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.
We present a versatile scheme for two-dimensional (2D) resolution enhancement in standing wave fluorescence
microscopy (SWFM). This SWFM scheme consists of an interferometer, where both beams are focused at the back focal
plane of the objective lens. Their position is controlled by a pair of acousto-optic deflectors (AODs). This results in two
collimated beams that interfere in the focal plane, creating a lateral periodic excitation pattern with controlled fringe
spacing and orientation. The phase of the standing wave (SW) pattern is controlled by the phase delay between two RF
sinusoidal signals driving the AODs. An enlarged fluorescence image formed using the same objective lens is captured
by a cooled CCD camera. Data collection involves acquiring images with excitation pattern of three equi-polar
orientations (0°, 60° and 120°) and three different phases (0°, 120°, 240°) for each orientation. The SWFM image is
algebraically reconstructed from these 9 acquired images. The SWFM image has enhanced 2D lateral resolution of about
100 nm with nearly isotropic effective point-spread function (PSF). As a result of the acousto-optic scanning, the total
acquisition time can be as short as 100 μs and is only further limited by the fluorescence intensity, as well as sensitivity
and speed of the CCD camera. Utilizing acousto-optic laser scanning for advanced SWFM provides the exceptional
precision and speed necessary for real-time imaging of subresolution processes in living biological systems.
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.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.