Efficient haploid cell sorting flow cytometer systems

a flow cytometer and haploid cell technology, applied in artificial cells, biochemistry apparatus and processes, instruments, etc., can solve the problems of reducing the net energy of the beam, cw lasers can have particular disadvantages for applications, and light may be affected, so as to reduce the residence time, improve the rate, and reduce the effect of cw lasers' net energy

Inactive Publication Date: 2006-11-23
XY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032] Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows differentiated particles to be separated into subpopulations at a greater rate than conventional particle analysis systems using a CW laser.
[0033] Another broad object of the invention can be to provide a particle analysis system having a pulsed laser which allows sperm cells of any species of

Problems solved by technology

Such light may also be affected by filters which may reduce the net energy of the beam.
However, in some instances, CW lasers can have particular disadvantages for applications as discussed here.
The beam can result in modification or destruction of the sample being observed.
For example, with respect to sperm cells, irradiation can result in lower fertility of the sperm cells.
This disclosure may teach the disadvantages of a laser diode apparatus.
Factors which can contribute to inaccurate measurements of single particles may include the saturation of a stain or even a conjugate to a particle, variation in the quanta of illumination light, effects from the shape of a particle, and perhaps even electronic noise in the detection apparatus.
An example of a particularly challenging problem is the sorting of X-chromosome bearing and Y-chromosome bearing sperm of mammals at high processing rates and high sorting purities.
There may be, however, a distinct problem in tha

Method used

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  • Efficient haploid cell sorting flow cytometer systems
  • Efficient haploid cell sorting flow cytometer systems
  • Efficient haploid cell sorting flow cytometer systems

Examples

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example 1

[0190] Purified fixed bull sperm heads (also described as bull sperm nuclei), stained in standard conditions with DNA binding stain Hoechst 33342, are used as a performance standard to calibrate a sperm sorting flow cytometer prior to the sorting of live sperm. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz illuminates the sample analysis stream in a flow cytometer operating at standard settings and provides the histogram plot shown in Fig. Ex 1. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to resolve bull sperm nuclei into X-chromosome bearing and Y-chromosome bearing populations using standard conditions.

example 2

[0191] A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz illuminates the sample analysis stream in a flow cytometer operating at standard settings sorting said sperm and provides the histogram plot shown in Fig Ex 2. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to resolve live sperm into X-chromosome bearing and Y-chromosome bearing populations under standard conditions.

[0192] The above sample is sorted for collection of X-chromosome bearing sperm, and the sort collection rate is 3800 live X-chromosome bearing sperm second. A resort analysis of the sample prepared in said manner measures the purity of said sorted sample to be 95%. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to enrich the content of a sperm population from one...

example 3

[0194] A sample of live bull sperm is stained in standard conditions with DNA binding stain Hoechst 33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is equipped with beam splitters and neutral density filters, in five separate conditions, to provide illumination energy beam levels of 160 mW (53% of beam power), 130 mW (43% of beam power), 90 mW (30% of beam power), 60 mW (20% of beam power), and 20 mW (6.6% of beam power), respectively, to illuminate the sample analysis stream of a sperm sorting flow cytometer operating at standard settings sorting said stained sperm and providing the 5 histogram plots shown in Fig Ex 3. This demonstrates that a standard sperm sorting flow cytometer equipped with the pulsed laser is able to clearly resolve live sperm with energies as low as 60 mW (20% of the beam).

[0195] The above samples are sorted at each of the 5 beam energy settings for collection of X-chromosome bearing sperm and Y-chromoso...

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Abstract

A flow cytometry system (1) for sorting haploid cells, specifically irradiatable sperm cells, with an intermittingly punctuated radiation emitter (56). Embodiments include a beam manipulator (21) and even split radiation beams directed to multiple nozzles (5). Differentiation of sperm characteristics with increased resolution may efficiently allow differentiated sperm cells to be separated higher speeds and even into subpopulations having higher purity.

Description

I. FIELD OF THE INVENTION [0001] The present invention relates to a flow system for particle analysis. More specifically, the invention relates to the use of a pulsed laser on a flow system for particle analysis which results in more accurate quantification of measurable properties of individual particles. It may be of particular interest in analyzing populations of very similar particles, at high speeds, allowing more efficient separation of particles into two or more different populations. The invention is particularly useful in the application of separating live X-chromosome bearing and Y-chromosome bearing sperm of all mammals at higher speeds, better purities and with equal or better sperm health outcomes, meaning less damage to sperm. The invention may contribute significant improvements to the economics of sperm sorting. II. BACKGROUND OF THE INVENTION [0002] Lasers can be used to deliver light to biological or non-biological particles and emission spectra can be used for the...

Claims

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Application Information

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IPC IPC(8): G01N33/567A01K67/027C12NC12N5/00C12N5/071G01N15/14G01N33/50
CPCC12N5/0612G01N15/147G01N15/1475Y10T436/12G01N2015/149C12Q3/00G01N33/5005
Inventor EVANS, KENNETH M.GILLIGAN, THOMAS B.SUH, TAE KWANGCOX, TODD A.
Owner XY
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