Simultaneous multiplane imaging with reverberation multiphoton microscopy

Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compromise in performance. Specifically, multiple planes are acquired by near-instantaneous axial scanning while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, is well adapted for large-scale imaging in scattering media with low repetition-rate lasers, and can be implemented with conventional MPM as a simple add-on.Accepted manuscrip

Closed loop adaptive optics with a laser guide star for biological light microscopy

We report on the development of a widefield microscope that achieves adaptive optics correction through the use of a wavefront sensor observing an artificial laser guide star induced within the sample. By generating this guide star at arbitrary positions and depths within the sample we allow the delivery of high-resolution images. This approach delivers much faster AO correction than image optimization techniques, and allows the use of AO with fluorescent imaging modalities without generating excessive photo-toxic damage in the sample, or inducing significant photo-bleaching in the flurophore molecules

Az ENSZ Környezetvédelmi Programjának környezetállapot-értékelési rendszere

The performance of adaptive systems that consist of microscale on-chip elements [microelectromechanical mirror (µ-mirror) arrays and a VLSI stochastic gradient descent microelectronic control system] is analyzed. The µ-mirror arrays with 5 × 5 and 6 × 6 actuators were driven with a control system composed of two mixed-mode VLSI chips implementing model-free beam-quality metric optimization by the stochastic parallel perturbative gradient descent technique. The adaptation rate achieved was near 6000 iterations/s. A secondary (learning) feedback loop was used to control system parameters during the adaptation process, further increasing the adaptation rate

Realtime wavefront sensing in a SPIM microscope, and active aberration tracking

Adaptive optics (AO) can potentially allow high resolution imaging deep inside living tissue, mitigating against the loss of resolution due to aberrations caused by overlying tissue. Closed-loop AO correction is particularly attractive for moving tissue and spatially varying aberrations, but this requires direct wavefront sensing, which in turn requires suitable "guide stars" for use as wavefront references. We present a novel method for generating an orthogonally illuminated guide star suitable for direct wavefront sensing in a wide range of fluorescent biological structures, along with results demonstrating its use for measuring time-varying aberrations, in vivo

Planarization of a CMOS die for an integrated metal MEMS

This paper describes a planarization procedure to achieve a flat CMOS die surface for the integration of a MEMS metal mirror array. The CMOS die for our device is 4 mm × 4 mm and comes from a commercial foundry. The initial surface topography has 0.9 μm bumps from the aluminum interconnect patterns that are used for addressing the individual micro mirror array elements. To overcome the tendency for tilt error in the planarization of the small CMOS die, our approach is to sputter a thick layer of silicon nitride (2.2 μm) at low temperature and to surround the CMOS die with dummy pieces to define the polishing plane. The dummy pieces are first lapped down to the height of the CMOS die, and then all pieces are polished. This process reduces the 0.9 μm height of the bumps to less than 25 nm

High throughput screening system for engineered cardiac tissues

Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging.Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol.Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats