Dejan Vučinić

Imaging brain activity with voltage- and calcium-sensitive dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license.

Anomalous Diffusion of Single Particles in Cytoplasm

The crowded intracellular environment poses a formidable challenge to experimental and theoretical analyses of intracellular transport mechanisms. Our measurements of single-particle trajectories in cytoplasm and their random-walk interpretations elucidate two of these mechanisms: molecular diffusion in crowded environments and cytoskeletal transport along microtubules. We employed acousto-optic deflector microscopy to map out the three-dimensional trajectories of microspheres migrating in the cytosolic fraction of a cellular extract. Classical Brownian motion (BM), continuous time random walk, and fractional BM were alternatively used to represent these trajectories. The comparison of the experimental and numerical data demonstrates that cytoskeletal transport along microtubules and diffusion in the cytosolic fraction exhibit anomalous (nonFickian) behavior and posses statistically distinct signatures. Among the three random-walk models used, continuous time random walk provides the best representation of diffusion, whereas microtubular transport is accurately modeled with fractional BM.

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task.

CMOS Descanning and Acousto-Optic Scanning Enable Faster Confocal Imaging

Fast pixel random access capabilities of CMOS imagers are exploited to create a virtual pinhole or slit in synchrony with acousto-optic laser beam scanning to significantly speed up confocal microscopy.

Hybrid reflecting objectives for deep-tissue functional two-photon imaging

Most microscope objectives commonly used for two-photon imaging of neurons are optimized for high image quality under wide-field illumination and for long working distance, constraints that are at odds with the need for high fluorescence collection efficiency and large field of view dictated by the low fluorescence intensity and the scattering properties of brain tissue. We present a de novo design of an objective intended specifically for deep-tissue functional two-photon imaging. Our design has separate imaging and non-imaging pathways for incident and emitted light, making use of the optical sectioning intrinsic in non-linear fluorescence excitation, which relaxes a number of design constraints. We show through modeling that a twofold to fourfold improvement in fluorescence collection efficiency over traditional objective designs is easily achievable while maintaining nearly diffraction-limited performance within a 200-micron field of view and a long working distance.

What the nose tells the brain about odors

Optical recording using calcium sensitive dyes was used to measure the input to the olfactory bulb from the nose. Because all of the receptor neurons projecting to one glomerulus in the bulb express the same receptor protein, the signal from each glomerulus represents the response properties of a single receptor protein. Individual receptors responded to a variety of odorants. In the mouse, the number of responding glomeruli increased with increasing odorant concentration. In the turtle the input maps for different odorant concentrations were similar; the signals were concentration invariant.