US 4,628,026 A · Method and apparatus for automated double fluorochromization analysis in lymphocytotoxicity testing

Abstract

An automated system for rapid sequential photometric analysis of a collection of double fluorochrome stained lymphocyte specimens, useful for antibody screening or lymphocytotoxicity analysis. The specimens are sequentially alternately irradiated with light of two distinguishable wavelengths, producing fluorescence at two distinguishable wavelengths. The fluorescent emission light intensity for each irradiation of each specimen is measured using a photometer and computer. The computer controls the synchronization of the irradiation through alternately selected condenser sets with the sequential movement of specimens into the optical path of the irradiating and detected light, and calculates the quotient of the light intensities emitted from each specimen at the two selected fluorescent light wavelengths. These quotients are compared against a control ratio (for lymphocytotoxicity analysis) to classify the specimen. Also described is a method of preparing specimens for such analysis, which requires that a complement be added to the first staining solution after the latter is applied to the specimens, then this combination agitated, and then the second staining solution added and the specimen incubated.

Claims24

Dependent claims

For use in fluorescence assays of specimens exhibiting fluorescence at two distinguishable wavelength bands when irradiated respectively by light of two pre-selected distinguishable wavelength bands;

apparatus comprising:
a movable specimen container having spaced compartments for a plurality of individual specimens to be assayed;
alignment means for moving the container to provide predetermined sequential centered alignment of individual compartments of the container with a predetermined optical path;
irradiating means for sequentially irradiating said specimens, as each is aligned in the optical path, alternately with light of the two pre-selected distinguishable wavelength bands beamed along the optical path;
light collection means for receiving and distinguishing light produced by fluorescence in each said specimen along the optical path;
detector means for detecting and recording the intensity of the light produced by fluorescence of each said specimen at each of said two distinguishable wavelength bands;
analyzing means for calculating the quotient of the intensities of the light sequentially received from each said specimen at the two distinguishable wavelength bands and for comparing such quotient with at least one control quotient thereby to classify such specimen according to such comparison;
and
synchronization means for controlling, coordinating and synchronizing the operation of the alignment means, irradiating means, light collection means, detector means and analyzing means.

6 more elements

Automated apparatus for observing fluorescence stimulated by a light source in a plurality of specimens in a transparent specimen holder containing said specimens in a planar array, each of said specimens exhibiting fluorescence at a first emission wavelength when irradiated with light of a first exciting wavelength and fluorescence at a second emission wavelength when irradiated with light of a second exciting wavelength, comprising:

(i) an inversion microscope including a scanning stage operable to retain said specimen holder and to be sequentially positioned to place each of said specimens sequentially at a selected centered position within the objective field of such microscope to collect for observation fluorescent radiation from such specimen;
(ii) power means operable to drive said scanning stage in orthogonal directions within a plane perpendicular to the focal axis of the objective of said microscope;
(iii) first and second optical systems operable when said first and second optical systems are respectively positioned in the path of light from said source, to direct sequentially light of said first incident wavelength and said second incident wavelength, sequentially to the specimen located at said selected position, and to transmit light of said first emission wavelength and said second emission wavelength, respectively, from said specimen located at said selected position to the ocular of said microscope for observation;
(iv) motorized means for alternately positioning said first optical system and said second optical system in the path of light from said source;
and
(v) control means operatively connected with each of said power means and said motorized means for moving said scanning stage in incremental steps to place each of said specimens sequentially at said selected position within the objective field of said microscope for a predetermined period of time and to actuate said motorized slide means during said predetermined period of time so that fluorescence light of said first emission wave length and of said second emission wave length is observed by means of said microscope for each of said specimens;
(vi) photodetector means coupled with said microscope and operable to produce an amplified signal proportional to the intensity of radiation emitted by a sample located at the selected position within the optical field of said microscope;
and
(vii) a computer programmed to collect and store signal data from said photodetector measuring the fluorescence light intensity at said first emission wavelength and at said second emission wavelength from each said sample, and to transform said data into quotients which are directly related to reference data for positive and negative control standards of a given double-stained sample, said computer being programmed to coordinate said control means so that each said specimen is successively placed within the optical field of said microscope where it is alternatively irradiated with light of said first and second incident wavelengths.

6 more elements

Automated apparatus for the photometric analysis of a plurality of specimens of cellular material, each such specimen having been stained with a first fluorochrome exhibiting fluorescence at a first emission wavelength when irradiated with light at a first incident wavelength and a second fluorochrome exhibiting fluorescence at a second emission wavelength contrasting with first emission wavelength when irradiated with light at a second incident wavelength, comprising:

(i) a microtest tray operable to hold said specimens and appropriate fluorescence control standards within an array of discrete wells;
(ii) an inverted epi-fluorescence microscope, comprising:
(a) a motorized scanning stage operable to retain said microtest tray and to be sequentially positioned to place each of said wells sequentially at a selected centered position within the optical field of said microscope to collect fluorescent radiation from the specimen in said well,
(b) first control means operatively connected with said motorized scanning stage for moving said scanning stage in incremental steps to place each of said wells sequentially at said selected position,
(c) a light source operable to irradiate any of said specimens located at said selected position within the optical field of the microscope,
(d) first and second epi-fluorescence condenser sets operable to admit light of said first incident wavelength and said second incident wavelength, respectively, and to be interposed between said light source and said specimen located at said selected position within the optical field of the microscope,
(e) motorized filter-changing means operable to alternate the interposition of said first condenser set and said second condenser set between said light source and said specimen,
(f) second control means operatively connected with said motorized filter-changing means for successively irradiating each sample located at said selected position in the optical field of said microscope first with light of said first incident wavelength and then with light of said second incident wavelength for predetermined intervals of time;
(iii) photodetector means coupled with said microscope and operable to produce an amplified signal proportional to the intensity of radiation emitted by a sample located at the selected position within the optical field of said microscope;
(iv) a computer programmed to collect and store signal data from said photodetector measuring the fluorescence light intensity at said first emission wavelength and at said second emission wavelength from each said sample, and to transform said data into quotients which are directly related to reference data for positive and negative control standards of a given double-stained sample, said computer being programmed to coordinate said first control means and said second control means so that each said specimen is successively placed within the optical field of said microscope where it is alternatively irradiated with light of said first and second incident wavelengths.

7 more elements

For use in fluorescence assays of specimens of cellular material exhibiting fluorescence at two distinguishable wavelength bands when irradiated respectively by light of two pre-selected distinguishable wavelength bands;

an automated method comprising:
selecting and ordering specimens containing substantially uniform quantities of cellular material;
sequentially aligning individual ones of said specimens with an optical path;
irradiating the specimens sequentially and alternately with light of the two pre-selected distinguishable wavelength bands beamed along the optical path;
receiving and distinguishing light produced by fluorescence caused by the sequential and alternate irradiation of each said specimen along the optical path;
and
detecting the intensity of the light produced by fluorescence of each said specimen at each of said two distinguishable wavelength bands;
calculating the quotient of the intensities of the light sequentially received from each said specimen at the two distinguishable wavelength bands;
and
comparing the said quotient for each specimen against a predetermined control quotient thereby to classify the specimen according to such comparison;
said method including automatically synchronizing and coordinating all of the foregoing steps other than the selection and ordering of specimens.

8 more elements

Dependencies are inferred from the claim text.

Method and apparatus for automated double fluorochromization analysis in lymphocytotoxicity testing

Life of this patent

Method and apparatus for automated double fluorochromization analysis in lymphocytotoxicity testing

Life of this patent

Abstract

Claims24