Computer interfaced scanning fluorescence lifetime microscope applied to directed evolution methodologies and methods for light-mediated patterning in cell selection

A method for imaging and analyzing fluorescence lifetime in cells, the method comprising the steps of:

• providing a sample of cells, the cells containing a fluorescent marker, wherein the sample is disposed on a positioning stage;
• scanning a light beam across the sample of cells through an objective of a scanning microscope, causing the cells to emit fluorescent light;
• detecting the fluorescent light emitted by the cells;
• measuring the lifetime of the fluorescent light;
• correlating the lifetime of the fluorescent light with the position of objective relative to the sample to thereby generate a high-resolution image map of cell fluorescence lifetime;
• and
• storing the high resolution image map.

A method for imaging and analyzing fluorescence lifetime and anisotropy in cells, the method comprising the steps of:

• providing a sample of cells, the cells containing a fluorescent marker, wherein the sample is disposed on a positioning stage;
• scanning a light beam across the sample of cells through an objective of a scanning microscope, causing the cells to emit fluorescent light;
• passing the fluorescent light through a polarizer, to thereby produce two perpendicular planes of polarized light;
• passing each plane of polarized light through a wavelength separator, to thereby produce four fluorescent signals;
• independently detecting each fluorescent signal;
• and
• measuring and recording the lifetime, wavelength region and polarization of each fluorescent signal.

An apparatus for detecting spatially imaged fluorescence lifetime, comprising:

• a high frequency pulsed laser;
• a light detector;
• a time correlated single photon counting board, the board interfaced with the laser and with the light detector;
• and
• a computer, the computer interfaced with the time correlated single photon counting board;
• wherein the laser emits radiation onto a sample thereby causing the sample to emit fluorescent light, the fluorescent light being sensed by the light detector thereby causing the light detector to generate electrical pulses, the electrical pulses being sensed by the time correlated single photon counting board to thereby measure the lifetime of the fluorescent light emitted by the sample, and the lifetime of the fluorescent light emitted by the sample being stored in the computer.

A computer interfaced scanning microscope system for detecting spatially imaged fluorescence lifetime, the system comprising:

• a high frequency pulsed laser;
• an objective lens;
• a positioner located adjacent to the objective lens, the positioner including a stage designed to hold a sample, the positioner designed to move the stage relative to the objective lens to thereby variably position the sample relative to the focal point of the objective lens, and the positioner further designed to sense and record the position of the sample relative to the focal point of the objective lens as a function of time;
• a light detector;
• a time correlated single photon counting board, the board interfaced with the laser and with the light detector;
• and
• a computer, the computer interfaced with the time correlated single photon counting board;
• wherein the laser emits a light beam directed through the objective lens, the objective lens focuses the light beam on the sample on the stage to thereby cause some or all of the molecules in the sample to emit fluorescence;
• wherein at least a portion of the fluorescence passes through the objective to the light detector, the light detector detecting the fluorescence to thereby generate electrical pulses;
• wherein the electrical pulses are sensed by the computer-interfaced time correlated single photon counting board to thereby determine a fluorescence lifetime of the sample, the fluorescence lifetime being the period of time between the emission of light by the pulse laser and the emission of the fluorescence from the sample, and to record the time at which the fluorescence was detected by the light detector;
• and
• wherein data regarding the fluorescence lifetime of the sample and the time at which the fluorescence was detected by the light detector are stored in the computer.

A computer interfaced scanning microscope system for detecting spatially imaged fluorescence lifetime and anisotropy, the system comprising:

• a high frequency pulsed laser;
• an objective lens;
• a positioner located adjacent to the objective lens, the positioner including a stage designed to hold a sample, the positioner designed to move the stage relative to the objective lens to thereby variably position the sample relative to the focal point of the objective lens, and the positioner further designed to sense and record the position of the sample relative to the focal point of the objective lens as a function of time;
• a polarizer;
• wavelength separators;
• light detectors;
• multiplexing electronics interfaced with the light detectors;
• a time correlated single photon counting board, the board interfaced with the laser and the multiplexing electronics;
• and
• a computer, the computer interfaced with the time correlated single photon counting board;
• wherein the laser emits a light beam directed through the objective lens, the objective lens focuses the light beam on the sample on the stage to thereby cause some or all of the molecules in the sample to emit fluorescence;
• wherein at least a portion of the fluorescence passes through the objective to the polarizer, wherein the fluorescence is split into two planes of polarized light, each of which passes through a wavelength separator, the wavelength separator separating each plane of polarized light into two fluorescent signals;
• wherein the light detectors detect the fluorescent signals to thereby generate electrical pulses, the electrical pulses being sensed by the computer-interfaced time correlated single photon counting board to thereby determine the fluorescence lifetime and anisotropy of the sample;
• and
• wherein the fluorescence lifetime and anisotropy of the sample are stored in the computer.

An automated method for screening and selecting cells, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells expressing an imagable property;
• detecting and recording the imagable property;
• identifying and recording locations containing cells expressing a desired characteristic of the imagable property and locations not containing cells expressing the desired characteristic of the imagable property;
• and
• scanning lethal irradiation across the substrate through a high speed shutter, wherein the shutter is open only when the lethal irradiation is positioned over locations not containing cells expressing the desired characteristic of the imagable property to thereby kill the cells in such locations.

An automated method for screening and selecting cells, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells expressing an imagable property;
• detecting and recording the imagable property;
• identifying and recording locations containing cells expressing a desired characteristic of the imagable property and locations not containing cells expressing the desired characteristic of the imagable property;
• applying a sensitizing agent to the substrate, wherein the sensitizing agent is selected to render the sample of cells sensitive to light;
• and
• scanning a light beam across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not containing cells expressing the desired characteristic of the imagable property to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent amplitude, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the amplitude of the fluorescent light;
• correlating the amplitude of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired amplitude;
• and
• scanning lethal irradiation across the substrate through a high speed shutter, wherein the shutter is open only when the lethal irradiation is positioned over locations not emitting fluorescent light with the desired amplitude to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent amplitude, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the amplitude of the fluorescent light;
• comparing the amplitude of the fluorescent light to a predetermined desirable fluorescent amplitude, to thereby determine whether the location contains a cell emitting fluorescent light with the desired amplitude;
• and
• directing lethal irradiation to those locations that do not contain a cell emitting fluorescent light with the desired amplitude to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent amplitude, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the amplitude of the fluorescent light;
• correlating the amplitude of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired amplitude;
• applying a sensitizing agent to the substrate, wherein the sensitizing agent is selected to render the sample of cells sensitive to light;
• and
• scanning a light beam across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not emitting fluorescent light with the desired amplitude to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent amplitude, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the amplitude of the fluorescent light;
• correlating the amplitude of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired amplitude;
• inducing the cells to synthesize an endogenous porphyrin precursor;
• and
• scanning a light beam across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not emitting fluorescent light with the desired amplitude to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent lifetime, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the lifetime of the fluorescent light;
• correlating the lifetime of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired lifetime;
• and
• scanning lethal irradiation across the substrate through a high speed shutter, wherein the shutter is open only when the lethal irradiation is positioned over locations not emitting fluorescent light with the desired lifetime to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent lifetime, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the lifetime of the fluorescent light;
• comparing the lifetime of the fluorescent light to a predetermined desirable fluorescent lifetime, to thereby determine whether the location contains a cell emitting fluorescent light with the desired lifetime;
• and
• directing lethal irradiation to those locations that do not contain a cell emitting fluorescent light with the desired lifetime to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent lifetime, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the lifetime of the fluorescent light;
• correlating the lifetime of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired lifetime;
• applying a sensitizing agent to the substrate, wherein the sensitizing agent is selected to render the sample of cells sensitive to light;
• and
• scanning a light beam across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not emitting fluorescent light with the desired lifetime to thereby kill the cells in such locations.

An automated method for screening and selecting cells based on fluorescent lifetime, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells containing a fluorescent marker;
• directing a light beam onto each location, thereby causing the fluorescent marker to emit fluorescent light;
• automatically detecting the fluorescent light;
• automatically measuring and recording the lifetime of the fluorescent light;
• correlating the lifetime of the fluorescent light with the location containing the cell with the fluorescent marker emitting the fluorescent light;
• generating a kill map of the substrate, the kill map indicating those locations not emitting fluorescent light with the desired lifetime;
• inducing the cells to synthesize an endogenous porphyrin precursor;
• and
• scanning a light beam across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not emitting fluorescent light with the desired lifetime to thereby kill the cells in such locations.

An automated method for screening and selecting cells, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells expressing an imagable property;
• detecting and recording the imagable property;
• identifying and recording locations containing cells expressing a desired characteristic of the imagable property and locations not containing cells expressing the desired characteristic of the imagable property;
• and
• projecting lethal irradiation only onto those locations not containing cells expressing the desired characteristic of the imagable property to thereby selectively kill those cells.

An automated method for screening and selecting cells, the method comprising the steps of:

• providing a substrate with multiple locations, at least some of which contain one or more cells expressing an imagable property;
• detecting and recording the imagable property;
• identifying and recording locations containing cells expressing a desired characteristic of the imagable property and locations not containing cells expressing the desired characteristic of the imagable property;
• applying a sensitizing agent to the cells, wherein the sensitizing agent is selected to render the cells sensitive to light;
• and
• projecting light only onto those locations not containing cells expressing the desired characteristic of the imagable property to thereby selectively kill those cells.

A method for selecting cells exhibiting desirable fluorescence properties, the method comprising the steps of:

• providing a sample of cells, wherein the cells contain fluorescent markers that emit fluorescent light when excited;
• exciting the sample of cells;
• sensing and recording the fluorescence properties of the fluorescent light emitted by the cells in the sample;
• generating a high resolution image map of the cells, based on the fluorescence properties of the cells, indicating those cells exhibiting desirable fluorescent properties as well as those cells not exhibiting desirable fluorescent properties;
• and
• projecting lethal irradiation only onto those cells not exhibiting desirable fluorescence properties to thereby selectively kill those cells.

A method for selecting cells exhibiting desirable fluorescence properties, the method comprising the steps of:

• providing a sample of cells, wherein the cells contain fluorescent markers that emit fluorescent light when excited;
• exciting the sample of cells;
• sensing and recording the fluorescence properties of the fluorescent light emitted by the cells in the sample;
• generating a high resolution image map of the cells, based on the fluorescence properties of the cells, indicating those cells exhibiting desirable fluorescent properties as well as those cells not exhibiting desirable fluorescent properties;
• applying a sensitizing agent to the sample of cells, wherein the sensitizing agent is selected to render the sample of cells sensitive to light;
• and
• projecting light only onto those cells not exhibiting desirable fluorescence properties to thereby selectively kill those cells.

A method for selecting cells exhibiting desirable fluorescence properties, the method comprising the steps of:

• providing a sample of cells, wherein the cells contain fluorescent markers that emit fluorescent light when excited;
• exciting the sample of cells;
• sensing and recording the fluorescence properties of the fluorescent light emitted by the cells in the sample;
• generating a high resolution image map of the cells, based on the fluorescence properties of the cells, indicating those cells exhibiting desirable fluorescent properties as well as those cells not exhibiting desirable fluorescent properties;
• inducing the cells to synthesize an endogenous porphyrin precursor;
• and
• projecting light only onto those cells not exhibiting desirable fluorescence properties to thereby selectively kill those cells.

A method for selectively killing cells, the steps of the method comprising:

• applying a diagnostic fluorophore to a population of cells, wherein some cells in the population show identifiable fluorescent characteristics compared to other cells in the population;
• scanning the population of cells with a light beam from an ultrafast laser, the light beam having a wavelength and intensity selected to detect multiphoton excitation of the fluorophore without causing substantial cell death, wherein the fluorophore in the cells is excited by multiphoton absorption at a focal point of the light beam, causing the fluorophore to emit fluorescent light;
• detecting the fluorescent light;
• generating a high resolution kill map of the population of cells, the kill map indicating those cells emitting fluorescent light in response to the light beam;
• scanning the population of cells with a high intensity light beam from the ultrafast laser, the high intensity light beam having a wavelength and intensity selected to kill the cells, through a high speed shutter, wherein the shutter is open only when the high intensity light beam is focused on cells emitting fluorescent light, to thereby selectively kill those cells.

A method for selectively killing cells, the steps of the method comprising:

• applying a diagnostic fluorophore to a population of cells, wherein some cells in the population selectively absorb the fluorophore at a higher rate than other cells in the population to thereby emit more fluorescent light when excited relative to the other cells;
• focusing a light beam from an ultrafast laser on the population of cells, the light beam having a wavelength and intensity selected to detect multiphoton excitation of the fluorophore without causing substantial cell death, wherein the fluorophore in the cells is excited by multiphoton absorption at the focal point of the light beam, causing the fluorophore to emit fluorescent light;
• detecting the fluorescent light and measuring the total quantity of fluorescent light emitted at the focal point of the light beam;
• comparing the total quantity of fluorescent light emitted at the focal point of the light beam to a pre-determined total quantity of fluorescent light emitted by cells not selectively absorbing the fluorophore to thereby determine whether the cells at the focal point of the light beam have selectively absorbed the fluorophore at a higher rate;
• and
• increasing the intensity of the light beam from the ultrafast light beam to thereby selectively kill only those cells that have selectively absorbed the fluorophore.