How to Use Three-Photon (3P) Microscopy for 3P Imaging

Non-invasive functional imaging is permitted by the three-photon (3P) microscopy; by making cells visible in the deeper tissues, and thus providing higher spatial resolution and increased contrast in comparison to the two-photon excitation.

Higher axial resolution is established by the optical design required for 3P as compared to that of two-photon microscopy. Three-photon excitation of a variety of fluorophores, which include the current generations of protein-based genetically encoded calcium indicators (e.g. GCaMP6), is enabled by the spectral window, and the repetition rate of the laser source is sufficient for imaging Ca2+ transients produced from neural activity.

three-photon (3P) microscopy


three-photon (3P) microscopy


3P Range Microscopy

FEMTOSmart Galvo is equipped with a 3P range microscopy optional module, which permits excitation in a certain wavelength range: namely between 1200-1700 nm. Within the biological tissues, the longer excitation wavelengths are scattered less, which thus permits the extension of the penetration depth, minimizes out-of-focus excitation, as well as increasing the signal-to-noise-ratio. The figure above demonstrates fluorescent activity measured on several somata from the V1 region of a GCaMP labeled mouse.

FEMTOSmart Galvo


Third Harmonic Generation

A specific effect of 3P excitation is Third Harmonic Generation or THG. THG results from the conversion of three incoming photons into one emitted photon with tripled energy and therefore, an emission of the light of one third the wavelength. THG occurs at structural interfaces which are formed between aqueous fluids and lipid-rich structures like biological membranes, as well as between water and large protein aggregates: e.g. collagen bundles or muscle fibers.

THG image of a section of mouse kidney


The figure above depicts a THG image of a section of mouse kidney: the kidney cells were excited at 1500 nm, and the photons which were subsequently emitted, were collected at the green channel (~500 nm) of the detector system.

About Femtonics Ltd.

Femtonics focuses on the research and development of two-photon laser scanning microscopes for the booming area of cutting-edge brain research and pharmaceutical development. Our specialty is represented by the acousto-optical scanner-based Femto3D Atlas microscope which takes the ability to scan the three-dimensional sample with astonishing speed and thereby it is unique on the market.

In the field of traditional galvanometric and resonant scanner-based systems, we present our customers the flexibility and freedom to customize their own products according to their vision and objective.

The high-valuable measurement and analysis solutions of our MES control software enable scientists to perform a wide variety of experiments. A well-selected microscope working together with the appropriate software modules shapes the customer’s idea into a remarkable product.

Our technology is pioneering and innovative in the field of microscopic imaging. It has been awarded at high levels namely by winning a number of grants and professional prizes and confirmed by our publications in the most prestigious international scientific journals and our presentations at the major conferences.

However, the main achievements for us are customer satisfaction and the scientific results and breakthroughs produced by our microscopes. We are proud of the fact that many results on the field of neuroscience have been published in the highest quality international scientific journals with the tools that we have designed.

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Last updated: Feb 7, 2020 at 7:47 AM


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