Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results

Cytometry. 1994 Jul 1;16(3):195-205. doi: 10.1002/cyto.990160303.

Abstract

A time-resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 microseconds to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 microsecond to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described. Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye-loaded liposomes. Quantitation of cell-associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA-associated ethidium bromide is shown. The background generated by instrument components in the time-gated measurement channel is characterized, and measures for its complete elimination are discussed.

MeSH terms

  • Chelating Agents
  • Ethidium
  • Flow Cytometry / instrumentation*
  • Fluorescent Dyes*
  • Humans
  • Lanthanum / chemistry
  • Liposomes
  • Lymphocytes / cytology
  • Metals, Rare Earth / chemistry*

Substances

  • Chelating Agents
  • Fluorescent Dyes
  • Liposomes
  • Metals, Rare Earth
  • Lanthanum
  • Ethidium