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A Bullen

Publications and source records attributed to A Bullen.

8 recordsLinked to original sources
PubMed1999

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes.

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.

Action Potentials↗
PubMed1998

Painful decisions

Explore the source record for details and available documents.

Journal Article↗
PubMed1998

Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy.

The instrumental design and experimental conditions for high-speed, simultaneous optical recording of membrane potential and intracellular Ca2+ with subcellular resolution are presented. This method employs an extended version of a high-speed, random-access, laser-scanning fluorescence microscope designed to record fast physiological signals from small neuronal structures with high spatiotemporal resolution (Bullen, Patel, Saggau, Biophys J 73:477-491, 1997). With this instrument, imaging and optical recording functions are conducted separately allowing frame rates up to 3 kHz. Individual scanning points are selected interactively from a reference image collected with differential interference contrast (DIC) optics. At each recording site, fluorescence from two indicators is measured simultaneously by independent photodetectors. To optimize signal strength, spectral separation and the achievable signal-to-noise ratio, several combinations of voltage-sensitive dye, Ca2+ indicator and optical elements (dichroic mirrors, filters, etc.) were considered. The best results were achieved from the combination of the intracellular voltage-sensitive dye Di-2-ANEPEQ and the Ca2+ indicator Calcium Green-1. These indicators have overlapping absorption spectra allowing simultaneous excitation with a single laser line (488 nm). Spectral separation of the fluorescence from these two indicators was accomplished using a secondary dichroic mirror (DCLP580) and emission filters (535/45 and OG590). Representative records obtained with this instrument and this combination of indicators demonstrate the feasibility of simultaneous high fidelity measurements of membrane potential and intracellular Ca2+ from the same point at high spatial (2 micrometer) and temporal (<ms) resolution without requiring signal averaging.

Animals↗
PubMed1998

Acousto-optic random-access laser scanning microscopy: fundamentals and applications to optical recording of neuronal activity.

A novel approach to laser scanning microscopy is presented that utilizes diffraction-based scanning principles to achieve fast random-access positioning of a focused laser beam. This non-imaging approach overcomes the speed limitation of present reflection-based scanning microscopes while maintaining high spatial resolution. The presented system combines conventional video microscopy with fast non-imaging scanning microscopy. Together with readily available optical indicators of neuronal activity, this system permits multi-site optical recording from living brain tissue. In this paper, we will review the underlying principles of laser scanning microscopy and the steps in development that led to the current acousto-optic scanning system. We will present typical signals recorded with the current system, and we will outline ongoing extensions of the system. We will also discuss the present limitation of this instrumentation and look into directions of future development.

Acoustics↗
PubMed1997

High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.

Animals↗
PubMed1982

Suppressor cell activity, splenic function and HLA B8 status in man.

The enhanced proliferative response of peripheral blood lymphocytes (P.B.L.) to Concanavalin A (Con A) occurring after a 24 hr period of incubation was used as the basis of a suppressor cell assay and the results expressed as a suppressor index (S.I.) which if low implies impaired suppressor cell activity. S.I. was measured on P.B.L. from 26 patients with coeliac disease and defined splenic function, 8 patients with splenic atrophy not associated with coeliac disease, 30 patients after surgical splenectomy, and 52 controls. S.I. was lower than control in the splenectomy group (P less than 0.01) and in the coeliac group, but coeliacs with splenic atrophy were no different from coeliacs with apparently normal splenic function, nor were the non-coeliac splenic atrophy group different from control. Coeliacs with the HLA B8 gene had lower S.I. than those without, as did controls. Suspensions of cells from spleens removed at laparotomy demonstrated suppressor activity but this was no more marked than in P.B.L. It is concluded that the intact spleen is required for normal activation of suppressor cells in man, but this function is retained in splenic atrophy, and that the HLA B8 gene is associated with low suppressor cell activity in coeliac disease and in health.

Celiac Disease↗