Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity.

Abstract

BACKGROUND:

Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.

RESULTS:

Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.

CONCLUSIONS:

Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.

• Authors: Margaret T Butko, Mikhail Drobizhev, Nikolay S Makarov, Aleksander Rebane, Brendan C Brinkman, Joseph G Gleeson
• Journal: BMC biotechnology (BMC Biotechnol) Vol. 11 Pg. 20 (Mar 02 2011) ISSN: 1472-6750 [Electronic] England
• PMID: 21366923 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural)

Topics

• Animals
• Cell Line
• Cells (metabolism)
• Humans
• Microscopy, Fluorescence, Multiphoton (methods)
• Sensitivity and Specificity