This review presents a selection of advanced microscope designs employing acousto-optical deflectors (AODs). In the designs covered, AODs are used as tunable diffraction gratings to control complex illumination patterns at the Fourier plane of an objective lens. This approach allows us to generate desired illumination patterns at the focal plane of a light microscope. In simple terms, I will describe two established designs, the 3D Random-Access Multi-Photon Microscope and the Standing-Wave Super-Resolution Microscope, as well as two new schemes including the Random-Access STED Microscope and the Frequency-Encoded Multi-Beam Microscope. All instruments mentioned here were designed to overcome the throughput limitations of previously used light microscopes in experimental Neuroscience.
A novel scheme for two-dimensional (2D) standing wave fluorescence microscopy (SWFM) using acousto-optic
deflectors (AODs) is proposed. Two laser beams were coupled into an inverted microscope and focused at the back focal
plane of the objective lens. The position of each of two beams at the back focal plane was controlled by a pair of AODs.
This resulted in two collimated beams that interfered in the focal plane, creating a lateral periodic excitation pattern with
variable spacing and orientation. The phase of the standing wave pattern was controlled by phase delay between two RF
sinusoidal signals driving the AODs. Nine SW patterns of three different orientations about the optical axis and three
different phases were generated. The excitation of the specimen using these patterns will result in a SWFM image with
enhanced 2D lateral resolution with a nearly isotropic effective point-spread function. Rotation of the SW pattern
relative to specimen and varying the SW phase do not involve any mechanical movements and are only limited by the
time required for the acoustic wave to fill the aperture of AOD. The resulting total acquisition time can be as short as
100 µs and is only further limited by speed and sensitivity of the employed CCD camera. Therefore, this 2D SWFM can
provide a real time imaging of subresolution processes such as docking and fusion of synaptic vesicles. In addition, the
combination of 2D SWFM with variable angle total internal reflection (TIR) can extend this scheme to fast microscopy
with enhanced three-dimensional (3D) resolution.
The dendritic processes of neurons have been shown to possess active and dynamic properties
that give them the ability to modulate synaptic integration and shape individual synaptic responses.
Effectively studying these properties at multiple locations on a live neuron in highly light scattering brain
tissue requires an imaging/recording mechanism with high spatio-temporal resolution as well as optical
sectioning and random access site selection capabilities. Our lab has made significant steps in developing
such a system by combining the spatial resolution and optical sectioning ability of advanced imaging
techniques such as confocal and multi-photon microscopy with the temporal resolution and random access
capability provided by acousto-optic laser scanning. However, all systems that have been developed to
date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which
many neurons can be studied since they represent complex three-dimensional (3D) structures. We have
previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of acoustooptic
deflectors and does not require axial movements of the objective lens. We have also shown how,
when used with the ultra-fast laser pulses needed in multi-photon microscopy, this scheme inherently
compensates for the spatial dispersion which would otherwise significantly reduce the resolution of
acousto-optic based multi-photon microscopy. We have now coupled this scanning scheme to a modified
commercial research microscope and use the combined system to effectively image user-defined sites of
interest on fluorescent 3D structures with positioning times that are in the low microsecond (μs) range. The
resulting random-access scanning mechanism allows for functional imaging of complex 3D structures such
as neuronal dendrites at several thousand volumes per second.
Over the past two decades, the dendritic processes of neurons have been shown to possess active and
dynamic properties that give them the ability to modulate synaptic integration and shape individual
synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly
scattering brain tissue requires an imaging/recording mechanism with high spatiotemporal resolution as
well as optical sectioning and random access site selection capabilities. Our lab has made significant steps
in developing such a system by combining the spatial resolution and optical sectioning ability of imaging
techniques such as confocal and multi-photon microscopy with the temporal resolution and random access
capability provided by acousto-optic laser scanning. However, all systems that have been developed to
date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which
many neurons can be studied since they represent complex three-dimensional (3D) structures. We have
previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of multiple
acousto-optic deflectors and does not require axial movements of the objective lens. Here we couple this
scanning scheme to a modified commercial research microscope and use the combined system to
effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are
in the low microsecond range. The resulting random-access scanning mechanism allows for functional
imaging of complex 3D cellular structures such as neuronal dendrites at frames rates on the order of tens of
kilohertz.
We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.
A scheme for fast 3-D laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counterpropagating acoustic waves that are both chirped and offset in their frequencies, we show that it is possible to simultaneously scan both axially and laterally with frame rates on the order of tens of kilohertz. This scheme was specifically designed for application with multiphoton imaging, particularly of neurons, where it will enable the concurrent monitoring of physiological signals at multiple locations within a microscopic 3-D volume (350×350×200 µm). When used for this purpose, we demonstrate how this scheme would also inherently compensate for spatial dispersion when ultrafast laser pulses are used in acousto-optic multiphoton microscopy.
A scheme for fast three-dimensional laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counter-propagating acoustic waves that are both chirped and offset in their frequencies, it is possible to simultaneously scan both in the axial and lateral directions. This scheme also has the added benefit of inherently compensating for spatial dispersion when ultra-fast laser pulses are used in acousto-optic multi-photon microscopy.
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