Abstract: Complex multidimensional datasets generated by digital imaging spectroscopy can be organized and analyzed by applying software and computer-based methods comprising sorting algorithms. Combinations of these algorithms to images and graphical data, allow pixels or features to be rapidly and efficiently classified into meaningful groups according to defined criteria. Multiple rounds of pixel or feature selection may be performed based on independent sorting criteria. In one embodiment sorting by spectral criteria (e.g., intensity at a given wavelength) is combined with sorting by temporal criteria (e.g., absorbance at a given time) to identify microcolonies of recombinant organisms harboring mutated genes encoding enzymes having desirable kinetic attributes and substrate specificity. Restriction of the set of pixels analyzed in a subsequent sort based on criteria applied in an earlier sort (“sort and lock” analyses) minimize computational and storage resources.

Abstract: The present invention provides an instrument and methods for a multispectral optical technique that can simultaneously classify individual biological cells within mixed populations. This invention, known as Multispectral Taxonomic Identification (MTID), shows that microscopy can be combined with a software analysis program to measure and categorize the fluorescence and other spectroscopically identifiable signals from complex populations of cells in situ, without cultivation. The invention thus enables high-throughput screening of cells for taxonomic classification.

Abstract: Improvements in calorimetric assays, surfaces for arraying microcolonies and instrument hardware for screening mutagenized enzymes and proteins in a solid phase format are presented. These improvements permit new enzyme activities to be screened. New filter membrane materials and formats for arraying the microcolonies provide higher throughput, better solvent resistance and ease of handling. Modifications to the instrument heating and illumination systems provide improved temperature control and a more compact, folded light path.

Abstract: Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red).

Abstract: The invention relates generally to the area of drug development and more specifically to screening novel compounds for antimicrobial activity. Methods are described for generating mutagenized peptide libraries expressed in colonies of cells. A fluorescent live/dead cell assay combined with a digital imaging spectrophotometer provides a high-throughput solid-phase screening method for colonies or arrayed synthetic libraries. The assay enables screening of antimicrobial peptide activity in at least 105-106 colonies per experiment. These methods generate and identify antimicrobial compounds.

Abstract: Complex multidimensional datasets generated by digital imaging spectroscopy can be organized and analyzed by applying software and computer-based methods comprising sorting algorithms. Combinations of these algorithms to images and graphical data, allow pixels or features to be rapidly and efficiently classified into meaningful groups according to defined criteria. Multiple rounds of pixel or feature selection may be performed based on independent sorting criteria. In one embodiment sorting by spectral criteria (e.g., intensity at a given wavelength) is combined with sorting by temporal criteria (e.g., absorbance at a given time) to identify microcolonies of recombinant organisms harboring mutated genes encoding enzymes having desirable kinetic attributes and substrate specificity. Restriction of the set of pixels analyzed in a subsequent sort based on criteria applied in an earlier sort (“sort and lock” analyses) minimize computational and storage resources.

Abstract: A MicroColonyImager instrument and solid phase methods to screen cells expressing mutagenized enzymes for enhanced activity. The MicroColonyImager instrument and methods permit high throughput screening of enzyme libraries by time course analyses of single-pixels, using either absorption, fluorescence or FRET. This high throughput assay can detect small differences in enzyme rates within microcolonies grown at a nearly confluent density on an assay disk. Each microcolony is analyzed simultaneously at single-pixel resolution, requiring less than 100 ml substrate/measurement. By simultaneously assaying different substrates tagged with spectrally distinct chromogenic or fluorogenic reporters, the substrate specificity of an enzyme can be changed.

Abstract: An optical instrument for use in collecting light from an object, the optical instrument having a post-objective waist with a variable optical filter positioned approximately at the post-objective waist. The invention also comprises an optical instrument for collecting light from an object, comprising an objective lens positioned to receive light from the object, an exit lens positioned to focus the light at a focal position, and a variable optical filter positioned between the objective lens and the focal position approximately at a waist of the light. The optical instrument may be an epifluorescence microscope for use in observing an image formed from light from an object under illumination by a light source. The light source may be, for example, a filtered light source or a monochromatic light source, preferably in an epifluorescence configuration. The variable optical filter may be a circular variable interference filter or a tilting interference filter.