Peter Saggau

Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy

We describe novel approaches for compensating dispersion effects that arise when acousto-optic (AO) beam deflection of ultrafast laser pluses is used for multiphoton laser-scanning microscopy (MPLSM). AO deflection supports quick positioning of a laser beam to random locations, allowing high frame-rate imaging of user-selected sites of interest, in addition to conventional raster scanning. Compared to standard line-scan approaches, this results in improved signal strength (and thus increased signal-to-noise) as well as reduced photobleaching and photodamage. However, 2-D AO scanning has not yet been applied for multiphoton microscopy, largely because ultrafast laser pulses experience significant spatial and temporal dispersion while propagating through AO materials. We describe and quantify spatial dispersion, demonstrating it to be a significant barrier to achieving maximal spatial resolution. We also address temporal dispersion, which is a well-documented effect that limits multiphoton excitation efficacy, and is particularly severe for AO devices. To address both problems, we have developed a single diffraction grating scheme that reduces spatial dispersion more than three-fold throughout the field of view, and a novel four-pass stacked-prism prechirper that fully compensates for temporal dispersion while reducing by two-fold the required physical length relative to commonly employed designs. These developments enable the construction of a 2-D acousto-optic multiphoton laser-scanning microscope system.

Three-dimensional mapping of microcircuit correlation structure

Great progress has been made toward understanding the properties of single neurons, yet the principles underlying interactions between neurons remain poorly understood. Given that connectivity in the neocortex is locally dense through both horizontal and vertical connections, it is of particular importance to characterize the activity structure of local populations of neurons arranged in three dimensions. However, techniques for simultaneously measuring microcircuit activity are lacking. We developed an in vivo 3D high-speed, random-access two-photon microscope that is capable of simultaneous 3D motion tracking. This allows imaging from hundreds of neurons at several hundred Hz, while monitoring tissue movement. Given that motion will induce common artifacts across the population, accurate motion tracking is absolutely necessary for studying population activity with random-access based imaging methods. We demonstrate the potential of this imaging technique by measuring the correlation structure of large populations of nearby neurons in the mouse visual cortex, and find that the microcircuit correlation structure is stimulus-dependent. Three-dimensional random access multiphoton imaging with concurrent motion tracking provides a novel, powerful method to characterize the microcircuit activity in vivo.

A high-speed confocal laser-scanning microscope based on acousto-optic deflectors and a digital micromirror device

Neuronal dendrites are known to possess active computational properties. An ideal method of investigating these properties would be to monitor the complete electrical and chemical behavior of a single cell; however, currently available recording techniques force a tradeoff between spatial and temporal resolution. The objective of this project is to develop a confocal microscope that can make multisite optical recordings of single neuron function in a living brain slice at a frame rate sufficient to measure neuronal events that occur on a 1 ms timescale. Specifically, we would like to measure membrane potential and calcium concentration changes using voltage and ion-sensitive fluorescent indicators at user-selected sites-of-interest (SOIs). To accomplish this, we use acousto-optic deflectors (AODs) as random-access beam positioners in the illumination path, along with a digital micromirror device (DMD) as an addressable spatial filter in the detection path. We have demonstrated that our device is capable of optical sectioning and therefore capable of imaging in light-scattering preparations such as brain slices. Furthermore, our system is capable of an aggregate frame rate of up to 25 kHz.

Development of fast two-dimensional standing wave microscopy using acousto-optic deflectors

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.

Development of a random access multiphoton microscope for fast three-dimensional functional recording of neuronal activity

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.

Acousto-optic multiphoton laser scanning microscopy (AO-MPLSM) for structural and functional imaging in living brain slices

The intrinsic optical sectioning, reduced light-scattering, and reduced photodamage of multiphoton laser-scanning microscopy (MPLSM) has generated great interest for this technique in experimental Neuroscience, as it enables to study both structure and function of fine neuronal processes within living brain tissue. At present, virtually all MPLSM systems employ galvanometric beam positioning. Due to this inertia-limited approach, single-dimension line scans are employed to achieve frame rates sufficient for functional imaging. Although such line scans allow adequate sampling rates (≤1kHz), two significant drawbacks remain. First, the majority of scan time is wasted by illuminating regions of no interest, while sacrificing signal integration time at sites-of-interest. Second, the sites from which signals can be recorded are limited to those along a single line. Alternatively, acousto-optic (AO) beam positioning with high-resolution TeO2 deflectors allows inertia-free skipping between arbitrary sites within the field-of-view in <15μs. This achieves high sampling rate recording at multiple, non-adjacent sites quasi-simultaneously (1-5kHz frame rate, 12-60 sites). Such a multi-site optical recording system would greatly advance studying complex neuronal function, by enabling membrane potential or calcium transients to be observed throughout the complex geometry of neuronal dendrites. This paper presents images and functional recordings from living neurons within brain slices, acquired with AO-MPLSM. Our novel imaging system allows a user to collect structural images first and subsequently select sites of interest for fast functional imaging. To demonstrate the system’s power, we present high-speed recordings (1kHz) from >10 sites within the dendrites of pyramidal neurons in acute brain slices, at signal-to-noise ratios comparable to line-scan systems.

Fast three-dimensional random access multi-photon microscopy for functional recording of neuronal activity

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.

Fiber-coupled non-descanned 4π detection with a commercial confocal microscope modified for multiphoton imaging

The extremely small (femtoliter) excitation volume of multiphoton (MP) microscopy renders all emitted photons useful in detecting fluorescence signals. Hence, multiphoton laser scanning microscopy (MPLSM) systems can collect fluorescence through the objective (epi-fluorescence), as well as the condenser (trans-fluorescence). For maximal collection efficiency, both optical paths can be used concurrently (4π detection). Most MPLSM systems incorporate photodetectors directly in or adjacent to the epi- and trans-fluorescence optical paths of the microscope, generally photomultiplier tubes with associated optics. These arrangements are optically straightforward, but are often bulky and difficult to reconfigure. Here, we demonstrate that all fluorescence from the specimen can be efficiently coupled into two multimode optical fibers -- one each for the epi- and trans-fluorescence pathways. Fiber-coupled detection enables a modular detection paradigm where light can be routed to easily reconfigurable and interchangeable detection module(s). A novel MPLSM system was constructed, which is readily switched between the original de-scanned detection path for confocal microscopy, and the newly added pathways supporting fiber-coupled non-descanned 4π detection for MP microscopy. Sample MP images of fluorescent beads and fluorescent-labeled hippocampal neurons are presented, demonstrating the viability of fiber-coupled detection.

Development of a novel confocal microscope for functional recording of fast neuronal activity

Currently available recording methods limit the study of fast neuronal signaling by forcing a tradeoff between spatial and temporal resolution. Fortunately, advanced optical imaging techniques can be used to overcome this limitation. Specifically, we are developing confocal microscopy schemes that allow multisite recordings of neuron function in live brain tissue with high spatial and temporal resolution. The first scheme that is currently being investigated involves the use of a digital micromirror device (DMD) to implement the light paths necessary for high-speed confocal imaging: addressable point illumination and spatial filtering via addressable point detection. The second scheme involves the use of acousto-optic deflectors (AODs) in the illumination path to increase the excitation intensity, along with the DMD or an addressable CMOS imager as the spatial filter in the detection path. Calculations of the signal-to-noise ratios and operating parameters of the three devices indicate that we will be able to study both calcium concentration and fast membrane potential changes at several sites within the dendritic tree of a neuron.

Compartmentalization of the Outer Hair Cell Demonstrated by Slow Diffusion in the Extracisternal Space

In the outer hair cell (OHC), the extracisternal space (ECiS) is a conduit and reservoir of the molecular and ionic substrates of the lateral wall, including those necessary for electromotility. To determine the mechanisms through which molecules are transported in the ECiS of the OHC, we selectively imaged the time-dependent spatial distribution of fluorescent molecules in a <100 nm layer near the cell/glass interface of the recording chamber after their photolytic activation in a diffraction-limited volume. The effective diffusion coefficient was calculated using the analytical solution of the diffusion equation. It was found that diffusion in the ECiS is isotropic and not affected by depolarizing the OHC. Compared with free solution, the diffusion of 10 kDa dextran was slowed down in both the ECiS and the axial core by a factor of 4.6 and 1.6, respectively.