Generalized cytometry instrument and methods of use

Claim 2

A generalized cytometry instrument for the repetitive measuring of properties of live cells comprising: (a) surface means for immobilizing the cells to be studied; (b) cell locating means for determining the cellular location of at least two cells on said surface means; (c) illumination means for providing illumination to a located cell on said surface means; (d) means for detecting a response to said illuminating means; (e) means for controlling coupled to said cell locating means and to memory means where said cellular location may be stored, said means for controlling further coupled to said illumination means and said means for detecting whereby, in response to said cell locating means, said illumination means is activated and the response from said means for detecting is recorded in said memory means respectively with said cellular location; and (f) display means for presenting data stored in said memory means.

Claim 3

The generalized cytometry instrument as provided in claim 1 further comprising translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 4

The generalized cytometry instrument as provided in claim 2 wherein the means for controlling further comprises signal conditioning means for altering signals to a form suitable for the respective locating, illuminating, detecting, memory and control means operations.

Claim 5

The generalized cytometry instrument as provided in claim 1 wherein the means for detecting detects an optical interaction selected from the group consisting of phase contrast, interference, light scatter, fluorescence, absorption, polarization, and any combination of the foregoing.

Claim 6

The generalized cytometry instrument as provided in claim 2 wherein the means for detecting detects an optical interaction selected from the group consisting of phase contrast, interference, light scatter, fluorescence, absorption, polarization, depolarization and any combination of the foregoing.

Claim 7

The generalized cytometry instrument as provided in claim 5 wherein the surface means further comprises an immobilizing agent selected from the group consisting of agarose and antibodies specific for a determinant associated with said cells to be immobilized.

Claim 8

The generalized cytometry instrument as provided in claim 6 wherein said cell locating means comprises a helium-neon laser, said illumination means comprises an argon-ion laser, and said means for controlling comprises a central processing unit.

Claim 9

The generalized cytometry instrument as provided in claim 1 wherein said display means presents cellular response data as a function of time.

Claim 10

A method for measuring the activation of a cell as an indication of sensitization to a substance comprising the steps of: (a) providing the cells whose sensitization is to be measured; (b) immobilizing said cells on surface means of a generalized cytometry instrument as described in claim 1; (c) reacting said cells with said substance; (d) locating a cell with said cell locating means; (e) illuminating said located cell with said illumination means and detecting a first activation response; (f) further contacting said cells with a general mitogen, locating said previously located cell, illuminating said cell with said illumination means and with said means for detecting measuring a second activation response; and (g) relating said first and said second activation responses to determine the state of sensitization of said cells to said particular substance.

Claim 11

The method of claim 9 wherein the generalized cytometry instrument further comprises translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 12

The method as presented in claim 10 further comprising the step of contacting said cells at least once with a dye or a labeled probe whereby illumination from said illumination means interacts with said dye or labeled probe to provide a detectable activation response.

Claim 13

A method for measuring the activation of a cell as an indication of sensitization to a substance comprising the steps of: (a) providing the cells whose sensitization is to be measured; (b) immobilizing said cells on surface means of a generalized cytometry instrument as described in claim 1; (c) reacting said cells with a general mitogen; (d) locating a cell with said cell locating means; (e) illuminating said located cell with said illumination means and detecting a first activation response; (f) washing said cells to substantially remove all of said general mitogen; (g) further contacting said cells with said specific substance, locating said previously located cell, illuminating said cell with said illumination means and with said means for detecting measuring a second activation response; and (h) relating said first and said second activation responses to determine the state of sensitization of said cells to said particular substance.

Claim 14

The method of claim 12 wherein the generalized cytometry instrument further comprises translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 15

The method as presented in claim 13 further comprising the step of contacting said cells at least once with a dye or a labeled probe whereby illumination from said illumination means interacts with said dye or labeled probe to provide a detectable activation response.

Claim 16

A method for measuring the presence and/or sensitizing effects of a specific substance in a sample fluid comprising the steps of: (a) providing reagent cells capable of being sensitized by the substance to be detected and immobilizing said cells on surface means of a generalized cytometry instrument as provided in claim 1; (b) contacting said cells with the fluid sample containing the specific substance to be detected; (c) locating a cell with said cell locating means; (d) illuminating said located cell with said illumination means and with means for detecting measuring a first activation response; (e) washing said cells to substantially remove substantially all of said sample; (f) further contacting said cells with a general mitogen; (g) locating said previously located cell; (h) illuminating said cell with said illuminating means and with said means for detecting measuring a second activation response; and (i) relating said first activation and second activation responses to determine the presence and/or sensitizing effect of said specific substance in said sample fluid.

Claim 17

The method of claim 15 wherein the generalized cytometry instrument further comprises translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 18

The method as presented in claim 16 further comprising the step of contacting said cells at least once with a dye or a labeled probe whereby illumination from said illumination means interacts with said dye or labeled probe to provide a detectable activation response.

Claim 19

A method for measuring the presence and/or sensitizing effects of a specific substance in a sample fluid comprising the steps of: (a) providing reagent cells capable of being sensitized by the substance to be detected and immobilizing said cells on surface means of a generalized cytometry instrument as provided in claim 1; (b) contacting said cells with a general mitogen; (c) locating a cell with said cell locating means; (d) illuminating said cell with said illumination means and with said means for detecting measuring a first activation response; (e) further contacting said cells with a fluid sample containing the specific substance to be detected; (f) locating said previously located cell, (g) illuminating said cell with said illuminating means and with said means for detecting measuring a second activation response; and (h) relating said first activation and second activation responses to determine the presence and/or sensitizing effect of said specific substance in said sample fluid.

Claim 20

The method of claim 18 wherein the generalized cytometry instrument further comprises translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 21

The method as presented in claim 19 further comprising the step of contacting said cells at least once with a dye or a labeled probe whereby illumination from said illumination means interacts with said dye or labeled probe to provide a detectable activation response.

Claim 22

A method for measuring the activation of cells contained in a sample comprising the steps of: (a) immobilizing the cells in said sample on surface means of a generalized cytometry instrument as provided in claim 1; (b) contacting said cells with synthetic peptides corresponding to activation sites of proteins coded for by oncogenes or virus; (c) locating a cell with said cell locating means; and (d) illuminating said cells with said illumination means and with said means for detecting, measuring a response of said cells for determining the level of cell activation.

Claim 23

The method of claim 21 wherein the generalized cytometry instrument further comprises translocating means for providing comparative motion between the illumination means, means for detecting and said surface means, said translocating means acting in cooperation with said means for controlling and said cell locating means whereby different immobilized cells may be illuminated and their respective responses detected.

Claim 24

The method as presented in claim 22 further comprising the step of contacting said cells at least once with a dye or a labeled probe whereby illumination from said illumination means interacts with said dye or labeled probe to provide a detectable activation response.

Claim 25

The method of claim 23 wherein the surface means further comprises an immobilizing agent selected from the group consisting of agarose and antibodies specific for a determinant associated with said cells to be immobilized; said cell locating means comprises a helium-neon laser, said illumination means comprises an argon-ion laser, and said means for controlling a central processing unit.

Claim 26

The method of claim 21 wherein said step of measuring a response is performed a plurality of times.

Claim 29

The method as provided in claim 26 further comprising after step (d) the step of further reacting said cells with another reagent different from said first reagent and then performing steps (e) and (f).

Claim 30

A generalized cytometry instrument for repetitively measuring properties of live cells comprising: (a) surface means for immobilizing the cells to be studied; (b) illumination means for determining the location of at least two cells on said surface means in one mode of operation, said illumination means capable of operating in a second mode whereby suitable illumination of located cells may be provided to illicit a detectable response from said located cell; (c) detector means for detecting said response from said located cell when said illumination means is operating in said second mode; (d) means for controlling coupled to said illumination means for storing said cellular positions determined during said first operating mode and for operating said illumination means in said second mode; said means for controlling also coupled to said detector means for recording said cellular detectable response when said illumination means is operated in said second mode; and (e) means for display coupled to said means for controlling whereby said cellular response may be provided as a function of time.

Claim 31

A generalized cytometry instrument for repetitively measuring properties of live cells comprising: (a) surface means for immobilizing the cells to be studied; (b) illumination means for determining the location of at least two cells on said surface means in one mode of operation, said illumination means capable of operating in a second mode whereby suitable illumination of located cells may be provided to illicit a detectable response from said located cell; (c) detector means for detecting said response from said located cell when said illumination means is operating in said second mode; (d) means for controlling coupled to said illumination means for storing said cellular positions determined during said first operating mode and for operating said illumination means in said second mode; said means for controlling also coupled to said detector means for recording said cellular detectable response when said illumination means is operated in said second mode; and (e) means for display coupled to said means for controlling whereby said cellular response may be provided as a function of time.

Claim 32

The generalized cytometry instrument as provided in claim 1 wherein the time length of illumination provided in said second operating mode is adjusted in response to the number of located cells to be studied whereby sensitivity is improved.

Claim 33

The generalized cytometry instrument as provided in claim 1 wherein the illumination provided by said illumination means is adjusted in response to the number of located cells to be studied whereby sensitivity is improved.

Claim 34

The method of claim 26 wherein said step of measuring at least one property comprises measuring fluorescence for a period of time until a given integral of photons is obtained and determining the inverse of said period of time required whereby said inverse of illumination time may be displaced as a representation of the number of fluorescent sites per cell.