神経科学

Brighten up your images!

神経科学における多光子顕微鏡

顕微鏡をシンプルにするフェムト秒ファイバーレーザー

T二光子蛍光顕微鏡法は、生体組織をミクロンスケールで三次元・非侵襲的に観察できる、生物学イメージングの主要技術です。従来の線形蛍光顕微鏡とは異なり、その非線形性と長波長励起により、より深い観察深度と低い光毒性が得られます。
これらの利点により、非線形蛍光顕微鏡は、生体動物におけるニューロンおよび神経活動の直接的な時空間可視化に最適な手法となっています。こうした実験で鍵となる蛍光タンパク質は、緑色蛍光タンパク質（GFP）および赤色蛍光タンパク質（RFP）であり、それぞれ 920 nm と 1050 nm の二光子吸収によって励起できます。
近年、780 nm、920 nm、1050 nm で発振する固定波長のモードロックフェムト秒ファイバーレーザーへの急速な移行が進んでいます。これらのレーザーシステムは、特に神経科学分野において、二光子顕微鏡用途に対してコスト効率が高く、扱いやすい代替手段を提供します。

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のソリューション

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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