Two-photon frequency division multiplexing for functional in vivo imaging: a feasibility study.

Recently, we presented a new approach to create high-speed amplitude modulation of femtosecond laser pulses and tag multiple excitation beams with specific modulation frequencies. In this work, we discuss the utility of this method to record calcium signals in brain tissue with two-photon frequency-division multiplexing (2P-FDM) microscopy. While frequency-multiplexed imaging appears slightly inferior in terms of image quality as compared to conventional two-photon laser scanning microscopy due to shot noise-induced cross-talk between frequency channels, applying this technique to record average signals from regions of interest (ROI) such as neuronal cell bodies was found to be promising. We use phase information associated with each pixel or waveform within a selected ROI to phase-align and recombine the signals into one extended amplitude-modulated waveform. This procedure narrows the frequency detection window, effectively decreasing noise contributions from other frequency channels. Using theoretical analysis, numerical simulations, and in vitro imaging, we demonstrate a reduction of cross-talk by more than an order of magnitude and predict the usefulness of 2P-FDM for functional studies of brain activity.

Task Learning Promotes Plasticity of Interneuron Connectivity Maps in the Olfactory Bulb.

UNLABELLED: Elucidating patterns of functional synaptic connectivity and deciphering mechanisms of how plasticity influences such connectivity is essential toward understanding brain function. In the mouse olfactory bulb (OB), principal neurons (mitral/tufted cells) make reciprocal connections with local inhibitory interneurons, including granule cells (GCs) and external plexiform layer (EPL) interneurons. Our current understanding of the functional connectivity between these cell types, as well as their experience-dependent plasticity, remains incomplete. By combining acousto-optic deflector-based scanning microscopy and genetically targeted expression of Channelrhodopsin-2, we mapped connections in a cell-type-specific manner between mitral cells (MCs) and GCs or between MCs and EPL interneurons. We found that EPL interneurons form broad patterns of connectivity with MCs, whereas GCs make more restricted connections with MCs. Using an olfactory associative learning paradigm, we found that these circuits displayed differential features of experience-dependent plasticity. Whereas reciprocal connectivity between MCs and EPL interneurons was nonplastic, the connections between GCs and MCs were dynamic and adaptive. Interestingly, experience-dependent plasticity of GCs occurred only in certain stages of neuronal maturation. We show that different interneuron subtypes form distinct connectivity maps and modes of experience-dependent plasticity in the OB, which may reflect their unique functional roles in information processing. SIGNIFICANCE STATEMENT: Deducing how specific interneuron subtypes contribute to normal circuit function requires understanding the dynamics of their connections. In the olfactory bulb (OB), diverse interneuron subtypes vastly outnumber principal excitatory cells. By combining acousto-optic deflector-based scanning microscopy, electrophysiology, and genetically targeted expression of Channelrhodopsin-2, we mapped the functional connectivity between mitral cells (MCs) and OB interneurons in a cell-type-specific manner. We found that, whereas external plexiform layer (EPL) interneurons show broadly distributed patterns of stable connectivity with MCs, adult-born granule cells show dynamic and plastic patterns of synaptic connectivity with task learning. Together, these findings reveal the diverse roles for interneuons within sensory circuits toward information learning and processing.

Amplitude modulation of femtosecond laser pulses in the megahertz range for frequency-multiplexed two-photon imaging.

We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric scheme employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond laser pulses with periodically varying phase shift. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies, and subsequently overlapping these matching patterns, results in multiple encoded excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.

Random-Access Multiphoton Microscopy for Fast Three-Dimensional Imaging.

Studies in several important areas of neuroscience, including analysis of single neurons as well as neural networks, continue to be limited by currently available experimental tools. By combining molecular probes of cellular function, such as voltage-sensitive or calcium-sensitive dyes, with advanced microscopy techniques such as multiphoton microscopy, experimental neurophysiologists have been able to partially reduce this limitation. These approaches usually provide the needed spatial resolution along with convenient optical sectioning capabilities for isolating regions of interest. However, they often fall short in providing the necessary temporal resolution, primarily due to their restrained laser scanning mechanisms. In this regard, we review a method of laser scanning for multiphoton microscopy that overcomes the temporal limitations of pervious approaches and allows for what is known as 3D Random Access Multiphoton (3D RAMP) microscopy, an imaging technique that supports full three dimensional recording of many sites of interest on physiologically relevant time scales.

Combining Membrane Potential Imaging with Other Optical Techniques.

Membrane potential imaging using voltage-sensitive dyes can be combined with other optical techniques for a variety of applications. Combining voltage imaging with Ca2+ imaging allows correlating membrane potential changes with intracellular Ca2+ signals or with Ca2+ currents. Combining voltage imaging with uncaging techniques allows analyzing electrical signals elicited by photorelease of a particular molecule. This approach is also a useful tool to calibrate the change in fluorescence intensity in terms of membrane potential changes from different sites permitting spatial mapping of electrical activity. Finally, combining voltage imaging with optogenetics, in particular with channelrhodopsin stimulation, opens the gate to novel investigations of brain circuitries by allowing measurements of synaptic signals mediated by specific sets of neurons. Here we describe in detail the methods of membrane potential imaging in combination with other optical techniques and discus some important applications.

Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers.

BACKGROUND: The hippocampal CA3 area contains large amounts of vesicular zinc in the mossy fiber terminals which is released during synaptic activity, depending on presynaptic calcium. Another characteristic of these synapses is the presynaptic localization of high concentrations of group II metabotropic glutamate receptors, specifically activated by DCG-IV. Previous work has shown that DCG-IV affects only mossy fiber-evoked responses but not the signals from associational-commissural afferents, blocking mossy fiber synaptic transmission. Since zinc is released from mossy fibers even for single stimuli and it is generally assumed to be co-released with glutamate, the aim of the work was to investigate the effect of DCG-IV on mossy fiber zinc signals. RESULTS: Studies were performed using the membrane-permeant fluorescent zinc probe TSQ, and indicate that DCG-IV almost completely abolishes mossy fiber zinc changes as it does with synaptic transmission. CONCLUSIONS: Zinc signaling is regulated by the activation of type II metabotropic receptors, as it has been previously shown for glutamate, further supporting the corelease of glutamate and zinc from mossy fibers.

Scan-less microscopy based on acousto-optic encoded illumination.

Several optical microscopy methods are now available for characterizing scientific and industrial processes at sub-micron resolution. However, they are often ill-suited for imaging rapid events. Limited by the trade-off between camera frame-rate and sensitivity, or the need for mechanical scanning, current microscopes are optimized for imaging at hundreds of frames-per-second (fps), well-below what is needed in processes such as neuronal signaling or moving parts in manufacturing lines. Here, we present a scan-less technology that allows sub-micrometric imaging at thousands of fps. It is based on combining a single-pixel camera with parallelized encoded illumination. We use two acousto-optic deflectors (AODs) placed in a Mach-Zehnder interferometer and drive them simultaneously with multiple and unique acoustic frequencies. As a result, orthogonal light stripes are obtained that interfere with the sample plane, forming a two-dimensional array of flickering spots - each with its modulation frequency. The light from the sample is collected with a single photodiode that, after spectrum analysis, allows for image reconstruction at speeds only limited by the AOD's bandwidth and laser power. We describe the working principle of our approach, characterize its imaging performance as a function of the number of pixels - up to 400 × 400 - and characterize dynamic events at 5000 fps.

Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity.

The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Substantial progress has been made by optical imaging systems that combine confocal and multiphoton microscopy with inertia-free laser scanning. However, all of the systems developed so far restrict fast imaging to two dimensions. This severely limits the extent to which neurons can be studied, as they represent complex three-dimensional structures. Here we present a new imaging system that utilizes a unique arrangement of acousto-optic deflectors to steer a focused, ultra-fast laser beam to arbitrary locations in three-dimensional space without moving the objective lens. As we demonstrate, this highly versatile random-access multiphoton microscope supports functional imaging of complex three-dimensional cellular structures such as neuronal dendrites or neural populations at acquisition rates on the order of tens of kilohertz.

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.

Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

Aberration-free multi-plane imaging of neural activity from the mammalian brain using a fast-switching liquid crystal spatial light modulator.

We report a novel two-photon fluorescence microscope based on a fast-switching liquid crystal spatial light modulator and a pair of galvo-resonant scanners for large-scale recording of neural activity from the mammalian brain. The spatial light modulator is used to achieve fast switching between different imaging planes in multi-plane imaging and correct for intrinsic optical aberrations associated with this imaging scheme. The utilized imaging technique is capable of monitoring the neural activity from large populations of neurons with known coordinates spread across different layers of the neocortex in awake and behaving mice, regardless of the fluorescent labeling strategy. During each imaging session, all visual stimulus driven somatic activity could be recorded in the same behavior state. We observed heterogeneous response to different types of visual stimuli from ∼ 3,300 excitatory neurons reaching from layer II/III to V of the striate cortex.

New methods and uses for fast optical scanning.

Advanced optical imaging techniques used in neurobiology commonly employ fluorescent molecules for studying the structure and function of neural tissue. To obtain adequate spatio-temporal resolution, sophisticated scanning schemes are used to manage the excitation light going to and emission light coming from objects under observation. Although the fundamental principles of these techniques remain the same, such as scanning point illumination and point detection for confocal imaging, their physical implementation is the subject of technological advance, for example, the advent of inertia-free discontinuous scanning schemes. In general, the aims of these technological advances are to improve the spatio-temporal resolution of and/or reduce potential photodamage caused by optical imaging in live neural tissue. The number of recent advances in scanning methods indicates their increasing importance in imaging techniques.

SmartScope2: Simultaneous Imaging and Reconstruction of Neuronal Morphology.

Quantitative analysis of neuronal morphology is critical in cell type classification and for deciphering how structure gives rise to function in the brain. Most current approaches to imaging and tracing neuronal 3D morphology are data intensive. We introduce SmartScope2, the first open source, automated neuron reconstruction machine integrating online image analysis with automated multiphoton imaging. SmartScope2 takes advantage of a neuron's sparse morphology to improve imaging speed and reduce image data stored, transferred and analyzed. We show that SmartScope2 is able to produce the complex 3D morphology of human and mouse cortical neurons with six-fold reduction in image data requirements and three times the imaging speed compared to conventional methods.

Developmental broadening of inhibitory sensory maps.

Sensory maps are created by networks of neuronal responses that vary with their anatomical position, such that representations of the external world are systematically and topographically organized in the brain. Current understanding from studying excitatory maps is that maps are sculpted and refined throughout development and/or through sensory experience. Investigating the mouse olfactory bulb, where ongoing neurogenesis continually supplies new inhibitory granule cells into existing circuitry, we isolated the development of sensory maps formed by inhibitory networks. Using in vivo calcium imaging of odor responses, we compared functional responses of both maturing and established granule cells. We found that, in contrast to the refinement observed for excitatory maps, inhibitory sensory maps became broader with maturation. However, like excitatory maps, inhibitory sensory maps are sensitive to experience. These data describe the development of an inhibitory sensory map as a network, highlighting the differences from previously described excitatory maps.

High-speed addressable confocal microscopy for functional imaging of cellular activity.

Due to cellular complexity, studying fast signaling in neurons is often limited by: 1. the number of sites that can be simultaneously probed with conventional tools, such as patch pipettes, and 2. the recording speed of imaging tools, such as confocal or multiphoton microscopy. To overcome these spatiotemporal limitations, we develop an addressable confocal microscope that permits concurrent optical recordings from multiple user-selected sites of interest at high frame rates. Our system utilizes acousto-optic deflectors (AODs) for rapid positioning of a focused laser beam and a digital micromirror device (DMD) for addressable spatial filtering to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current system has an adjustable spatial resolution of approximately 0.5 to 1 microm. Furthermore, we show that recordings can be made at an aggregate frame rate of approximately 40 kHz. The system is capable of optical sectioning; this property is used to create 3-D reconstructions of fluorescently labeled test specimens and visualize neurons in brain slices. Additionally, we use the system to record intracellular calcium transients at several sites in hippocampal neurons using the fluorescent calcium indicator Oregon Green BAPTA-1.

Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells.

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.

Photometric recording of transmembrane potential in outer hair cells.

Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 microm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

Facilitation of L-type Ca2+ channels in dendritic spines by activation of beta2 adrenergic receptors.

We studied the contribution of L-type Ca2+ channels to action potential-evoked Ca2+ influx in dendritic spines of CA1 pyramidal neurons and the modulation of these channels by the beta2 adrenergic receptor. Backpropagating action potentials (bAPs) (three at 50 Hz) were evoked by brief somatic current injections, and Ca2+ transients were recorded in proximal basal dendrites and associated spines. The R- and T-type Ca2+ channel blocker NiCl2 (100 microm) significantly reduced Ca2+ transients in both spines and their parent dendrites (approximately 50%), suggesting that these channels are the major source of bAP-evoked Ca2+ influx in these structures. The L-type Ca2+ channel blockers nimodipine and nifedipine (both 10 microm) reduced spine Ca2+ transients by approximately 10%, whereas the L-type Ca2+ channel activators FPL 64176 (2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methylester) and Bay K 8644 ((+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester) (both 10 microm) significantly enhanced the spine Ca2+ transients by 40-50%. Activation of beta2 adrenergic receptors with salbutamol (40 microm) or formoterol (5 microm) resulted in significant enhancements of the spine (40-50%) but not dendritic Ca2+ transients. This increase was prevented when L-type Ca2+ channels were blocked with nimodipine (10 microm) or when cAMP-dependent protein kinase A (PKA) was inhibited with KT5720 (3 microm), Rp-cAMPS (Rp-adenosine cyclic 3',5'-phosphorothioate) (100 microm), or PKI (100 microm). The above data suggest that L-type Ca2+ channels are functionally present in dendritic spines of CA1 pyramidal neurons, contribute to spine Ca2+ influx, and can be modulated by the beta2 adrenergic receptor through PKA in a highly compartmentalized manner.

Modulation of presynaptic calcium transients by metabotropic glutamate receptor activation: a differential role in acute depression of synaptic transmission and long-term depression.

Activation of group I metabotropic glutamate receptors (mGluRs) can induce acute depression of excitatory synaptic transmission and long-term depression (LTD) in area CA1 of the rat hippocampus. The underlying mechanisms for both forms of depression are unknown. By measuring presynaptic calcium transients, we show that a reduction in the stimulation-induced presynaptic calcium rise that triggers vesicular release causes the acute depression of transmission by group I mGluRs. In contrast, the mechanism underlying mGluR-induced LTD does not involve a persistent change in stimulation-induced calcium influx. However, analysis of paired-pulse facilitation experiments suggests a presynaptic location for expression of this form of LTD. Furthermore, we show that mGluR-induced LTD can be completely blocked by a specific mGluR5 antagonist, whereas mGluR1 antagonists strongly attenuate the acute depression of transmission. These results support the hypothesis that the acute depression of transmission caused by activation of group I mGluRs involves regulation of stimulation-induced presynaptic calcium transients, whereas mGluR-induced LTD involves a distinct presynaptic modulation downstream of calcium influx.

Fast three-dimensional laser scanning scheme using acousto-optic deflectors.

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 x 350 x 200 microm). 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.