Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems

E. Wang, C. M. Babbey, Kenneth Dunn

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system with several conventional single-point scanning confocal (SPSC) microscopes, using the relationship between image signal-to-noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal-to-noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high-speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three-dimensional motion of endosomes moving up to several micrometres per second.

Original languageEnglish
Pages (from-to)148-159
Number of pages12
JournalJournal of Microscopy
Volume218
Issue number2
DOIs
StatePublished - May 2005

Fingerprint

Lighting
metal spinning
high speed
Photobleaching
Scanning
illumination
scanning
Fluorophores
Fluorescence
fluorescence
Microscopes
microscopes
Cells
Endosomes
three dimensional motion
Signal-To-Noise Ratio
saturation
Imaging techniques
Fluorescence Microscopy
Fluorescence microscopy

Keywords

  • Photobleaching
  • Single-point scanning confocal
  • Spinning disc confocal

ASJC Scopus subject areas

  • Instrumentation

Cite this

Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems. / Wang, E.; Babbey, C. M.; Dunn, Kenneth.

In: Journal of Microscopy, Vol. 218, No. 2, 05.2005, p. 148-159.

Research output: Contribution to journalArticle

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