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Faster and deeper imaging with acoustic liquid lenses

10 Apr | By Biophotonics.World
Faster and deeper imaging with acoustic liquid lenses
By: Simonluca Piazza and Marti Duocastella

By Simonluca Piazza and Marti Duocastella

Two-photon microscopy (2PE) is arguably the tool of choice for the in-vivo characterization of biological processes as relevant as neuronal signaling or cell interaction dynamics. Based on the focusing ultrashort laser pulses to excite fluorescence molecules from a confined volume via a nonlinear process, 2PE enables three-dimensional (3D) sub-cellular imaging over penetration depths in the order of millimeters. However, volumetric information is normally reconstructed from a focal stack of 2D images acquired from multiple planes in a sequential manner, an intrinsically slow process that strongly limits 3D acquisition speed. Indeed, the shifting of the laser focus between planes is typically achieved by mechanical translation of the sample or objective, a step that requires long idle times (in the order of 10s of milliseconds) for proper repositioning to occur. Thus, volumetric rates currently achievable by 2PE are insufficient to unmask the dynamics of fast and important events such as brain activity, in which neurons organized in complex 3D networks communicate through a burst of pulses with a duration below 50 ms.

On a new perspective, researchers from the Istituto Italiano di Tecnologia (IIT) have developed a novel 2PE architecture capable of volumetric imaging at millisecond timescales. The approach is based on the use of a liquid lens, named TAG lens, which uses sound to shape light at unprecedented speeds. In particular, a piezoelectric transducer driven with a high-frequency signal is utilized to generate sound waves in a liquid, causing a periodic variation in its refractive index. Consequently, light passing through the lens can be modulated with different phase profiles selectable by the simple adjustment of the driving frequency. Thus, when the liquid lens is driven with a parabolic phase profile, high-speed axial focus shifting occurs, and continuous volumetric imaging can be achieved. Notably, such focus translation does not involve any mechanical moving parts, enabling axial scanning on a pixel by pixel basis. As such, volumetric imaging above ten times faster than traditional 2PE is now possible. More importantly, the pixel-based focus control represents a radically new concept for 3D imaging, in which the user can select how many planes are acquired at each pixel, or reconstruct a volume from a series of axial slices. In the case the liquid lens is driven with other phase profiles, controlled aberrations can be induced in the image. After a straightforward post-processing step, images with an improved signal to noise ratio can be restored, which can help in reduce the effects of scattering, namely the deviation of light from straight trajectories. Note that scattering is the main factor that limits depth penetration of light into tissues. Therefore, the liquid lens can also help improve the imaging depth capabilities of existing 2PE systems.

In summary, the innovative two-photon microscope developed at IIT labs opens the door to the investigation of complex biological phenomena with an unparalleled temporal resolution and improved penetration depth, while maintaining sub-cellular resolution. This development is expected to have a major impact in live brain imaging, and it will hopefully shed some light on brain plasticity, neuronal activity or many of the other fascinating events that dictate brain function.

Related journal article:  http://onlinelibrary.wiley.com/doi/10.1002/jbio.201700050/full

By: Simonluca Piazza and Marti Duocastella

   

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