Extended depth of field imaging for high speed cell analysis

Ortyn, William E.; Perry, David J.; Venkatachalam, V.; Liang, Luchuan; Hall, Brian E.; Frost, Keith; Basiji, David

doi:10.1002/cyto.a.20370

Cited by 83 publications

(76 citation statements)

References 16 publications

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“…32 The EDF version of the ImageStream system incorporates a specialized optical element in the standard optical system that causes light from widely different focal positions in the object to be imaged on the detector plane simultaneously in a process referred to as Wavefront Coding™ by its developer, CDM Optics, Inc. (Boulder, CO). 33 The modified imagery is post-processed to recover image sharpness while preserving the increased depth of focus that comes from the modification of the wavefront during data acquisition.…”

Section: High Throughput Extended Depth Of Field Imaging Of Cells Submentioning

confidence: 99%

Cellular Image Analysis and Imaging by Flow Cytometry

Basiji

Ortyn

Liang

et al. 2007

Clinics in Laboratory Medicine

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369

288

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SynopsisImaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high throughput quantitation of FISH probes in human peripheral blood mononuclear cells. The FISH application will be further enhanced by the integration of extended depth of field imaging technology with the current optical system. Keywordsimaging; flow; cytometry; fluorescence; brightfield; darkfield; multispectral Quantifying Cellular Structure in Health and DiseaseThe eukaryotic cell is a highly structured, three-dimensional object containing a wide range of molecular species. The size, shape, and structure of the cell, as well as the abundance, location, and co-location of any of these constituent biomolecules may be of significance in any given clinical situation or research application. For instance, in hematopoiesis, as cells differentiate and mature, different subsets of molecules are expressed that reflect a specialized functional capacity for that unique cell type (e.g., granulocytes vs. lymphocytes). In general the characterization of this array of constituent molecules by imaging or flow cytometry provides insight into the physiological function of any particular cell or alternatively, pathological changes that may have occurred or accrued. In clinical practice and in research settings, cellular evaluation by imaging technologies and flow cytometry provides significant information reflecting the particular cellular phenotype, both normal and pathological. Microscopy provides a wealth of information, but data acquisition rates are slow and analysis is generally subjective. In flow cytometry, data acquisition is rapid and better suited for the evaluation of pathologies present in low frequency, but the data are only intensity-based, thus lacking the morphology that truly lends credence to the analysis.In addition, the assessment and evaluation of cell samples by imaging and flow cytometric techniques is complicated by a number of factors. For instance, changes in a cell type or aCorresponding author for proofs and reprints:

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Section: High Throughput Extended Depth Of Field Imaging Of Cells Submentioning

confidence: 99%

Cellular Image Analysis and Imaging by Flow Cytometry

Basiji

Ortyn

Liang

et al. 2007

Clinics in Laboratory Medicine

Self Cite

369

288

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“…Single-plane images were analyzed, as opposed to images obtained from an extended depth of field (EDF) (as described by Ortyn et al 2007;Hassman et al 2011), because the use of EDF is best suited for analysis of cells with limited numbers of punctate foci each with distinct x-and y-coordinates. If, instead, signals in each cell are either too numerous such that they share x-and y-coordinates and are separated only in the z-axis (depth of cell) and/or are dimensionally large, EDF compression results in the merging of signals.…”

Section: Data Collection and Analysismentioning

confidence: 99%

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

Borah¹,

Nichols²,

Hassman³

et al. 2012

RNA

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We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.

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“…Carles presented an analytical approach to determine the optimal CPM strength, and Liu made the stationary phase analysis of CPM. However, most optimized CPMs in microscopy systems are only applied to one power micro-objective [13]. That means micro-objectives with different powers each need a specific CPM, which is not practical and economical.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Wavefront Coding Phase Mask Applied to 5X-40X Micro-Objectives Simultaneously

Liu

Miao

Sui

et al. 2015

Journal of the Optical Society of Korea

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A wavefront coding (WFC) technique provides an extension of the depth of field for a microscopy imaging system with slight loss of image spatial resolution. Through the analysis of the relationship between the incidence angle of light at the phase mask and the system pupil function, a mixing symmetrical cubic phase mask (CPM) applied to 5X-40X micro-objectives is optimized simultaneously based on point-spread function (PSF) invariance and nonzero mean values of the modulation transfer function (MTF) near the spatial cut-off frequency. Optimization results of the CPM show that the depth of field of these micro-objectives is extended 3-10 times respectively while keeping their resolution. Further imaging simulations also prove its ability in enhancing the defocus imaging.

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Extended depth of field imaging for high speed cell analysis

Cited by 83 publications

References 16 publications

Cellular Image Analysis and Imaging by Flow Cytometry

Cellular Image Analysis and Imaging by Flow Cytometry

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

Optimization of Wavefront Coding Phase Mask Applied to 5X-40X Micro-Objectives Simultaneously

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