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Measuring DNA content in live cells by fluorescence microscopy.

Cecil J Gomes1,2, Michael W Harman3,4, Sara M Centuori1

  • 11University of Arizona Cancer Center, University of Arizona, 1515 N. Campbell Ave, Tucson, AZ 85724 USA.

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|September 12, 2018
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Summary

This study introduces a novel, non-toxic method for measuring DNA content in live cells using fluorescence microscopy. This technique allows researchers to correlate cellular dynamics with DNA content and cell cycle stage without dye-induced toxicity.

Keywords:
DNA contentHoechst 33342ImagingLive-cell microscopy

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Area of Science:

  • Cell Biology
  • Microscopy
  • Genetics

Background:

  • Live-cell fluorescence microscopy (LCFM) is crucial for studying cellular dynamics.
  • Existing methods for measuring DNA content during LCFM are limited by dye toxicity.
  • A non-toxic method to measure DNA content in live cells is needed to couple imaging with ploidy and cell cycle analysis.

Purpose of the Study:

  • To develop and validate a widely applicable, non-toxic method for measuring DNA content in live cells using fluorescence microscopy.
  • To enable the correlation of real-time cellular dynamics with DNA content and cell cycle stage.
  • To overcome limitations of dye-associated toxicity in live-cell imaging.

Main Methods:

  • Introduce a live-cell, membrane-permeant DNA fluorophore (e.g., Hoechst 33342) at the end of live-cell imaging.
  • Measure integrated nuclear fluorescence to quantify DNA content per cell.
  • Utilize an automated algorithm for nuclear fluorescence measurement and cell cycle histogram plotting.

Main Results:

  • Demonstrated tracking of chromosomal segregation errors and identification of cell ploidy (diploid/polyploid).
  • Developed and validated an automated algorithm for accurate DNA content measurement.
  • Validated algorithm performance through parallel flow cytometry studies.

Conclusions:

  • The method enables high-throughput correlation of single-cell dynamics with cellular stage and ploidy.
  • Applicable to standard epifluorescence microscopes with incubation capabilities.
  • Facilitates high-resolution microscopic analysis of cell cycle progression, DNA replication, and cell division.