Apparatus and method for multidimensional characterization of objects in real time

An apparatus for making classification decisions in real time on events and sorting in real time said events based upon said classification decisions comprising:

• means for asynchronously collecting multidimensional data about an event through a plurality of sensors providing analog signals at analog signal outputs, each said analog signal from one of said sensors having a peak for the corresponding event, said multidimensional data defining a space having more than three dimensions, each said analog signal output being coupled to an input of a corresponding channel means, each said channel means including means for sampling the corresponding analog signal and converting said corresponding analog signal to a digital data sample, each said channel means having a separate means for sampling each said analog signal corresponding to an event at the peak of said analog signal corresponding to said event and for converting each said sample to a digital data sample;
• first means for temporarily storing said digital data samples in a FIFO buffer along with data by which data samples corresponding to any particular event may be correlated to that event, and where said FIFO buffer includes a separate FIFO buffer for each said channel means, said FIFO buffer storing said digital data samples in first-in-first-out fashion, and wherein each said separate FIFO buffer includes means to generate a control signal when data is present in said separate FIFO buffer;
• means for synchronizing the access of all said digital data samples which pertain to a particular event by retrieving said data from said FIFO buffer when said control signal indicates that data from selectable ones of said channel means is present in all the FIFO buffers for the selected channels, and for comparing in real time said digital data samples so accessed to the node values of a binary classification decision tree and for generating classification decision data in real time based upon said comparisons;
• second means for temporarily storing said classification decisions for a predetermined time;
• and
• sorting means for retrieving from said second means the classification decision pertaining to each event at a predetermined time and for sorting said event based upon the classification decision pertaining to said event.

A cell-sorting decision-making apparatus comprising:

• a laser for generating a light beam;
• a plurality of scattered light and fluorescent light detectors;
• nozzle means for causing a stream of conductive liquid in which have been entrained a plurality of cells to which fluorescing dye has been attached to flow through said light beam, said cells to be sorted after they pass through the beam of said laser and for causing said stream to break into a plurality of droplets containing said cells to be sorted;
• means for sensing when a cell has passed through said laser beam and for generating a first signal indicating the time of said passage for each said cell so detected;
• means for receiving said first signal for each cell so detected and for generating a second signal in response to said first signal, each said second signal being delayed by a programmable amount from the time of occurrence of said first signal;
• a plurality of channel means, each coupled to one of said scattered light or fluorescent light detectors, each said channel means comprising means for sampling and digitizing the analog signal generated by the corresponding detector as a cell passes through said laser beam, and each said channel means further comprising attenuation means for generating a compensation signal which is an attenuated copy of the signal being sampled by said channel means wherein said attenuation means is programmable, and each said channel means further comprising compensation means for receiving said compensation signals from selected ones of said other channel means and for compensating the signal being sampled for crosstalk between said channel means which could generated errors, said means for sampling and digitizing further comprising sampling means for receiving a corresponding one of said second signals for each event defined as a cell passing through said light beam passage of said cell through said light beam being detected and for sampling the analog signal generated by the corresponding one of said light detectors caused by each said event at the time of receipt of the corresponding one of said second signals, and, said means for sampling and digitizing further comprising digitizing means for converting the samples analog signal from said corresponding light detector to a digital value representing one characteristic of said event;
• a FIFO storage means coupled to each said channel means for asynchronously receiving said digital value corresponding to said one characteristic of each event detected by the light detector coupled to said corresponding channel means and for storing said digital data in first-in-first-out order;
• means in said plurality of channel means to generate a third signal when an event has been detected and for storing said third signal in said FIFO storage means with said digital data characterizing the event which caused the generation of said third signal, said third signal including event number data and channel number data;
• means for receiving said third signal from each said FIFO storage means and for comparing said third signal to sort mode data supplied from an external source indicating which channels are involved in a particular sort mode, and when said third signal has been received from each said FIFO storage means for all the channels involved in a particular sort mode, for sequentially reading the digital values from only the FIFO storage means for the channels involved in a particular sort mode and outputting said digital values on a bus;
• second FIFO means for receiving and storing the digital data from said means for receiving said third signal;
• classification means for reading the data from said second FIFO means and for comparing the digital values representing all the characteristics of an event that were gathered for the current sort mode and for comparing said data to the nodes of a binary classification tree and for traversing through said binary classification tree on a path based upon the results of said comparisons and for making a sort decision based upon the path taken through said binary classification tree.

A method of sorting objects comprising the steps of:

• gathering light scatter and fluorescence data in a multidimensional space having more than three dimensions by passing said objects through one or more laser beams and collecting scattered and fluorescent light using a plurality of detectors and an event detector coupled to one of said detectors to generate an event signal and a plurality of signal conditioning channels, each said channel including a sample and hold circuit and a FIFO buffer, with each said channel coupled to one of said detectors, by generating a sample signal using said event detector and delaying said sample signal a selectable delay interval for each said channel, the amount of delay for each channel being selected to correspond to the time delay from the time an event is detected at the detector to which said event detector is coupled to the time when the signal peak occurs in the signal corresponding to the same event as detected by a detector coupled to that channel, and for coupling said peak in said signal using said sample and hold circuit as triggered by said delayed sample signal, and converting the sampled signal value to digital data, and storing all said digital data corresponding to a particular event in the same relative position in all said FIFO buffers in said plurality of channels along with data indicating the event to which each piece of digital data in said FIFO buffers corresponds;
• resynchronizing all said digital data corresponding to a particular event by retrieving all said data corresponding to a particular event from the same relative locations in said FIFO buffers and comparing said light scatter and fluorescence data in real time to a binary classification tree having node values which define the various classes of events to be separated and making a sort decision in real time regarding substantially all events;
• and
• using said sort decisions to sort said objects in real time.

A method of processing data elements describing a plurality of characteristics of a plurality of events for purposes of sorting said events into classes comprising the steps of:

• asynchronously gathering a plurality of data elements regarding a plurality of different characteristics of each of a plurality of different events to be sorted using a plurality of different channels of signal processing circuitry where each channel gathers data elements regarding one characteristic of each event to be sorted by sampling a signal corresponding to said event when the peak of said signal occurs to generate a data element corresponding to one particular characteristic of the event, and electronically compensating said data elements for crosstalk between all other channels that cause errors in the data elements by receiving compensation signals in each channel from all the other channels and subtracting in each channel the sum of these compensation signals from said data element generated by said channel;
• temporarily storing said data elements as they are gathered until all the data elements regarding a particular event are gathered;
• and
• reading the data regarding an event and comparing the data to the nodes of a binary classification tree and making a sort decision based upon the path taken through said binary classification tree based upon the results of each comparison.

An apparatus for making classification decisions in real time regarding a plurality of events comprising:

• data gathering means including a plurality of light detectors for asynchronously gathering a plurality of digital data derived from signals generated by said light detectors from scattered and fluorescent light caused by objects tagged with fluorescing dyes which have been passed through a plurality of laser beams using a plurality of channel means for storing said data, said digital data defining a multidimensional space describing each said event and said data gathering means including means for temporarily storing said digital data in a separate FIFO buffer for each channel means, said data gathering means including an event detection means for detecting when an event occurs which will cause a pulse signal to occur in each said channel means, and for generating a sample signal upon occurrence of said event, and, after a delay tailored to match the individual delay for any particular channel before said pulse signal in that particular channel peaks, for sending said event signal to that particular channel, and for repeating this process for each said channel means, each said channel means including means to sample said pulse signal upon receipt of said event signal and for converting said sample to said digital data and storing said digital data in said separate FIFO buffer for that channel means, all said channel mean storing digital data pertaining to the same event in the same relative positions in said FIFO buffers, said data gathering means including attenuation means for attenuating the data gathered by each said channel means by a programmable amount and for outputting said attenuated data from each said channel means as a compensation signal, and further comprising compensation means in each said channel means for receiving said compensation signals from predetermined ones of said attenuation means in the other channel means and for summing the compensation signal received and further attenuating the data gathered by said channel means by an amount proportional to the sum of said compensation signals received from said predetermined ones of said attenuation means in said other channel means;
• comparing means for resynchronizing the digital data pertaining to a particular event by retrieving the digital data pertaining to one event from selected ones of said FIFO buffers in said channel means and for comparing in real time said digital data regarding each said event to the nodes of a binary classification tree and including means for making a classification decision based upon the comparison results at each said node.

An apparatus for making classification decisions in real time comprising:

• means for collecting multidimensional analog data about an event;
• means for subtracting out noise from said data, and filtering said data to maximize the signal to noise ratio;
• means for converting said data to digital data by sampling said analog data at its peak and doing an analog to digital conversion on it;
• first means for temporarily storing said data while awaiting a classification decision;
• means for retrieving said selected data from said storage means and for comparing it to a classification decision tree and for generating a classification decision based upon said comparison;
• second means for temporarily storing said classification decisions until they are needed;
• and
• means for retrieving said classification decisions from said second means and for classifying said event based upon the classification decision retrieved from said second means pertaining to said event.

A method for classifying events in real time comprising the steps of:

• asynchronously, electronically gathering a plurality of data about an event, said data defining a multidimensional space having more than three dimensions;
• resynchronizing said asynchronously gathered data by storing said data in a plurality of first-in-first-out buffers, one such buffer being dedicated to each dimension of the multidimensional space such that all said data pertaining to a single event in said multidimensional space can be retrieved at substantially the same time when said event is to be classified;
• reading data about said event out of said first-in-first-out buffers and comparing said data in real time to data organized in the form of a binary classification tree;
• formulating in real time a classification decision based upon the results of the comparison against said binary classification tree;
• storing the classification decision in a buffer such that said classification decision can be retrieved later when said classification decision is needed to take action regarding the event to which it pertains;
• and
• retrieving in real time the classification decision regarding a particular event to be acted upon, and taking the action specified in the classification decision in real time.