Kirk S. Schroeder

FLIPR: A New Instrument for Accurate, High Throughput Optical Screening

Modern optical screening assays demand high data throughput along with uncompromised data fidelity. FLIPR (Fluorescent Imaging Plate Reader) was developed to perform quantitative optical screening for cell-based kinetic assays. FLIPR incorporates an integrated design, including low-level optical detection, precise temperature control, and precise fluid handling, all in one package. The unique aspect of FLIPR is that all 96 wells of a standard microplate are stimulated and optically measured simultaneously. Kinetic updates on all 96 wells can be obtained in under 1 sec, allowing for transient signals to be quantified. Demonstrated applications include measurements of intracellular calcium, intracellular pH, and membrane potential.

IonWorks™ HT: A New High-Throughput Electrophysiology Measurement Platform

To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a plate-based electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate™). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate™, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks™ HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening. (Journal of Biomolecular Screening 2003:50-64)

High Throughput Ion-Channel Pharmacology: Planar-Array-Based Voltage Clamp

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

Three-dimensional lensless imaging using laser frequency diversity.

A laser radar system for three-dimensional (3-D) lensless imaging is analyzed in theory and experiment. 3-D imaging is accomplished by making use of the relationship between the angular and wavelength dependence of the scattered light and an objects 3-D Fourier transform. The concept is demonstrated by obtaining a 3-D image of an extended object by using a charge-coupled device detector array and an argon-ion laser with a tunable intracavity étalon.

Holographic laser radar

Experimental results from a fine-resolution three-dimensional (3-D) imaging method are presented. An object is flood illuminated with coherent light from a frequency-tunable laser. Electronic holograms are then recorded for a series of laser frequencies. These recordings are digitally assembled into a 3-D data array that is Fourier transformed to yield a 3-D image. 3-D imaging with 4.2-microm range resolution using a broadly tunable dye laser is demonstrated.

Speckle from rough rotating objects

Dynamic speckle from rough rotating objects with nonplanar underlying shape is considered in theory and experiment. The theoretical treatment is based on modeling the optical field as a sum of contributions from discrete scatterers on the surface of the object. It follows that computation of the speckle correlation function requires knowledge of the objects average scattering strength as a function of position and the underlying shape of the object. Calculation of the speckle correlation function reduces to tracing a series of rays that coarsely sample the object and for each ray computing the relative phase shift resulting from object rotation. This method is quite simple compared to previous analytic techniques. Theory and experiment are compared for cylinders with a variety of surface coatings. Dynamic speckle from multiple rotating objects and objects with complicated underlying shape is also considered.