Slit Scanner Research Articles

Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS

Imaging, optical mapping, and optical multisite recording of transmembrane potential (Vm) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in Vm. Accordingly, they have been used for monitoring Vm in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER—the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands—was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast “slit scanner” increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.

4-D Scanning of Calcium Sparks in Cardiomyocytes Reveals their In-Focus Amplitude

Ca sparks, puffs and related discrete events of intracellular Ca release have so far been studied by imaging fluorescence of a suitable monitoring dye over one or two spatial coordinates and time (xt or xyt modes). By missing the vertical (z) coordinate either technique is susceptible to the out-of-focus error. This error corrupts every measurement, especially altering the measurement of amplitude, which is crucial for evaluation of numbers of contributing channels and other features with mechanistic implications. Existing theory of spark scanning (Izu et al.1998; Ríos et al. 2001) allow correction of the out-of-focus error, but only in a statistical sense, leading to correct distributions rather than accurate amplitudes of individual sparks. We now use a fast confocal slit scanner (5-LIVE; Zeiss) to image sparks in x, y and z as they evolve in time, and take advantage of the added dimension to characterize those sparks that are in focus. The distribution of amplitudes of 1196 such sparks (33 cat atrial cells) was not a sum of decaying exponentials, indicating that their Ca sources are non-Markovian channels (in agreement with Ríos et al. 2001, Wang et al. 2002), due for instance to interactions within the cluster. The directly determined distribution of amplitudes was similar to that obtained by correction, according to the theory of spark scanning, of the distribution in a larger sample imaged in xt linescans (xt-to-xyzt correction). The theory is generalized to provide the relationship between the distribution of amplitudes in focus and in xyt images (xyt-to-xyzt correction), thus unifying the three possible modes of spark sampling. The relative merits and shortcomings of the three modes will be discussed from this unified viewpoint. Funded by NIAMS, NHLBI and NCRR (NIH).

Flux in Artificial Ca Sparks Generated by 2-Photon Release from a Novel Cage Confocally Imaged at Microsecond Resolution

Control of calcium signaling in striated muscle relies on concurrent actions of calcium ions to promote and inhibit release channel opening. To understand these actions we developed artificial Ca sparks generated by 2-photon (2P) release from NDBF-EGTA (Momotake, Nature Methods 2006) as quantifiable local stimuli. A “Dual Scanner” (Zeiss) delivers IR laser flashes through a LSM 510 scanner, while rapidly imaging fluorescence of a [Ca2+] monitor via a slit scanner (5-LIVE; ca 100 μs/line). Ca sparks of 0.1 to 10 μM (A, B) are elicited in a droplet after microseconds of 2P irradiation at 720 nm and imaged with the low affinity dye fluo 4FF. Reaction-diffusion analysis (Rios, JGP 1999) yields the flux of Ca photorelease (C). This flux, which initially reaches several hundred mM/s, decays with τ of 2-3 ms. The technique is used to measure physico-chemical properties of calcium ligands, including bio-sensors. Applied inside muscle fibers (Figueroa, this meeting) it serves to quantitatively characterize calcium control in cells.Instrument purchased with a S10 NCRR award and Hasterlik Family matching funds. Supported by NIAMS, NHLBI/NIH and MDA.View Large Image | View Hi-Res Image | Download PowerPoint Slide

3D Slit Scanning with Planar Constraints★

Abstract We present a planarity constraint and a novel three‐dimensional (3D) point reconstruction algorithm for a multiview laser range slit scanner. The constraint is based on the fact that all observed points on a projected laser line lie on the same plane of laser light in 3D. The parameters of the plane of laser light linearly parametrize a homography between a pair of images of the laser points. This homography can be recovered from point correspondences derived from epipolar geometry. The use of the planar constraint reduces outliers in the reconstruction and allows for the reconstruction of points seen in only one view. We derive an optimal reconstruction of points subject to the planar constraint and compare the accuracy to the suboptimal approach in prior work. We also construct a catadioptric stereo rig with high quality optical components to remove error due to camera synchronization and non‐uniform laser projection. The reconstruction results are compared to prior work that uses inexpensive optics and two cameras.

Imaging of heavy-ion beams on kapton film

Kapton film is used to image K+ ion beams in the 0.4 to 2 MeV energy range. Dose response is shown to be linear and to follow a simple model for a range of exposures. The measured profiles agree with profiles obtained with a slit scanner. Kapton has excellent spatial resolution, dynamic range, and discrimination against stray low energy and low mass particles.

Use of a charge coupled device-camera system for optical diagnostics of intense ion beams

Among the various possibilities for investigating the properties of ion beams, two of the most important principles for determining the beam quality are profile measurements using wires or slit apertures, and different kinds of emittance measurement devices. However, most of the commonly used systems have the disadvantage of being either destructive to the beam, or at least to have a considerable influence on the further beam propagation downstream of the point of measurement. Also, for the investigation of intense ion beams, thermal problems on the scanners themselves might arise. Observation of particles emitted from the ion beam in radial direction allow an indirect but nondestructive observation of the beam. Of the different optical methods that exist to accomplish this, we recently conducted an investigation on the possibilities to employ a charge coupled device-camera system for such nondestructive beam diagnostics in our space-charge compensation test stand. First, experimental results on optical profile measurements are presented and compared to results obtained by a slit scanner and a wire scanner. Additionally, to gain a better understanding of the measured data, optically obtained sectional profiles behind a slit and a circular aperture were analyzed. Possibilities for gaining information on the two-dimensional beam profile and the beam emittance are explored.

Shape Measurement and Estimation of Flexible Structures Using a Laser Scanner.

This paper presents a method for estimating the shape of flexible structures from multipoint spatially distributed measurements of structural deflections. The estimation algorithm is based on an infinite-dimensional approach, where the estimation errors are reduced by introducing the necessary finite element truncation and approximation after solving the estimation problem for the distributed parameter system. This paper also proposes a remote optical sensing system for fast and precise measurements of multipoint structural deflections. The optical measurement system consists of a laser slit scanner and corner-cube reflectors distributed on the flexible structure. An experimental measurement system has been implemented for one-dimensional deflection estimation and tested on a simple structure in the laboratory. The results of the numerical simulations and experiments show the practical usefulness of the proposed shape estimation method.

An inexpensive reading instrument with a sound output for the blind.

An alternative auditory code is suggested to alleviate the reading problem presented by the Optophone in its older form. A ``musical'' code is discussed which has a high degree of coalescence. Physically it takes the form of an FM squarewave. As for the Optophone, a slit scanner is employed to scan along the line of print. Using N y photocells, the same number of binary signals b 1 . . . b n . . . b n y are generated by the print patterns. Photocell 1 is located at the bottom of the scanning slit; the cell with n = N y is at the top. When the cell is over black, b n = 1; over white, b n = 0. Two modulating functions have been used. The first is the familiar binary digit to analog transformation. It is easy to instrument. The disadvantage is the large number of values produced. The second coding function produces fewer f values by discarding information mainly connected with letter thickness. Using either version of the musical code, certain general features in print letters can be recognized. Single straight lines are easy; repeated or symmetrical print shapes, e.g., the letter ``w,'' are again easily identified. The code is weakest at representing curves. For example, the letters ``o'' and ``c'' sound nearly the same. Features used to recognize the lower-case alphabet letters are given. All letters of the alphabet (both lower and upper case) and numbers can be identified by their corresponding code sounds, which are reported to be quite pleasant.