Multiphoton Microscopy III: A Novel Concept in Multiphoton Microscopy

EXCITE THEM ALL!

Imagine simultaneously imaging a wide range of fluorophores without having to think about selecting the optimum excitation wavelength of your laser. In two-photon microscopy, this is often a complex and sometimes an impossible task when using traditional 100fs excitation lasers.
Transform-limited 100fs solid-state lasers emitting in the IR have a spectrum in the range of 10 – 20nm and as a result, they can only simultaneously excite fluorophores whose excitation spectra falls within this 10-20nm spectrum.
To simultaneously excite a larger variety of fluorophores with a single laser, lasers with broader bandwidths and shorter pulse durations are required.
For example, a laser with 15 fs pulses, such as FYLA´s SCH laser, centered at 1050nm, delivers 15fs pulses and a bandwidth of 200nm. All green and red fluorophores within this bandwidth (which extends across 900 to 1200nm) can be excited simultaneously by such laser.

Fyla_graphic

This makes simultaneous imaging of multiple fluorophores a viable, practical and simple alternative for two-photon microscopy.

Fyla_graphic2

Two-Photon Fluorescence Microscopy Image of a Mouse Intestine. Section stained with
Sytox Green: labelling the nuclei (magenta). FITC Filter
Alexa Fluor 568 Phaloidin: labelling the actin filaments (green). TRITC Filter
Both fluorescent markers were simultaneously excited with FYLA´s SCH and fluorescence was filtered with Nikon´s fluorescence cubes.
Image taken at ICFO-SLN the Super-Resolution Light Microscopy at ICFO-Institute of Photonics Sciences, Barcelona, Spain.
Acknowedgements to Sphere Photonics for the D-SCAN pre-compressor

You can find the presentation of SPAOM2020, about this topic here.

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