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.
The 2P-AO Microscope incorporates Thorlabs' MPM-2PKIT Multiphoton Essentials Kit and a gold-coated, 140 actuator, Multi-Deformable Mirror (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.
The two-photon emission light from the sample is diverted to a two-channel, non-descanned, detection module via a dichroic mirror cube. This module incorporates GaAsP PMTs, which are connected to a control/interface box and computer. The PMT and scanner signals are used to produce high-sensitivity two-photon images. These images are then used in a closed-loop feedback fashion to drive the necessary wavefront correction. The feedback system enables continuous compensation of the wavefront between acquired image frames.
By combining deep imaging via two-photon microscopy and aberration correction by incorporating adaptive optics, improved imaging deep into live tissue samples is possible.
Results Without Adaptive Optics
When the adaptive optics system is off, the deformable mirror in the figure above is flat. Input laser light reflects from the deformable mirror with an unchanged wavefront. The light is directed onto the sample by relay lenses, galvo steering mirrors, and the objective. The focused spot size within the sample will be larger than the theoretical minimum due to aberrations caused by the optical system as well as the sample itself, resulting in the low-resolution image shown on the left.
Results with Adaptive Optics
As the Adaptive Optics system engages, the deformable mirror's actuators deform the surface of the mirror until the intensity measured by the PMT module is at a maximum. The deformed mirror yields a wavefront which is corrected to form a minimized spot size in the sample. The image to the right shows the results: both the contrast and brightness are increased when compared to the same image without the adaptive optics system.
For More Information
For more information on deformable mirrors, including detailed tutorials on adaptive optics and aberrations, lists of publications, and detailed specifications for the deformable mirrors, please visit our deformable mirrors product presentation.
Products in Development
These products were featured in the adaptive optics in two photon microscopy demonstration and are still under development. For more information on these products, please click on the Web Page links below.
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
Products Available Now
The images below link to items which may be useful in constructing an adaptive optics system for a two-photon microscope similar to the demonstration model presented on this page. For more information about Thorlabs offerings of imaging systems, including two-photon microscopes and adaptive optics systems, please email ImagingSales@thorlabs.com.