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Novel millimeter-wave-based method for in situ cell isolation and other applications.

ABSTRACT: As an alternative to laser-based methods, we developed a novel in situ cell isolation method and instrument based on local water absorption of millimeter wave (MMW) radiation that occurs in cellular material and nearby culture medium while the cultureware materials (plastic and glass) are transparent to MMW frequencies. Unwanted cells within cell population are targeted with MMWs in order to kill them by overheating. The instrument rapidly (within 2-3?seconds) heats a cell culture area of about 500?µm in diameter to 50?°C using a low-power W-band (94?GHz) MMW source. Heated cells in the area detach from the substrate and can be removed by a media change leaving a bare spot. Hence we named the instrument "CellEraser". Quick, local and non-contact heating with sharp boundaries of the heated area allows elimination of the unwanted cells without affecting the neighboring cells. The instrument is implemented as a compact microscope attachment and the selective hyperthermic treatment can be done manually or in an automated mode. Mammalian cells heated even momentarily above 50?°C will not survive. This "temperature of no return" does not compromise cellular membranes nor does it denature proteins. Using the CellEraser instrument we found that the key event that determines the fate of a cell at elevated temperatures is whether or not the selectivity of its nucleus is compromised. If a cell nucleus becomes "leaky" allowing normally excluded (cytoplasmic) proteins in and normally nuclear-localized proteins out, that cell is destined to die. Quick heating by MMWs to higher temperatures (70?°C) denatures cellular proteins but the cells are not able to detach from the substrate - instead they undergo a phenomenon we called "thermofixation": such cells look similar to cells fixed with common chemical fixatives. They remain flat and are not washable from the substrate. Interestingly, their membranes become permeable to DNA dyes and even to antibodies. Thermofixation allows the use of western blot antibodies for immunofluorescence imaging.

SUBMITTER: Boyce B

PROVIDER: S-EPMC6170430 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Project description:Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH techniques have visualized intra-nuclear genomic structure and sub-cellular transcriptional dynamics of many genes and revealed their functions in various biological processes.

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Novel millimeter-wave-based method for in situ cell isolation and other applications.

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