Abstract
Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.
Highlights
Over the past 25 years [1], two-photon microscopy (2PM) has become a gold standard for various biomedical studies [2,3,4]
Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them
We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions
Summary
Over the past 25 years [1], two-photon microscopy (2PM) has become a gold standard for various biomedical studies [2,3,4]. The main challenges toward this goal are: 1) adding multiple NDDs with suited band pass filters or using detector arrays to acquire and analyze photons over the whole spectrum, 2) labeling cells of interest by transgenic expression of new fluorescent proteins with non-overlapping emission spectra [10], 3) finding optimal excitation strategies to reveal the fluorescence of all markers [11, 12] Used in combination, these approaches have allowed the rise of multicolor intravital 2PM and we reported the observation of up to 6 different structures in the living animal [11]
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