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http://www.google.com/patents/US4765737

The present invention relates generally to the field of cell flow cytometry using light measurement and cell sorting which includes the measurement of the size of selected cells, and more particularly with the making of the measurement of the size of the cell when the cell is the only cell passing at right angles through a light beam and the cross section of the light beam is larger than the cell size to be measured.

There are many publications relating to the measurement of sizes of cells in the field of flow cytometry and cell sorting. As the field of biotechnology expands, the application to biological cells will be manifold.

An article by J. A. Steinkamp and H. A. Crissman in the Journal of Histochemistry and Cytochemistry, Vol. 22, No. 7, pp. 616-621 (1974) entitled "Automated Analysis of Deoxyribonucleic Acid, Protein and Nuclear to Cytoplasmic Relationships in Tumor Cells and Gynecologic Specimans" is representative of the prior art. In that publication, cells to be analyzed are stained with fluorescent material and passed in a stream through a laser beam at right angles where both low angle forward scatter of light and fluorescent emmissions are measured at right angles from both the axis of flow and the laser beam. Red and green fluorescent signals are amplified and then integrated by electronic integrators to provide an output signal with an amplitude proportional to total DNA and protein content respectively. One color measured DNA and the other color measured protein content. The integrators contained additional circuitry to detect the time duration in which fluorescence signals were above a threshold level (i.e. cross-over timing). These time spans were then converted to signal amplitudes which are proportional to nuclear and cytoplasmic diameters using time-to-voltage height converters. Steinkamp describes his system as another technique for determining nuclear and cytoplasmic size relationships of cells stained with two fluorochromes specific to the nucleus and cytoplasm. He calls his technique "time of flight" as measuring the time it takes for the cell nucleus and cytoplasm to cross through a narrow laser beam. He states that if the cell flow rate (velocity) is constant, then the nuclear and cytoplasmic diameters are proportional to the time of flight across the beam. He concludes that a new methodology is demonstrated in which the duration of time of flight across a narrow laser beam is used to determine nuclear and cytoplasmic sizes and that improvements in laser beam shaping optics are being incorporated into the system to provide even narrower beams to measure cell dimensions more accurately.

Later, an article by T. K. Sharpless and M. R. Melamed in the Journal of Histochemistry and Cytochemistry, Vol. 24, No. 1, pp 257-264 (1976) entitled "Estimation of Cell Size from Pulse Shape in Flow Cytofluorometry" confirms that as long as the beam of light traversed by the cell is not broad compared to the cell diameter, the time course of fluorescence emission will be a blurred one-dimensional image of the cell and from such an image one might expect at least a good approximation of cell size. Moreover, the publication goes on to describe two alternate amplitude-independent estimates of pulse width. The first is based on a threshold at some fraction of pulse height, or on a pair of thresholds scaled to some fixed central fraction of the total intensity. The second is based on the ratio of pulse area to peak height. Both of these methods require that the pulse shape be stored in a high quality delay line until the peak height or total intensity has been measured and held.

Later, a textbook was published by Wiley & Sons, Inc. (1979) entitled Flow Cytometry and Sorting and edited by Melamed et al. Chapter 6 of that book entitled "Slit-scanning and Pulse Width Analysis" by Leon L. Wheeless, Jr. is pertinent background to the teaching of the present invention in that it examines and reinforces the custom and practice of using the benefits of the narrow laser beam requirements for the measurement of the size of cells in flow cytometry equipment wherein the phrase one-dimensional slit-scan system is used to characterize the narrow laser beam impinging on the path of flowing cells at right angles and the course of either the forward light scatter signal or the fluorescence emmission is a blurred one dimensional image of the cell being measured. Based on the technique described in Chapter 6, laser resolution slit-scan systems are widely used in present day cell flow cytometry and cell sorting systems, and narrow laser beams (when compared with the size of the cells being measured) are widely used as an accepted technique. This is true despite the fact that the use of the narrow laser beam gives rise to significant disadvantages. They are: (1) The construction of and maintenance of the very narrow beam is critical to the operation of the equipment; (2) The narrow laser beam is applied to only a small fraction of each cell at any one time, and the light scatter or secondary light emission from the fluorescent material on the cell does not depict the nature of the cell with fidelity without the use of additional electronic circuitry including a high quality delay line for the purpose of piecing the signal from plural cross sections of the cells together as required for the measurement of, or sampling of, the total cell under inspection; (3) When the aforesaid additional electronic circuitry, including an electronic delay line and computational devices and or circuits, are used, the output signal from either the forward light scatter detection or the secondary emission detectors cannot be used to initiate the sorting function for each cell of the stream as it passes beyond the flow chamber unless the number of cells passing through the laser in the stream per unit time is relatively low. For example, when the electronic delay lines and the resulting computational complexity are made necessary by the use of the narrow laser beam, the maximum rate of introduction of stained cells into the stream through the flow chamber is about 1000/sec, thereby placing great limitations on the operation of the equipment and particularly the sorting operation.

On the other hand, dropping the electronic delay time from the operating system and relying on a signal from forward light scatter or secondary fluorescent emission from a part of a cell was not acceptable as a basis for sorting (size or other parameters) unless all parts of each of the cells were uniform in their optical characteristics or their fluorescence.

When Becton Dickinson, Inc. of Mountainview, Calif. designed its Fluorescence Activated Cell Sorter, Model 440, it clearly decided to market a piece of equipment which would sort a stream of cells one at a time at a stream rate of up to 5000 cells per second. Therefore, the complications and limitations arising out of narrow laser beams were not acceptable. In fact, the cell size limits for the operation of the FACS 440 is the range from 2u (microns) to 30u, and the laser beam is designed to have a larger cross section of 50u. As the cell passes through the larger beam, the system measures both front scatter of the laser beam light energy as the cell passes through the beam, as well as orthogonal secondary emissions from the cell during that time reflecting characteristic fluorescence from the cell and its nucleus as desired. The method used for measuring cell size under these circumstances, if any, is to assume that the amplitude of the forward light scatter by the cell is directly proportional to cell size and a cell sorting signal is selected as a measure of the amplitude. The amplitude of the forward light scatter signal can only approximate the cell size when the cells do not vary from cell to cell in their spectral characteristics. It is impossible to determine whether a decreased peak scatter signal is the result of smaller size or light absorption of the cell or particle. The amplitude of the signal derived from the secondary emission from fluorescence from the cell or its nucleus similarly lacks accuracy as a measure of the size of the cell and its nucleus because the peak amplitude reflects only the amount of fluorochrome. The system included no functional capability to accurately measure the size of each cell in the stream and sort on that information.

The present invention was made in the circumstance that the present inventor had a Model 440 Fluorescent Activated Cell Sorter with all of the benefits of the laser beam being larger than the cells and a high operating rate well in excess of 1000 cells/sec but no accurate capability to measure size of the cell passing through the laser beam, as well as sort the cells one from another, based on the size measurements. The size of the cells being smaller than the cross section laser beam allowed the cell to be examined as to front scatter and secondary emission in total and not in segments. The time of flight measurement described in the three different scientific papers described herein above identified the measurement of the time it took for a cell (whose size is to be measured) nucleus or cytoplasm to cross through a narrow laser beam. The problem solved by the present invention is to measure the size of a cell by measuring its time of flight passing through a beam which is larger than the cross section of the cell because in that context the cell can be examined in total as well as provide "immediate" information as to the size for recording and/or sorting.

It is a primary object of the present invention to provide a new and improved method for measuring the size of a cell flowing at a constant velocity by measuring its time of flight at a right angle through a light beam which is larger in cross section than the cross section of the flowing cell.

It is another object of the present invention to provide a new and improved method for measuring the size of a cell flowing in a column forming a stream of plural cells at a constant velocity by measuring the cells' time of flight at a right angle, one cell at a time, through a light beam which is larger than the cross section of each of the flowing cells so that each of the cells can be examined in total and provide immediate information as to size for recording and/or sorting.

It is still another object of the present invention to provide a new and improved method for measuring the size of a cell flowing in a column as a stream of plural cells at a constant velocity by measuring its time of flight at a right angle one cell at a time through a light beam which is larger than the cross section of each of the flowing cells so that each of the cells can be examined in total by the low angle forward light scatter in the direction of flow and/or the secondary emmission from the fluorescent material on the surface of the cell as seen from a direction which is orthogonal to the direction of flow of the column of cells and the light from the cells to provide immediate information as to the size of each cell for recording and/or sorting.

It is another object of the present invention to provide a new and improved method for measuring the size of a cell flowing in a column in a stream of plural cells at a constant velocity by measuring its time of flight at a right angle one cell at a time through a light beam which is larger than the cross section of each of the cells such that each cell can be examined in total by the low angle forward light scatter to provide immediate information as to the size of the cell for recording and/or sorting and the flow rate of the cells through the light beam is substantially in excess of 1,000 cells/sec.

It is another object of the present invention to provide a new and improved method for measuring the size of a cell flowing in a column in a stream of plural cells at a constant velocity by measuring its time of flight at a right angle one cell at a time through a light beam which is larger than the cross section of each of the cells such that each cell can be examined in total by the secondary emmission from the fluorescent material on the surface of the cell as seen from a direction which is orthogonal to the direction of flow of the column of cells and the light beam to provide immediate information as to the size of each cell for recording and/or sorting at a flow rate of the cells through the light beam is substantially in excess of 1,000 cells/sec.