SBIR-STTR Award

Fluorescence-activated-cell-sorting (FACS) or flow cytometry enables clinicians and researchers to quantitatively characterize the physical (cell size, shape, and granularity) and biochemical (DNA content, cell cycle distribution, cell surface markers, and viability) properties of cells, however FACS devices do not produce an image of the cell. Increasing sophistication of research assays now rely on the collection of cells based on their phenotypical and spatial characteristics; with the capabilities offered only by microscopic imaging cytometers having severe limits in throughput and lacking cell isolation. We propose an innovative, low cost design to combine the merits of FACS and microscopic imaging cytometry without the limits of each, offering the biomedical research and clinical community a unique tool to address the needs for current and emerging applications. The key innovation is based on a significant extension of the spatial-coding algorithms our team demonstrated in the past years. In the proposed design, we create a special filter with a matrix of periodic slits in front of each PMT detector. The resulting PMT signal is composed of the multiplexed cell signals modulated by the filter, which can subsequently be deconvolved to produce fluorescence and scatter generated from different areas of the cell: the image. In this Phase I program, we will integrate imaging technology into our existing WOLF Cell Sorter to produce the very first imaging cytometer with cell sorting capabilities. Since we use conventional, non-pixelated detectors (e.g. PMTs) found in conventional flow cytometers, this technology is compatible with existing flow cytometer architectures allowing for wide use. Equipped with cell imaging capabilities, researchers can track many important biological processes by analyzing not only the intensity but the localization of certain proteins within cytosolic, nuclear, or cell membrane domains and subdomains. With the rapidly developing capabilities of handling "big data", images of millions of single cells in a flow cytometer provide vast resources for research and disease analysis, and rapid growth has been predicted in the market of such high-content imaging cytometer cell sorters for emerging applications such as precision medicine. We believe the proposed design is a major breakthrough that can potentially revolutionize the field of flow cytometry, and its impact and ramifications on both fundamental biomedical research and clinical applications can be tremendous.

Public Health Relevance Statement:


Public Health Relevance:
The advances proposed here will allow NanoCellect to achieve its mission: to make conventional and image- based cell-sorting flow cytometry an everyday lab tool by making our device portable, affordable, and easy-to- use. We will develop a simple spatial-frequency optical filter to encode image information with conventional flow cytometry sensors.

NIH Spending Category:
Bioengineering; Drug Abuse (NIDA only); Substance Abuse

Project Terms:
Address; Algorithms; Architecture; Area; Back; base; Benchmarking; Big Data; Biochemical; Biological Assay; Biological Process; Biomedical Research; Cell Cycle; Cell membrane; cell motility; Cell Separation; Cell Size; Cell surface; Cells; cellular imaging; Characteristics; Clinical; clinical application; Code; Collection; Communities; Complex; cost; design; Detection; detector; Devices; Disease; Flow Cytometry; Fluorescence; fluorescence imaging; fluorescence microscope; Fluorescence-Activated Cell Sorting; Frequencies; Image; Image Cytometry; Imaging Techniques; Imaging technology; innovation; instrument; Label; light scattering; Location; macrophage; Marketing; Masks; Methods; Microfluidics; microscopic imaging; Microscopy; Mission; Mus; Nuclear; Nuclear Protein; optic flow cytometer; Optics; particle; Performance; Phagocytosis; Phase; photomultiplier; Ploidies; precision medicine; Process; programs; Property; Proteins; public health relevance; Publishing; rapid growth; Research; Research Personnel; Resources; sensor; Shapes; Side; Signal Transduction; Sorting - Cell Movement; Staining method; Stains; Stream; System; Technology; Time; tool; Tube; Zymosan

Advances in reagents (e.g. CRiSPR) and analytical tools (e.g. flow cytometers) have improved the ability to alter and characterize cellular phenotypes. Ultimately, many key applications in biomedicine require efficient and accurate isolation of cell populations according to features contained in high content images. Unfortunately, microscopic laser microdissection systems have a throughput that is too slow to be practical in many applications; while the existing flow cytometers that can sort cells (fluorescence-activated cell sorters or FACS) provide only size, internal complexity, and fluorescence intensity information and lack the rich data of imaging. Another critical limitation is that the existing flow cytometers that can image, cannot sort cells. NanoCellect has made a highly affordable FACS to increase access to this important high-throughput tool for cell analysis. Here we propose to enhance our existing low-cost FACS with the ability to image cells and sort them based on image features. This will allow users to pursue new strategies in drug screening and mechanism of action research; as well as work with suspension cell lines, such as those that dominate the recent advances in immuno-oncology. In Phase I research, we have demonstrated the world's first imaging flow cytometer with cell sorting capabilities (iFACS) in a unique design of space-time coding with an optical spatial filter. The approach adds negligible cost to the system for the desirable features of cell imaging and sorting. To fully realize the enormous potential of the design and to meet the demands for most applications, in Phase II we will develop high-throughput image-based cell sorting with innovative image-guided gating schemes supported by machine learning and interactive user/machine interface. Essentially, image-based flow cytometry gating uses similar cell isolation criteria as the techniques of laser capture microdissection or cell aspiration to isolate cells of interest, with 10,000X throughput improvements to 1000+ cells per second. We envision such unique capabilities will become common, default features for tomorrow's users as the tool becomes as intuitive and ubiquitous as fluorescent microscopy. The proposed iFACS will be transformative and benefit numerous biomedical applications, such as isolation of cells based on organelle translocation, cell cycle analyses, detection and counting of phagocytosed particles, and protein co-localization, to name just a few.

Public Health Relevance Statement:
iFACS: Imaging Florescence Activated Cell Sorter to sort cells based on images NanoCellect Biomedical, Inc. RESEARCH & RELATED Other Project Information 8. PROJECT NARRATIVE The advances proposed here will allow NanoCellect to achieve its mission: to advance image-based fluorescence-activated cell sorting technology that can be placed in an affordable bench-top device. This will be achieved by integrating cell-imaging and cell-sorting, and relevant software into an easy-to-use package that extends the features of the existing WOLF® Cell Sorter.

Project Terms:
Action Research; Adopted; Algorithmic Software; analytical tool; Back; base; Benchmarking; Cell Cycle; Cell Line; Cell Separation; Cell Size; Cells; Cellular biology; cellular imaging; Chromatin; Code; commercialization; Computer software; cost; Data; design; Detection; detector; Devices; Dexamethasone; drug mechanism; experience; experimental study; Flow Cytometry; Fluorescence; fluorescence activated cell sorter device; Fluorescence-Activated Cell Sorting; Frequencies; gene therapy; Glucocorticoid Receptor; high throughput analysis; Histones; Image; Image Analysis; image guided; image processing; imaging capabilities; Immunooncology; improved; Individual; innovation; interest; Intuition; laser capture microdissection; Lasers; Liquid substance; Machine Learning; Measures; Membrane; Methodology; Microdissection; Microfluidics; Microscopic; Microscopy; Mission; Mitosis; Morphology; Motion; Names; Nuclear; optical imaging; Optics; Organelles; particle; Patients; Pharmacologic Substance; Phase; Phenotype; Phorbol Esters; photomultiplier; Photons; Physiological; Population; Preclinical Drug Evaluation; prevent; protein kinase C gamma; Proteins; prototype; Reagent; Reporter; Research; response; Sampling; Scheme; screening; sensor; Sorting - Cell Movement; Structure; Suspensions; System; Techniques; Technology; Testing; Time; tool; Tube; Validation; Work