# US 6,256,096 B1 · Flow cytometry apparatus and method

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- Number: US 6,256,096 B1
- Document ID: US6256096B1
- Status: Granted
- Kind: B1
- Application number: 09/228,247
- Filed: 1999-01-11
- Granted: 2001-07-03
- Earliest claimed: 1999-01-11
- Assignees: SoftRay
- Inventors: Paul E. Johnson
- Web page: https://patentgrep.com/patent/US6256096B1

## CPC

- G01N 15/14: Optical investigation techniques, e.g. flow cytometry
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A flow cytometer for detecting target particles such as microorganisms including biological cells and viruses as well as molecular species. The flow cytometer includes a detection system involving a CCD having a time delay integration capability to thereby increase the signal from the target particle and decrease the noise detected by the CCD. Calibration particles can be included in the sample stream of the flow cytometer for coordinating the readout of the CCD with the rate of flow of the sample stream to improve the detection capability of the CCD. Statistical analysis techniques can also be used to determine the rate of flow of target particles in the sample stream to thereby coordinate the readout rate of the CCD with the rate of flow of the target particles.

## Claims

### Claim 1

Apparatus for identifying particles in a sample stream moving through a flow zone at a rate relative to the flow zone, the sample stream containing target particles, the apparatus comprising:
means for illuminating the sample stream within the flow zone;
a detector for detecting light emitted or scattered from illuminated target particles within the flow zone;
a time delayed integration element for integrating the detected light, the time delayed integration synchronized to the sample stream flow rate; and
means for identifying target particles responsive to the time delayed integration element.

### Claim 2 (depends on 1)

The apparatus of claim 1 wherein the sample stream is contained in a flow cytometer.

### Claim 3 (depends on 1)

The apparatus of claim 1 wherein the illuminating means comprises a laser.

### Claim 4 (depends on 1)

The apparatus of claim 1 wherein the detector comprises a charge coupled device (CCD).

### Claim 5 (depends on 4)

The apparatus of claim 4, wherein the time delayed integration element includes means for shifting CCD pixel rows in synchronization with the sample stream flow rate.

### Claim 6 (depends on 5)

The apparatus of claim 5 wherein the means for identifying target particles includes means for reading pixels the CCD one row at a time.

### Claim 7 (depends on 5)

The apparatus of claim 5 wherein the means for identifying target particles includes means for applying statistical analysis to data collected by the pixels of the CCD to determine pixels related to a single target particle.

### Claim 8 (depends on 1)

The apparatus of claim 1, further including means for measuring the sample stream flow rate including:
a calibration particle in the sample flow; and
means for measuring the velocity of the calibration particle.

### Claim 9 (depends on 8)

The apparatus of claim 8, wherein the sample stream rate is measured in real time while target particles are being detected.

### Claim 10 (depends on 8)

The apparatus of claim 8 wherein the calibration particle is a target particle.

### Claim 11 (depends on 10)

The apparatus of claim 10, wherein the sample stream rate is measured in real time while target particles are being detected.

### Claim 12 (depends on 1)

The apparatus of claim 1, further including means for measuring the sample stream flow rate including:
means for applying statistical analysis to data collected by pixels of the CCD to determine pixels related to a single target particle; and
means for measuring velocity of at least one target particle.

### Claim 13 (depends on 1)

The apparatus of claim 1, further including means for synchronizing the time delayed integration element and the sample stream flow rate by adjusting the time delayed integration element until target particles appear substantially as dots.

### Claim 14 (depends on 1)

The apparatus of claim 1, further including means for detecting two different target particle species, wherein each species produces a unique frequency or phase shift in its scattered or emitted light, wherein the detector includes means for differentiating frequency of phase shift.

### Claim 15 (depends on 1)

The apparatus of claim 1, further including a notch filter between the sample stream and the detector for filtering out undesired light.