Boaz A. Nemet

A two-photon and second-harmonic microscope

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.

Second harmonic imaging of membrane potential of neurons with retinal.

We present a method to optically measure and image the membrane potential of neurons, using the nonlinear optical phenomenon of second harmonic generation (SHG) with a photopigment retinal as the chromophore [second harmonic retinal imaging of membrane potential (SHRIMP)]. We show that all-trans retinal, when adsorbed to the plasma membrane of living cells, can report on the local electric field via its change in SHG. Using a scanning mode-locked Ti-sapphire laser, we collect simultaneous two-photon excited fluorescence (TPEF) and SHG images of retinal-stained kidney cells and cultured pyramidal neurons. Patch clamp experiments on neurons stained with retinal show an increase of 25% in SHG intensity per 100-mV depolarization. Our data are the first demonstration of optical measurements of membrane potential of mammalian neurons with SHG. SHRIMP could have wide applicability in neuroscience and, by modifying rhodopsin, could in principle be subject for developing genetically engineered voltage sensors.

Imaging microscopic viscosity with confocal scanning optical tweezers

The techniques of confocal microscopy and optical tweezers have shown themselves to be powerful tools in biological and medical research. We combine these methods to develop a minimally invasive instrument that is capable of making hydrodynamic measurements more rapidly than is possible with other devices. This result leads to the possibility of making scanning images of the viscosity distribution of materials around biopolymer-producing cells. 100 x 100 images can be taken with 0.5-microm spatial resolution in 3 min. An image of the viscosity distribution around a pullulan-producing cell of Aureobasidium pullulans is shown as an example.

Microscopic flow measurements with optically trapped microprobes

The use of optical tweezers to measure micrometer-resolution velocity fields in fluid flow is demonstrated as an extension of a scanning confocal viscosity microscope. This demonstration is achieved by detection of the motion of an optically trapped microsphere in an oscillating laser trap. The technique is validated by comparison with an independent video-based measurement and applied to obtain a two-dimensional map of the flow past a microscopic wedge. Since the velocity is measured simultaneously with the trap relaxation time, the technique requires no fluid-dependent calibration and is independent of the trap stiffness and the particle size.

Measuring microscopic viscosity with optical tweezers as a confocal probe

We demonstrate, what is to the best our knowledge, a new method for studying the motion of a particle trapped by optical tweezers; in this method the trapping beam itself is used as a confocal probe. By studying the response of the particle to periodic motion of the tweezers, we obtain information about the medium viscosity, particle properties, and trap stiffness. We develop the mathematical model, demonstrate experimentally its validity for our system, and discuss advantages of using this method as a new form of scanning photonic force microscopy for applications in which a high spatial and temporal resolution of the medium viscosity is desired.

Imaging Brain Slices

Brain slices are convenient preparations to study synapses, neurons, and neural circuits because, while they are easily accessed by experimental manipulations such as drug applications, intracellular recordings, and optical imaging, they preserve many of the essential functional properties of these circuits. In this chapter, we describe techniques of live brain-slice imaging used in our laboratory. We cover in detail experimental protocols and know-how acquired over the years about preparing neocortical and hippocampal slices and slice cultures, loading neurons with dyes or using biolistic transfection techniques, two-photon and second harmonic imaging, morphological reconstructions, and image processing and analysis. These techniques are used to study the functional or morphological dynamics of synaptic structures, including dendritic spines and axon terminals, and to characterize circuit connectivity and dynamics.

Microscopic flow measurements using optically trapped microprobes

Summary from only given. In previous work, we used a confocal detection modification of a method to measure fluid viscosity by recording the response of a microsphere in a viscous medium under the action of rapidly oscillating photonic force. We showed that the viscosity of the fluid can be accurately and rapidly measured by detecting the phase lag of the motion. In this paper, we extend this technique to measure local fluid flow. A spherical micron-sized silica bead is trapped in a flow cell on an inverted microscope by a strongly focused beam from a Ti-sapphire laser. An acousto-optic deflector nudges the particle periodically by moving the laser tweezers back and forth.

Imaging microscopic fluid viscosity and velocity fields using confocal scanning optical tweezers

Confocal microscopy and optical tweezers were combined to develop a minimally invasive instrument capable of making hydrodynamic measurements more rapidly than is possible with other devices. This result leads to the possibility of making scanning images of the viscosity distribution of materials around bipolymer producing cells. An image of the viscosity distribution around a pullulan producing cell of Aureobasidium pullulans is shown as an example. We present results from experiments supporting a linearized model for the motion of a trapped bead in an oscillating harmonic potential. Fluid velocity measurements are tested by comparing to an independent video based measurement. We apply the technique to obtain a 2-D map of the flow past a microscopic wedge and compare to a theoretical solution for the stream lines assuming potential flow. Since the velocity is measured simultaneously with the trap relaxation time, it requires practically no calibration and is independent of the trap stiffness and the particle size.

Local probing of medium properties using optical tweezers and a confocal setup

Summary form only given. In the study of colloidal suspensions, polymer solutions and in cell and micro-biology it is often desirable to have a direct observation in real time of the changes in the local viscosity, which affect the dynamics of objects like colloidal particles, cells and organelles. For example, the cells transport and diffusion of macro-molecules both depend on the local environment. It is especially important to have high spatial resolution in complex environments in which local inhomogeneities are present. We describe a method to probe the environment of such objects in a fairly simple way that is both sensitive and fast. We use the probe beam of an inverted confocal microscope as optical tweezers so that only light scattered from the focal point of the tweezers is confocally detected. In our experiment we nudge a particle periodically by moving the laser tweezers back and forth across it, with an oscillation amplitude that is slightly larger than the particle diameter.