Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Margaret T Butko1, Mikhail Drobizhev2, Nikolay S Makarov2, Aleksander Rebane2, Brendan C Brinkman1, Joseph G Gleeson1
- چاپ و سال / کشور: 2011
Description
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.
Butko et al. BMC Biotechnology 2011, 11:20 http://www.biomedcentral.com/1472-6750/11/20
