ChIP-seq Flow cytometry MRI-based neuroimaging Eukaryotic cell lines Policy information about cell lines Cell line source(s) Jurkat cells for nanoparticle uptake measurement cells gift from D Yablonski. More information in Methods section. AuthenticationThe Jurkat cells used were authenticated to be those from which the Jurkat Clone E6.1 was originated (Cellosaurus no. CVCL_0367). Authentication was performed using the Promega GenePrint 24 System in order to determine short tandem repeat (STR) profile of 23 loci plus Amelogenin for gender determination (X or XY) and analyzed using the 3500xl Genetic Analyzer (Life Technologies) and GeneMapper IDX software. Mycoplasma contaminationCell lines tested were confirmed not to contain mycoplasma contamination. Commonly misidentified lines (See ICLAC register)No commonly misidentified cell lines were used.

Three-dimensional spatiotemporal tracking of microscopic particles in multiple colors is a challenging optical imaging task. Existing approaches require a trade-off between photon efficiency, field of view, mechanical complexity, spectral specificity, and speed. Here, we introduce multiplexed point-spread-function engineering that achieves photon-efficient, 3D multicolor particle tracking over a large field of view. This is accomplished by first chromatically splitting the emission path of a microscope to different channels, engineering the point-spread function of each, and then recombining them onto the same region of the camera. We demonstrate our technique for simultaneously tracking five types of emitters in vitro as well as colocalization of DNA loci in live yeast cells.

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Imaging flow cytometry replaces the canonical point-source detector of flow cytometry with a camera, unveiling subsample details in 2D images while maintaining high-throughput. Here we show that the technique is inherently compatible with 3D localization microscopy by point-spread-function engineering, namely the encoding of emitter depth in the emission pattern captured by a camera. By exploiting the laminar-flow profile in microfluidics, 3D positions can be extracted from cells or other objects of interest by calibrating the depth-dependent response of the imaging system using fluorescent microspheres mixed with the sample buffer. We demonstrate this approach for measuring fluorescently-labeled DNA in vitro and the chromosomal compaction state in large populations of live cells, collecting thousands of samples each minute. Furthermore, our approach is fully compatible with existing commercial apparatus, and can extend the imaging volume of the device, enabling faster flowrates thereby increasing throughput. RESULTS Device and functionalityAn illustration of the device is shown in Figure 1 A. Samples are loaded into the instrument and then directed through a multilaser-illuminated imaging volume. Emission light from the sample is captured by a 60X objective, relayed through a series of lenses, divided into separate spectral channels, and directed onto a camera, whose readout rate is synchronized with the flow speed. As a first demonstration, we show how an astigmatism can be incorporated into the system. By inserting a cylindrical lens between two of the aforementioned relay lenses (see Methods), the axial and lateral focal planes of the instrument are bifurcated, thus an object closer to the objective will appear focused laterally, but defocused axially and vice versa (Figures 1 B, C). The extent of the astigmatism contains information about the depth of the emitter, but is not a direct measurement of the z position. The typical 3D calibration used in microscopy (Huang et al., 2008) is therefore inapplicable and the incorporation of PSF engineering into flow imaging necessitates a novel 3D calibration procedure, which we describe below.