Automated and accurate drop delay for flow cytometry

Disclosed is an automated method and apparatus for automatically setting a drop delay period by detecting calibration particles in a waste stream. The drop delay is incremented over a series of drop delays and the number of calibration particles in the waste stream is detected for each drop delay. The drop delay is selected which has the least number of calibration particles in the waste stream.

BACKGROUND

Flow cytometers are useful devices for analyzing and sorting various types of particles in fluid streams. These cells and particles may be biological or physical samples that are collected for analysis and/or separation. The sample is mixed with a sheath fluid for transporting the particles through the flow cytometer. The particles may comprise biological cells, calibration beads, physical sample particles, or other particles of interest, which are collectively referred to herein as “particles.” Sorting and analysis of these particles can provide valuable information to both researchers and clinicians. In addition, sorted particles can be used for various purposes to achieve a wide variety of desired results.

SUMMARY

An embodiment of the present invention may therefore comprise a method of automatically selecting a drop delay period in a flow cytometer comprising: operating the flow cytometer with calibration particles using a plurality of different drop delay periods; deflecting droplets that are charged using the plurality of different drop delay periods; collecting non-deflected droplets in a waste stream for each drop delay period of the plurality of drop delay periods; interrogating the waste stream to cause fluorescent emissions of the calibration particles in the waste stream for each drop delay period; sensing the fluorescent emissions from the calibration particles with a detector for each drop delay period; collecting data regarding a number of calibration particles sensed in the waste stream for each drop delay period; selecting a desired drop delay period from the plurality of different drop delay periods that has a substantially minimal number of the number of calibration particles sensed in the waste stream.

An embodiment of the present invention may further comprise a system for selecting a drop delay period using calibration particles in a flow cytometer comprising: a waste stream that is generated from droplets that are not deflected in the flow cytometer using a plurality of drop delay periods; a waste stream receiver that receives the waste stream; a waste stream interrogation optical source that interrogates the waste stream in the waste stream receiver for each drop delay period of the plurality of drop delay periods; a detector that senses fluorescent emissions from the calibration particles in the waste stream for each drop delay period; an analyzer that determines an amount of the calibration particles in the waste stream for the each drop delay period and selects a drop delay period based upon the amount of the calibration particles in the waste stream.

DETAILED DESCRIPTION OF THE EMBODIMENTS

FIG. 1is a schematic isometric view of an embodiment of the present invention. As illustrated inFIG. 1, a nozzle102receives a sample, which includes particles, e.g., biological cells, calibration beads or other particles (collectively referred to herein as “particles”) from a sample reservoir104. The particles are mixed with a sheath fluid from sheath reservoir106in the nozzle102. This creates a stream111which comprises a liquid sheath that is formed around the particles from the sample reservoir104that helps to impart a substantially uniform velocity and flow of the particles in stream111. The particles in the stream111intersect a laser beam109from the interrogation laser108. The point at which the laser beam109intersects the stream111is referred to as interrogation point113. As particles move through the interrogation point113, the particles may cause the laser beam109to scatter. In addition, the laser beam may excite the particles, which have fluorescent properties, such as fluorescent markers, that adhere to or are contained within certain particles of interest. Alternatively, calibration beads that have fluorescent properties may be included in the stream111. In this manner, the drop delay time (explained below) can be determined using calibration beads, rather than sample particles. In addition, the calibration beads may provide a more accurate way of setting the drop delay time because the fluorescent response of the calibration beads is strong.

The scattered light is normally detected by detector110while the fluorescent emissions are detected by detector112. Detectors110,112are normally photomultiplier tubes that can be quite expensive. Detectors110,112can also comprise photodiodes or other light detecting devices, which receive light emitted at the interrogation point113. Controller/analyzer140is connected to detectors110,112, which analyzes the data generated by detectors110,112to generate a control signal to activate the charging unit142to sort cells based upon physical and fluorescent properties, as detected by detectors110,112. The process of sorting is explained in more detail below.

The embodiment ofFIG. 1illustrates an example of a method for electrostatically separating droplets115using charged plates114,116. The sheath fluid and sample that exit the nozzle102initially form a stream111, which separates into individual droplets115. To assist in causing the droplets to consistently form at a selected and consistent location along the stream111, the stream111is vibrated, typically by a piezoelectric device (not shown). The droplets115form at a droplet separation point117, which is a certain consistent distance along the stream111, depending upon the velocity, viscosity and size of the stream111, as well as the intensity and frequency of the piezoelectric vibrator. For each new nozzle102, the distance between the interrogation point113and the droplet separation point117varies. The distance between the interrogation point113and the droplet separation point is referred to as the drop delay distance. The drop delay distance can be varied by varying the piezoelectric vibration. The time that it takes for a cell to move from the interrogation point113to the droplet separation point117, i.e., the drop delay distance, is referred to as the drop delay time. Also, the sheath fluid from sheath reservoir106is electrically conductive, since it contains electrolytes, such as various salts.

The controller/analyzer140ofFIG. 1functions to generate a control signal that is applied to the charging unit142to charge the electrically conductive stream111with either a positive or negative charge. If a droplet is being formed that includes a particle of interest, a positive or negative charge is applied to stream111, depending upon the type of particle, which is isolated into a droplet115. Accordingly, when a particle of interest is detected by detectors110,112, the drop delay time between the travel of a cell between the interrogation point113and the droplet separation point117must be known very accurately so that the proper droplet is charged. If the proper droplet is charged, the droplet will be deflected either into deflected droplet stream118or deflected droplet stream120, depending upon the charge of the droplet. If the droplet is uncharged, the droplet will fall directly downwardly in the waste droplet stream122and not be deflected into either deflected droplet stream118,120. In other words, when the stream111is charged by the charging unit142through the nozzle102, just before the droplet115separates from the stream111at the droplet separation point117, the charge on the stream111will be isolated in a droplet115. The isolated charge in the droplet115will then cause the droplet to be deflected by charged plates114,116, so that the droplet is deflected into either deflected droplet stream118or deflected droplet stream120, based upon the isolated charge of the droplet.

As can be readily appreciated, the travel time delay of particles from the interrogation point113to the droplet separation point117(droplet delay distance), which is referred to as the “drop delay time” or “drop delay period,” is critical in the process of charging the proper droplet that contains the particles of interest. If a correct drop delay time is not used, a cell of interest may end up in the waste droplet stream, or in an incorrect deflected droplet stream118,120.

The deflected droplet streams118,120are collected in deflected droplet stream reservoirs (not shown). The waste droplet stream122is collected in a waste stream collector124. The waste stream collector124is connected to a waste tubing126. The waste tubing126, in one example, may comprise FEP tubing with an internal diameter of 0.5 mm and an external diameter of 1/16 inch. Pump136pumps the collected waste from the waste stream collector124through the waste tubing126to a waste disposal138. To keep the collected waste stream from backing up in the waste tubing126, pump136pumps the collected waste stream123at a rate that is slightly faster than the rate at which fluid enters the waste tubing126. For example, in one embodiment, fluid enters the waste tubing126at approximately 8 mL/min and the waste pump136is operated at approximately 12 mL/min. Optical source128generates a beam130that interrogates the collected waste stream123in the waste tubing126at a wavelength of approximately 532 nm. The optical source may comprise a laser, an LED, or other optical source. The beam130is focused on a waste interrogation point144to interrogate the waste droplet stream collected in the waste tubing126. The FEP material of the waste tubing126is substantially transparent to the beam130at 532 nm, so that the laser beam130can interrogate the collected waste stream123in the waste tubing126at the waste interrogation point144. In addition, the waste tubing126is substantially transmissive to the fluorescent emissions of calibration particles interrogated by the beam130at waste interrogation point144. The fluorescent emissions132are gathered and focused on the detector134, which detects the presence of fluorescent emissions of calibration particles in the collected waste stream123in the waste tubing126.

When the detector134detects the presence of fluorescent emissions, as shown inFIG. 1, the detector134generates a signal that is applied to the controller/analyzer140. Fluorescent emissions are created by calibration particles that have fluorescent markers, or are made with a fluorescent material, including calibration beads that are typically used for calibrating the drop delay. It should be noted that the drop delay can also be calculated with actual samples that have fluorescent markers, or by detecting scattered light from samples or calibration particles. Pump136draws the collected waste stream123through the waste tubing126at a substantially constant rate, which allows the detector134to detect calibration particles in the collected waste stream123at a substantially constant rate. Each time the detector134detects a fluorescent emission, an electrical signal is transmitted to the controller/analyzer140. The controller/analyzer140collects and analyzes the data, as explained in more detail below. The existence of calibration particles in the collected waste stream123is an indication of whether the drop delay time is properly set in the flow cytometer100. As explained below, data relating to the existence of calibration particles in the collected waste stream123is an indication that the drop delay time is incorrect. As also disclosed below, the drop delay time is varied and data is collected for various drop delay times, so that a proper drop delay time can be selected.

FIG. 2is a schematic isometric view of another embodiment of a flow cytometer200. As illustrated inFIG. 2, the waste droplet stream222is collected in the waste stream collector224in a manner similar to that shown inFIG. 1. Waste tubing226transports the collected waste stream228via pump230. The waste interrogation laser generates a laser beam that interrogates the collected waste stream228at the waste interrogation point204. Fluorescent emissions212are generated by calibration particles that have been tagged with a fluorescent marker or calibration beads that have fluorescent properties. The collection optics206collects the fluorescent emissions212and generates a focused fluorescent emission214that is applied to the input of a fiber optic cable208. En route to fiber optic cable208, the focused fluorescent emissions212are filtered by the fluorescent filters207, such that the focused fluorescent emissions214do not substantially include the scattered light from laser beam205. The fiber optic cable208transmits the focused fluorescent emissions214to the output of the fiber optic cable208. The fiber optic output216, which comprises the focused fluorescent emissions214, are directed to a focusing reflector210. The focusing reflector210generates a reflected fiber optic output218that is applied to the input of detector220. Detector220is used to detect both the fluorescent emissions212from the collected waste stream228and fluorescent emissions from particles in the stream238. The output of the detector220is applied to an analyzer/controller232that separates data from the detector220, as explained in more detail below. The analyzer/controller232is coupled to a charging unit234to charge the nozzle236in the same manner as described above with respect toFIG. 1.

During the process of calibrating the flow cytometer100for sorting particles based upon drop delay time, calibration beads are typically utilized that are provided in a steady stream from the sample reservoir104. As indicated above, the calibration beads may be constructed, at least in part, by a fluorescent material that provides consistent and strong fluorescent emissions. Of course, other ways and other types of particles can be used for calibration purposes, including particles that create a scattered emission that can be detected by a detector such as detector134. In addition, other techniques can be used comprising but not limited to visible camera based detection and impedance methods, such as the Counter Principle.

Since the calibration particles from the sample reservoir104are supplied at a substantially constant flow rate, the calibration particles in the collected waste stream123flow through the waste tubing126at a substantially constant rate. As explained in more detail below, the substantially constant rate of flow of calibration particles in the collected waste stream123allows for an accurate method of determining the number of calibration particles in the waste stream for each drop delay period.

The embodiment ofFIG. 2uses only a single detector220to detect both fluorescent emissions from stream238and fluorescent emissions212from the collected waste stream228. Since the photomultiplier tube detectors can be expensive, the cost of the overall flow cytometer200can be reduced by utilizing the single detector220for detecting both the fluorescent emission of particles from the stream238(primary event) and the fluorescent emissions212from the collected waste stream228.

The calibration particles in the collected waste stream228have a velocity that is substantially less than the velocity of the calibration particles in stream238. For example, calibration particles in stream238may move at a velocity of approximately 20 m/s based on driving pressure of 30 PSI and a nozzle exit orifice of 100 μm. On the other hand, calibration particles in the collected waste stream228move at a velocity of only approximately 1.25 m/s due to the 500 μm internal diameter of the waste tubing126which is much larger than the nozzle exit orifice. In this example the calibration particles in the stream238may be moving at 16 times the velocity of those in the waste stream228. The fluorescent emissions212are generated while the calibration particle is interrogated by the waste interrogation laser202at the waste interrogation point204. After the calibration particles move through the interrogation laser beam205, the fluorescent emissions cease. Of course, the same is true for calibration particles that are interrogated by interrogation laser241in stream238. The difference is that the calibration particles in stream238pass through the laser beam from interrogation laser241at a much higher speed and, as such, create a much narrower time pulse that is detected by detectors220,240, than the broader pulse that is generated by the detector220as waste particles pass through the waste interrogation point204at a much slower speed (e.g. 16 times less). Some prior art methods attempt to use high speed, expensive cameras to detect the presence of calibration particles in deflected droplet streams, such as deflected droplet streams118,120. Because of the high speed at which the droplets are moving, high speed, expensive cameras are required to detect calibration particles in the deflected droplet streams118,120and may produce incorrect results if the emissions are missed because of the short duration of the pulses that are produced.

Because of the differences in the pulse width, the output at detector220can easily distinguish between pulses generated by calibration particles in the collected waste stream228and pulses generated by calibration particles in stream238. For example, and not by way of exclusion, electronic exclusion techniques can be used by simply comparing the outputs of detector220and detector240to exclude the narrower pulses generated by calibration particles in stream238. As another example, electronic filtering can be used to separate the narrower pulses from the broader pulses. Further, the broader pulses, generated by the calibration particles in the collected waste stream228, can be distinguished from noise by determining if the pulse has a sufficient magnitude for a sufficient amount of time to qualify as an emission from a calibration particle. For example, the analyzer/controller232may analyze the pulse to determine if the pulse has a magnitude greater than a predetermined threshold for a predetermined period, as explained in more detail with respect toFIG. 7.

FIG. 3is a schematic side view illustrating an embodiment in which fluorescent emissions308can be collected, filtered and focused into a fiber optic cable314. As shown inFIG. 3, collector optics306are disposed in a location to receive the fluorescent emissions308from the waste tubing302that are generated by particles that are interrogated by laser beam316. As explained above, the collected waste stream304in waste tubing302is interrogated by an interrogation laser beam316, so that fluorescent emissions308are generated by calibration particles. The collector optics306collects and focuses the fluorescent emissions308to generate focused fluorescent emissions310. During transit, the fluorescent emissions are filtered by the filter optics318so that substantially only fluorescent light is included in the focused fluorescent emissions. The filtered, focused fluorescent emissions310are focused on the opening312of the fiber optic cable314. Fiber optic cable314then transmits the focused fluorescent emissions310to a focusing reflector406, as illustrated inFIG. 4.

FIG. 4is a schematic isometric view illustrating an embodiment in which the fluorescent emissions from the fiber optic cable402can be focused by the focusing reflector406on a detector404. As shown inFIG. 4, the fluorescent emissions are transmitted by the fiber optic cable402to a location proximate to the focusing reflector406. The laser emissions exiting the fiber optic cable402are focused by the focusing reflector406to a detector404. The projection of light from focusing reflector406onto a plane, as illustrated inFIG. 3, matches the optical detection area of the photomultiplier tube detector404. In this manner, the detector can produce a reliable and consistent output from the fluorescent emissions of waste calibration particles in the waste stream. Collected light, which constitutes fluorescent emissions from the interrogation point of primary stream238, is shown as also impinging on detector404.

FIG. 5is a graph500illustrating the number of detected calibration particles in the waste stream versus the drop delay period.FIG. 5illustrates the various data points, as well as a smoothing curve, which is a simple average of the various data points. As shown inFIG. 5, data is collected multiple times for each drop delay period. For example,FIG. 5illustrates five different calibration particles counts (waste particle detection periods) for each drop delay period, which are shown as small Xs inFIG. 5, that are averaged to create the darker curve, illustrated inFIG. 5. For example, for each drop delay period ofFIG. 5, there are five samples that are taken. Each of the Xs that are vertically aligned for that drop delay period indicate the number of calibration particles that were detected over a predetermined sampling period. For example, a sampling period (waste particle detection period) may comprise 250 milliseconds, during which a detector detects the number of particles in the waste stream. This process is repeated five different times for each drop delay period, in the example illustrated inFIG. 5, to determine the number of calibration particles in each of the five different 250 millisecond periods (waste particle detection periods). In this manner, multiple data points can be collected and averaged to provide more accurate data. Of course, a fewer or larger number of waste particle detection periods can be used for each drop delay period depending upon the desired speed and accuracy of the calibration process. The averaging of the data, as shown by the solid line inFIG. 5, clearly indicates the minimal point in the curve, which is 32.59 drops. The minimal point is the drop delay period that results in the fewest number of calibration particles in the waste stream and indicates the optimal drop delay period. Of course, other techniques can be used, other than just detecting the minimum value of an average signal, including using derivatives to determine curve slopes, curve fitting, cluster analysis and other techniques. One example of a method of determining the optimal drop delay period, is to use a two pass system, where the goal of the first pass is to ascertain an approximate value for the drop delay. Once the approximate value of the drop delay is determined, a finer sweep can be performed to hone in on the precise value. The coarse drop delay operates by sweeping a range of potential drop delays determined by the drop delay frequency, amplitude and sheath pressure. The number of calibration particles in the waste stream is sampled five times per second, for a total of one second, for incrementing the drop delay by the configured coarse step size, which is typically one-half drop. The five readings at each drop delay period are averaged (mean) and the drop delay in the lowest mean is used to perform the fine sweep. The fine sweep is centered on the coarse drop delay, ranging from minus one-half drop to plus one-half drop, i.e., the coarse drop delay increment. The fine sweep increment can be tailored to the desired precision, but is typically set to 0.05 drops. Once again, the waste particle detection period is repeated five times for each drop delay period and the readings are averaged. The ideal drop delay is then determined by smoothing the curve and picking the global minimum.

FIG. 6is a flow diagram600of the sampling and calibration techniques used in accordance with one embodiment of the present invention. As illustrated inFIG. 6, the process starts at step602. The flow cytometer100is loaded with calibration particles in the sample reservoir104. The calibration particles flow through the nozzle102at a substantially constant rate and are surrounded by a sheath fluid. At step604, the drop delay is set at one end of the range of suitable drop delay periods. The system then waits a length of time, referred to as the transit time, which is the time that it takes for a calibration particle to travel from the interrogation point113to the waste interrogation point144. By waiting a time period that is equal to the transit time, or a time period that is slightly greater than the transit time period, from the start of each drop delay period, it is assured that particles for that drop delay period are being detected at the waste interrogation point144, assuming that the waste particle detection period is less than the drop delay period. The waste particle detection period is the period of time during which particles are detected at the waste interrogation point144. There may be multiple waste particle detection periods for each drop delay period so that multiple data points can be collected for each drop delay period. The drop delay period is the period of time during which a selected drop delay is utilized by the flow cytometer100to generate data for that selected drop delay.

At step608ofFIG. 6, the calibration particles are detected and counted during a waste particle detection period. The particle count is then recorded for that waste particle detection period at step710. The waste particle detection period for the data illustrated inFIG. 5is 0.25 seconds. The same drop delay is maintained for the entire drop delay period, which includes multiple waste particle detection periods. The process then proceeds to step612, where it is determined whether more samples are desired for the current drop delay period. If it is determined that more samples are desired for the drop delay period, the process returns to step608and another waste particle detection period is started. The calibration particles are then detected during the next waste particle detection period, at step608, and the number of detected calibration particles are recorded at step610. The process then proceeds again to step612, to determine if more samples are desired. In the data illustrated inFIG. 5, five samples (i.e., five waste particle detection periods) were recorded for each drop delay period. If it is determined at step612that additional samples are not desired, the process proceeds to step614to determine if all of the drop delay periods have been incremented over the full range of the drop delay periods. For example, in the data illustrated inFIG. 5, there are multiple drop delay periods, i.e., on the order of eighty drop delay periods, as indicated along the axis ofFIG. 5. If all of the drop delay periods have not been incremented, the process proceeds to step616, where the drop delay period is incremented. The process then returns to step606, so that new data can be recorded for the next drop delay period. At that point, a new drop delay period is initiated. If the drop delay period has been incremented over the full range of periods, at step614, the process proceeds to step618, where the flow cytometer100is calibrated to operate at the optimal drop delay period.

FIG. 7is a state diagram700illustrating the process for counting pulses as particles in accordance with the detection of particles at the waste interrogation point144and to distinguish calibration particles from noise. The process starts at step702. Detector134generates an electronic signal from the fluorescent emissions132and transmits that signal to the controller/analyzer140. At step704, the controller/analyzer140waits for data that is greater than a threshold value and which is not a primary event. A primary event is a detection by detector220of calibration particles in stream238of the embodiment ofFIG. 2. In the embodiment ofFIG. 1, a separate detector134detects the fluorescent emissions132, so that an exclusion of a primary event is not needed. At step706, a counter is incremented when the magnitude of the fluorescent emission is above a threshold value. In accordance with the embodiment ofFIG. 2, if a primary event occurs, the counting is paused at step708and the system waits until the primary event has finished. Once the magnitude of the fluorescent emission drops below the threshold value, a comparator is used to compare the count in the counter with the count for a minimal pulse. If it is less than the number of counts for a minimal pulse, the process returns to step704. If it is greater than the number of counts for a minimal pulse, the process proceeds to step712and a separate counter, that counts the number of pulses, is incremented to indicate the number of particles. The process then returns to step704.

Accordingly, the embodiments of the present invention provide an accurate way of automatically calibrating the drop delay by counting the selected particles in the waste stream. The drop delay is incremented over a number of different drop delays and the system determines the drop delay for which the least number of calibration particles are present in the collected waste stream123. The system analyzes this data and selects the drop delay period having the minimal number of calibration particles. In addition, a single detector220, in the embodiment ofFIG. 2, can be used to count primary events, as well as waste stream events, to further reduce cost, by using a single detector220to perform both operations.