Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Aug 2;7:7101. doi: 10.1038/s41598-017-07165-0

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Principle of wavelength mixing two-photon microscopy: setup and characterization. (a) Imaging setup. A femtosecond laser (Ti:Sa) beam is divided in two fractions by a beam splitter. One is adjusted in a microscope, the second pumps an OPO. Pulses from Ti:Sa and OPO are synchronized using a delay stage (t) and spatially coaligned. GVD compensation is achieved using a conventional prism-based and a single-prism pulse compressors for Ti:Sa and OPO, respectively. The relative divergence of two laser beams is controlled using telescopes. Galvanoscan mirrors provide raster scanning the area up to 500 × 500 um². Chromatic aberration between NIR and IR wavelength of two lasers is corrected by the high numerical aperture objective. The non-descanned detection system is equipped with six PMTs. (b) Single-prism pulse compressor. Negative dispersion is accumulated on the four-pass travel through the main prism. The distance between a corner cube and the main prism is half that of the conventional design due to the image inversion of the corner cube. Pulse compression at different wavelengths can be achieved by rotating only one prism. (c) Spatially separated (left) and overlapping (right) Ti:Sa (red) and OPO (blue) foci in the microscope, measured on a 100 nm fluorescent bead (λ_emission = 605 nm). Scale bars, 0.2 μm. (d) Optimization of the ATPE. The mOrange2 fluorescent signal can be independently controlled by adjusting the delay between pulses. The insets represent images of HEK cells expressing mOrange2 at different delay times. Scale bar, 50 μm. (e) Spatiotemporal overlapping of two laser pulses. Left side: unsynchronized pulses (850 nm and 1230 nm) provide dual two-photon excitation, i.e. two parallel symmetric two-photon excitation processes. Right side: The wavelength mixing appears only if the pulses are synchronized in time and the two foci are matched in space. A third, asymmetric two-photon excitation process additionally takes place, making further fluorophores visible. Hence, simultaneous triple two-photon excitation of a broad set of chromophores is achieved.