Nonlinear excitation fluorescence microscopy: Source considerations for biological applications

Research output: Chapter in Book/Report/Conference proceedingConference contribution

2 Scopus citations


Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard twophoton dose is defined by 800nm, 1OmW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ∼830nm, ∼960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

Original languageEnglish (US)
Title of host publicationSolid State Lasers XVII
Subtitle of host publicationTechnology and Devices
StatePublished - May 7 2008
EventSolid State Lasers XVII: Technology and Devices - San Jose, CA, United States
Duration: Jan 20 2008Jan 24 2008


OtherSolid State Lasers XVII: Technology and Devices
Country/TerritoryUnited States
CitySan Jose, CA


  • Excitation wavelengths
  • Harmonic generation microscopy
  • Laser scanning microscopy
  • Two-photon excitation fluorescence
  • Ultra-short pulse lasers

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics


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