Neuroscience

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Multi-photon microscopy in neuroscience

Femtosecond fiber lasers for simplifying microscopy

Two-photon fluorescence microscopy is a key technology in biological imaging that enables three-dimensional, non-invasive studies of tissue on the micrometer scale. In contrast to conventional linear fluorescence microscopy, the nonlinear character and the longer excitation wavelength offer larger probing depths and lower phototoxicity.
Both advantages make non-linear fluorescence microscopy an ideal choice for the direct spatiotemporal visualization of neurons and neuronal activity in living animals. The key fluorescent proteins for such experiments are based on green (GFP) and red fluorescent proteins (RFP) that can be excited using 2-photon absorption at 920 nm and 1050 nm, respectively.

Over the last few years, a rapid shift has occurred towards fixed-wavelength mode-locked femtosecond fiber lasers emitting at 780 nm, 920 nm or 1050 nm. These laser systems provide a cost-efficient and easy-to-use alternative for two-photon microscopy applications, especially in neuroscience.

Miniaturized two-photon microscopes

Recently, light-weight, miniaturized two-photon microscopes (e.g. Mini2P) have been developed specifically for the neuroscience community. These tiny microscopes can be mounted on the heads of freely moving animals to study neuronal activity while minimizing interference with the animals' natural behavior. Femtosecond fiber lasers are commonly used as the excitation source for miniaturized two-photon microscopes because the most common fluorophores in neuroscience can be addressed with 920 and 1050 nm wavelengths. Fiber-based designs are compact, robust, and energy-efficient, making them well-suited for portable or head-mounted systems. The combination of miniaturized optics and femtosecond fiber laser sources enables high-resolution, deep-tissue imaging in mobile experimental setups. This greatly expands the range of biological questions that can be studied in living systems.

TOPTICA's offer

TOPTICA’s femtosecond fiber lasers provide optimal performance while being easy-to-use. In particular for neuroscience applications, TOPTICA’s FemtoFiber ultra lasers are ideally suited to address biological questions by imaging green (GFP) and red fluorescent proteins (RFP), and are already widely adopted in non-linear microscopes.

In order to achieve the best image quality, TOPTICA’s FemtoFiber ultra is equipped with Clean Pulse Technology that reduces pedestals and side-wings on the temporal pulse profile. Hence, the full laser power contributes to two-photon excitation. Clean Pulse Technology provides highest fluorescence image brightness and contrast while keeping thermal heating of the sample to a minimum.

TOPTICA’s FemtoFiber ultra provides also outstanding usability to scientists: The laser includes beam conditioning to adapt the input laser beam parameters to the microscope. Firstly, software-control of the group delay dispersion (GDD) pre-compensation offers user-friendly optimization of fluorescence signal strength. Secondly, an integrated acousto-optic modulator (AOM) enables fast power-modulation and flyback blanking in synchronization with the beam scanner. This minimizes sample damage and photo-bleaching. Thirdly, the lasers come in an extremely compact, passively cooled package, offering much reduced operating and maintenance cost.

The FemtoFiber ultra can now be equipped with femtosecond fiber delivery. This opens new possibilities for integration into modern microscopy set-ups. By eliminating the need for free-space pulse delivery between laser and microscope on an optical table with a simple FC/APC fiber connection, our solution further boosts usability while still providing high performance and maintaining the best fluorescence image quality. Especially, TOPTICA’s proprietary COOL technology offers automated fiber coupling which makes exchange of the hollow-core fiber or Mini2P-fiber easy and fast, and the fiber coupling very robust and reliable.

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