Photon-sorting spectroscopic microscope system

A spectroscopic microscope system comprising:

• (a) a light source providing a source beam of light that includes a chosen wavelength;
• (b) a microscope objective lens receiving the beam from the light source, the objective lens focussing the beam to a narrow point at which a specimen may be positioned;
• (c) a dichroic mirror positioned in a first beam path to direct light from the light source to the objective lens and to direct fluorescent light from a specimen passed back through the objective lens on a second beam path away from the source;
• (d) a spectral dispersive element comprising a concave curved holographic diffraction grating positioned in the second beam path to receive the fluorescent light and to spread the light according to its spectral content;
• (e) a multiple channel detector having multiple detector elements positioned in an array to receive the light spread by the spectral dispersive element and to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (f) discriminator electronics connected to receive the output signals from the multiple channel detector wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator;
• wherein the laser source is a pulsed source providing short pulses of light at a selected repetition rate, and including means operably connected to the laser source for enabling the counters in each series of counters in sequence at selected times after initiation of a light pulse from the laser source;
• and
• wherein the means for enabling the counters includes a tapped delay line connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, each tap of the tapped delay line connected to an input of an AND gate the other input of which is connected to the comparator and the output of which is connected to one of the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse.

A two dimensional photon sorting spectrometer system comprising:

• (a) a laser source providing a source beam of light in short pulses at a selected repetition rate;
• (b) a microscope objective lens receiving the beam from the light source, the objective lens focussing the beam to a narrow point at which a specimen may be positioned;
• (c) optical elements positioned in a first beam path to direct light from the light source to the objective lens and to direct fluorescent light from a specimen passed back through the objective lens on a second beam path away from the source;
• (d) a spectral dispersive element positioned in the second beam path to receive the fluorescent light from the specimen and to spread the light according to its spectral content;
• (e) a multiple channel array detector having multiple detector elements positioned to receive the light spread by the spectral dispersive element and to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (f) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a and providing a pulse output signal when the detector signal is above the threshold reference and a series of counters each connected to the comparator, and including means operably connected to the laser source for enabling the counters in each series of counters in sequence at selected times after initiation of a light pulse from the laser source.

A two dimensional photon sorting spectrometer system comprising:

• (a) a laser source providing a source beam of light in short pulses at a selected repetition rate;
• (b) a microscope objective lens receiving the beam from the light source, the objective lens focussing the beam to a narrow point at which a specimen may be positioned;
• (c) optical elements positioned in a first beam path to direct light from the light source to the objective lens and to direct fluorescent light from a specimen passed back through the objective lens on a second beam path away from the source;
• (d) a spectral dispersive element positioned in the second beam path to receive the fluorescent light from the specimen and to spread the light according to its spectral content;
• (e) a multiple channel array detector having multiple detector elements positioned to receive the light spread by the spectral dispersive element and to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (f) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator, and including means operably connected to the laser source for enabling the counters in each series of counters in sequence at selected times after initiation of a light pulse from the laser source;
• wherein the means for enabling the counters includes a tapped delay line connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, each tap of the tapped delay line connected to an input of an AND gate the other input of which is connected to the comparator and the output of which is connected to one of the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse.

A spectrometer detector system for use with a laser source for illuminating a specimen wherein the laser source is a pulsed source providing short pulses of light at a selected repetition rate, comprising:

• (a) a multiple channel detector having multiple detector elements that can receive the light spread by a spectral dispersive element to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (b) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator, and including a tapped delay line having multiple taps which is connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, the taps of the tapped delay line and the output of the comparator connected to the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse.

A spectrometer detector system for use with a laser source for illuminating a specimen wherein the laser source is a pulsed source providing short pulses of light at a selected repetition rate, comprising:

• (a) a multiple channel detector having multiple detector elements that can receive the light spread by a spectral dispersive element to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (b) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator, and including a tapped delay line having multiple taps which is connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, the taps of the tapped delay line and the output of the comparator connected to the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse;
• the detector has 32 detector elements and the discriminator electronics has 32 channels, each channel connected to receive the output of a detector element.

A spectrometer detector system for use with a laser source for illuminating a specimen wherein the laser source is a pulsed source providing short pulses of light at a selected repetition rate, comprising:

• (a) a multiple channel detector having multiple detector elements that can receive the light spread by a spectral dispersive element to provide output signals indicative of photon events detected by each detector element in the array;
• and
• (b) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator, and including a tapped delay line having multiple taps which is connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, the taps of the tapped delay line and the output of the comparator connected to the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse;
• wherein there are 32 counters in the series of counters in each channel.

A spectrometer detector system for use with a laser source for illuminating a specimen wherein the laser source is a pulsed source providing short pulses of light at a selected repetition rate, comprising:

• (a) a multiple channel detector having multiple detector elements that can receive the light spread by a spectral dispersive element to provide output signals indicative of photon events detected by each detector element in the array;
• (b) discriminator electronics connected to receive the output signals from the multiple channel array detector, wherein the discriminator electronics includes multiple channels, each channel connected to receive the output of a detector element, each channel including a comparator comparing the output of the detector to which it is connected to a reference and providing a pulse output signal when the detector signal is above the threshold and a series of counters each connected to the comparator, and including a tapped delay line having multiple taps which is connected to receive an enable signal corresponding to the initiation of a light pulse from the laser source and propagating the enable signal along the delay line, the taps of the tapped delay line and the output of the comparator connected to the counters in the series of counters such that the series of counters are enabled to begin counting in sequence at selected periods of time after the initiation of the laser light pulse;
• and
• (c) a series of AND gates the outputs of which are connected each to one of the counters, the inputs of each AND gate connected to one of the taps of the delay line and the output of the comparator.

A method for analyzing fluorescence in a specimen comprising:

• (a) providing a source beam of light that includes a chosen wavelength, the light in the beam being provided in pulses, and directing the beam to a microscope objective lens that focuses the beam to a narrow point in the specimen;
• (b) receiving the fluorescent light that is emitted from the specimen and passed back through the objective lens and separating the fluorescent light from the source beam light;
• (c) spatially dispersing the fluorescent light in accordance with the wavelengths of the fluorescent light and detecting the dispersed fluorescent light with multiple detector elements each of which detects fluorescent light in a different wavelength range, each detector element providing an output signal which is indicative of photon events detected by the detector element;
• and
• (d) counting the photon events detected by each detector element as a function of time over a selected period of time following a pulse of light in the source beam whereby fluorescence lifetime information can be obtained concurrently with spectral information.

A method for analyzing fluorescence in a specimen comprising:

• (2) providing a source beam of light that includes a chosen wavelength, the light in the beam being provided in pulses, and directing the beam to a microscope objective lens that focuses the beam to a narrow point in the specimen;
• (b) receiving the fluorescent light that is emitted from the specimen and passed back through the objective lens and separating the fluorescent light from the source beam light;
• (c) spatially dispersing the fluorescent light in accordance with the wavelengths of the fluorescent light and detecting the dispersed fluorescent light with multiple detector elements each of which detects fluorescent light in a different wavelength range, each detector element providing an output signal which is indicative of photon events detected by the detector element;
• and
• (d) counting the photon events detected by each detector element as a function of time over a selected period of time following a pulse of light in the source beam whereby fluorescence lifetime information can be obtained concurrently with spectral information;
• wherein the step of counting the photon events as a function of time in each channel comprises comparing the output signal from the detector element for the channel to a reference and providing a pulse output signal when the detector signal exceeds the reference to provide a series of pulses corresponding to the photon events detected by the detector element, and further including counting the pulses in the series corresponding to photon events in a series of counters connected to receive the series of pulses, each counter in the series beginning counting at a different period of time after the pulse of light in the source beam is incident upon the specimen, and ending the count in each counter a selected period of time after the rulse of light in the source beam is incident upon the specimen, whereby the value of the counts in the counters is indicative of the photon events as a function of time after the light pulse in the source beam as a function of time for the range of wavelengths detected by the detector for the spectral channel;
• and
• wherein there are 32 or more counters in each spectral channel.