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Two-Photon Microscope with Adaptive Optics Demonstration
Two-photon, or multiphoton microscopy, is a nonlinear fluorescence imaging technique that enables optical sectioning, potentially up to 1 mm deep, in living samples. In two-photon microscopy, fluorescence excitation and emission only occurs within the focal volume of interest. This localization of excitation is the key advantage compared to single-photon excitation microscopes, which need to employ additional elements such as pinholes to reject out-of-focus fluorescence. Localization also minimizes the potential photobleaching or photodamage to the sample because it is only localized to within the focal volume. Background noise created from signal generated from outside the focal region is also eliminated, making it possible to image deeper within the sample.
The Two-Photon Adaptive Optics (2P-AO) Microscope is a demonstration of combining Thorlabs' Multiphoton Essentials Kit with our Deformable Mirror adaptive optic to correct aberrations that result from imaging deep within a sample. The main element of an adaptive optics system is a deformable mirror, whose shape is controlled to compensate or reshape a wavefront.
Adaptive optics systems often incorporate a wavefront sensor to measure the wavefront and provide feedback on image quality. However, for the 2P-AO microscope, we are developing an iterative sensor-less correction system that utilizes a proprietary algorithm to determine the image quality and degree of wavefront correction necessary to improve the image. That metric is translated into drive voltages that control the shape of the deformable mirror. With each iteration, the deformable mirror is further adjusted until the image quality is optimized.
The two images below show the advantages that can be gained when using an adaptive optics system in a two-photon microscope.
Adaptive Optics and Two-Photon Microscopy
The 2P-AO Microscope incorporates Thorlabs' MPM-2PKIT Multiphoton Essentials Kit and a gold-coated, 140 actuator, Multi-Deformable Mirror (former Item # DM140-35-UM01). As shown on the right two-photon excitation light from an OCTAVIUS-2P femtosecond laser (not included) is directed towards the deformable mirror via a polarizing beamsplitter and λ/4 wave plate. The excitation light reflects off of the deformable mirror, passes back through the wave plate and beamsplitter, and is coupled to the Multiphoton Scan Head of the Essentials Kit.
Results Without Adaptive Optics
Results with Adaptive Optics
For More Information
Products in Development
Thorlabs' multiphoton essentials kit is a collection of products that are commonly needed when building a custom multiphoton imaging system. The MPM-2PKIT contains a scan head, a near infrared optics tube, and a dual PMT and filter block unit.
Contact: Enrique Chang for Availability Information
Thorlabs' has developed a laser specifically for two-photon microscopy. This laser's 10 fs pulses provide excellent signal, yet minimize photodamage to the sample. With peak power exceeding 500 kW and a bandwidth greater than 100 nm, it is an excellent choice for applications that require deep imaging with multiple fluophores excited simultaneously.
Contact: Jens Schumacher
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