along with computational acceleration with graphical processing units (GPUs), all these approaches reduces the plate-to-image turnover from hours to minutes – an eight-fold reduction in time over existing mechanical-scanning plate readers. In addition to providing phase imaging, the system is also capable of fluorescence imaging at the native resolution of the objectives. We anticipate that our high-throughout 96-camera imaging system will help advance the high content analysis of cell cultures beyond hundreds of test conditions, thus facilitates more in-depth characterization of biological screens and drug testing.
Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes, imaging an ANSI standard 6-well plate at the same time. By using the same low magnification objective lenses (NA of 0.1) as the objective and the tube lens, the EmSight is configured as a 1:1 imaging system that, providing large field-of-view (FOV) imaging (5.7 mm × 4.3 mm) onto a low-cost CMOS imaging sensor. The EmSight improves the image resolution by capturing a series of images of the sample at varying illumination angles; the instrument reconstructs a higher-resolution image by using the iterative Fourier ptychographic algorithm. In addition to providing high-resolution brightfield and phase imaging, the EmSight is also capable of fluorescence imaging at the native resolution of the objectives. We characterized the system using a phase Siemens star target, and show four-fold improved coherent resolution (synthetic NA of 0.42) and a depth of field of 0.2 mm. To conduct live, long-term dopaminergic neuron imaging, we cultured ventral midbrain from mice driving eGFP from the tyrosine hydroxylase promoter. The EmSight system tracks movements of dopaminergic neurons over a 21 day period.
We present a method to acquire both fluorescence and high-resolution bright-field images with correction for the spatially varying aberrations over a microscope’s wide field-of-view (FOV). First, the procedure applies Fourier ptychographic microscopy (FPM) to retrieve the amplitude and phase of a sample, at a resolution that significantly exceeds the cutoff frequency of the microscope objective lens. At the same time, FPM algorithm is able to leverage on the redundancy within the set of acquired FPM bright-field images to estimate the microscope aberrations, which usually deteriorate in regions further away from the FOV’s center. Second, the procedure acquires a raw wide-FOV fluorescence image within the same setup. Lack of moving parts allows us to use the FPM-estimated aberration map to computationally correct for the aberrations in the fluorescence image through deconvolution. Overlaying the aberration-corrected fluorescence image on top of the high-resolution bright-field image can be done with accurate spatial correspondence. This can provide means to identifying fluorescent regions of interest within the context of the sample’s bright-field information. An experimental demonstration successfully improves the bright-field resolution of fixed, stained and fluorescently tagged HeLa cells by a factor of 4.9, and reduces the error caused by aberrations in a fluorescence image by 31%, over a field of view of 6.2 mm by 9.3 mm. For optimal deconvolution, we show the fluorescence image needs to have a signal-to-noise ratio of ~18.
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