Abstract
We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.
Highlights
High precision structural and functional images of the living brain have been a dream for neuroscientists for a long time
We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research
Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend is achieved with the combination of a motorized high precision bearing and gearing
Summary
High precision structural and functional images of the living brain have been a dream for neuroscientists for a long time. More than two decades ago, Denk and colleagues introduced twophoton laser scanning fluorescence microscopy (2PLSM) for biological applications [2]. Considerable methodological progress has been made (see reviews [3, 4]) to the point where 2PLSM has become one of the most important in vivo techniques for the neurosciences. The importance for other organ systems continues to increase [5]. These experiments are facilitated with the use of dedicated in vivo two-photon instruments that differ significantly from standard upright microscopes. Confining excitation to the focal volume, better tissue penetration and reduced phototoxicity out of the focal plane - due to the near infrared excitation through pulsed laser light - are the major advantages of 2PLSM over confocal fluorescent microscopy
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