Abstract
BackgroundMultiphoton 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.ResultsHere 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.ConclusionsSimilar 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.
Highlights
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
multiple-excitation MPM (ME-MPM) was conducted on an upright Olympus confocal point scanning system equipped with FluoView 300 software (FV300) or an inverted Olympus spectral deconvolution confocal point scanning system equipped with variable band-pass filters (VBFs) and FluoView 1000 software (FV1000) using Spectra Physics Mai Tai broad-band (BB, 710 nm - 990 nm) and Mai Tai high-power (HP, 690 nm - 1040 nm) two-photon excitation sources (Figure 1a)
Three dimensional z-stacks were collected on the FV300 upright microscope using simultaneous CLSM at 488 nm and 543 nm, ME-MPM at 920 nm and 1040 nm, and single-excitation MPM (SE-MPM) at 950 nm, which we empirically found to be the optimal intermediate excitation wavelength and which roughly corresponds to the intersection point of the two-photon absorption (TPA) spectra (Figure 1c) for this pair of fluorophores (Figure 3)
Summary
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. Multiphoton microscopy (MPM) has gained popularity because it offers decreased phototoxicity and increased sample viability while preserving the CLSM still remains the most widely used technique for multi-color imaging because single-photon absorption peaks of common biological fluorophores are relatively narrow and usually non-overlapping. This characteristic makes it easy to differentially excite a set of spectrally distinct fluorophores in a given sample by incorporating standard excitation laser lines for multi-color CLSM. MPM offers many advantages over CLSM, it has not replaced CLSM for multi-color measurements that are critical to most fields of biology
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