Monitoring and control of droplet sorting

A method for sorting particles in a stream of a sample in a flow cytometer, comprising:

• (a) producing from a stable sort having desirable sort characteristics multiple images of a portion of said stream, each said image of said stream by
• i. positioning said stream between an image capturing device and an illumination field, said illumination field comprising a light source and an opaque mask of a pre-determined pattern interposed between said light source and the stream, wherein said pre-determined pattern includes an elongate slit extending substantially parallel to the stream, and wherein said elongate slit has a width of about 3 to 30 times a width of the stream being imaged providing a pattern of contrasting light and dark areas, and
• ii. obtaining images of at least a portion of the stream backlit by said illumination field such that said images of the stream include an image of said pattern of contrasting light and dark areas as imaged within the fluid stream itself;
• (b) generating from said multiple images an averaged numerical reference standard representative of said stable sort;
• (c) continuously collecting during the sorting multiple series of consecutive images of the same portion of said stream;
• (d) generating from each said multiple series of consecutive images (c) at least one numerical sample average representative of the sample sort of each collection of said multiple running images;
• and
• (e) comparing each numerical sample average (d) to said numerical reference standard (b) and determining whether average (d) exhibits a deviation from said reference standard that requires an adjustment of said sort,
• wherein said process continually corrects for any said deviation.

A method for sorting particles in a stream of a sample in a flow cytometer, comprising:

• (a) producing from a stable sort having desirable sort characteristics multiple images of a portion of said stream, each image of said stream being captured by
• i. positioning said stream between an image capturing device and an illumination field, said illumination field having a pattern of contrasting light and dark areas, and said image capturing device having an angle of light acceptance that captures a full width of the stream and light from said illumination field projecting externally and adjacent the lateral edges of the stream;
• and
• ii. obtaining images of at least a portion of the stream backlit by said illumination field such that said images of the stream include an image of said pattern of contrasting light and dark areas as imaged within the fluid stream itself, wherein said dark areas mark the lateral edges of the stream as seen in said images, and
• (b) generating from said multiple images an averaged numerical reference standard representative of said stable sort;
• (c) continuously collecting during the sorting multiple series of consecutive images of the same portion of said stream;
• (d) generating from each said multiple series of consecutive images (c) at least one numerical sample average representative of the sample sort of each collection of said multiple running images;
• and
• (e) comparing each numerical sample average (d) to said numerical reference standard (b) and determining whether average (d) exhibits a deviation from said reference standard that requires an adjustment of said sort,
• wherein said process continually corrects for any said deviation.

A method for sorting particles in a stream of a sample in a flow cytometer, comprising:

• (a) producing from a stable sort having desirable sort characteristics multiple images of a portion of said stream;
• (b) generating from said multiple images an averaged numerical reference standard representative of said stable sort;
• (c) continuously collecting during the sorting multiple running images of said portion of said stream;
• (d) generating from said multiple running images (c) at least one numerical sample average representative of the sample sort of each collection of said multiple running images;
• and
• e) comparing each numerical sample average (d) to said numerical reference standard (b) and determining whether average (d) exhibits a deviation from said reference standard that requires an adjustment of said sort,
• wherein said numerical reference standard and said numerical sample average are selected from the group consisting of:
• i. the averaged slope of the upscam edge of an undulation appearing downstream of the neck region of histograms generated from each multiple of images (a) or (c);
• ii. the averaged slope of the downstream edge of the undulation appearing upstream of the neck region of said histograms;
• iii. the average of the sum of the absolute values of the slopes (i) and (ii) for said histograms;
• iv. the averaged midpoint of the segment defining the slope of the downstream undulation of said histograms;
• v. the averaged integral of the area of said histograms between the lower of the slope valves of slopes (i) and (ii);
• and
• vi. combinations thereof.

A system for storing particles in a stream of a sample in a flow cytometer comprising:

• a) an imaging means for producing multiple images of a portion of said stream;
• and
• b) at least one information processing device for
• i. generating from multiple images of a stable sort having desirable characteristics an averaged numerical reference standard representative of said stable sort;
• ii. continuously collecting during the sorting multiple running images of said portion of said stream and generating from said multiple running images at least one numerical sample average representative of the sample sort of each collection of said multiple running images;
• and
• iii. comparing each numerical sample average to said numerical reference standard and determining whether any of said sample averages exhibits a deviation from said reference standard requiring an adjustment to said sort;
• wherein said device (b) converts said multiple images of said stable sort into a single average histogram having a downstream and upstream direction of the X axis, at least one neck region, and at least one undulation downstream of said neck region, said undulation comprising a positive slope and a negative slope;
• determines from each histogram a selected numerical value corresponding to the reference standard;
• and averages said selected numerical values from the multiple sample histograms of the multiple sample images to create at least one numerical sample average of the sample sort of each collection of multiple running images,
• wherein said numerical value is selected from the group consisting of:
• iv. the slope of the upstream edge of an undulation appearing downstream of the neck region of each histogram;
• v. the slope of the downstream edge of the undulation appearing upstream of the neck region of each histogram;
• vi. the average of the sum of the absolute values of the slopes (iv) and (v) for each histogram;
• vii. the midpoint of the segment defining the slope of the downstream undulation of each histogram;
• viii. the integral of the area of the histogram between the lower of the slope values of slopes (iv) and (v);
• and
• ix. combinations thereof.

A system for sorting particles in a stream of a sample in a flow cytometer comprising:

• a) an imaging means for producing multiple images of a portion of said stream;
• b) an illumination field comprising a light source and an opaque mask of a pre-determined pattern of light and dark areas interposed between said light source and the stream, wherein said pre-determined pattern includes an elongate slit extending substantially parallel to the stream, and wherein said elongate slit has a width of about 3 to 30 times a width of the stream being imaged, and wherein said imaging means is positioned to obtain images of at least a portion of the stream backlit by said illumination field such that said images of the stream include an image of said pattern of contrasting light and dark areas as imaged within the fluid stream itself;
• and
• c) at least one information processing device for
• i. generating from multiple images of a stable sort having desirable characteristics an averaged numerical reference standard representative of said stable sort;
• ii. continuously collecting during the sorting multiple series of consecutive images of said portion of said stream and generating from said multiple series of consecutive images at least one numerical sample average representative of the sample sort of each collection of said multiple series of consecutive images;
• and
• iii. comparing each numerical sample average to said numerical reference standard and determining whether any of said sample averages exhibits a deviation from said reference standard requiring an adjustment to said sort, wherein said system continually corrects for any said deviation.

A system for sorting particles in a stream of a sample in a flow cytometer comprising:

• a) an imaging means for producing multiple images of a portion of said stream;
• b) an illumination field having a pattern of contrasting light and dark areas, and wherein said imaging means is positioned to obtain images of at least a portion of the stream backlit by said illumination field such that said images of the stream include an image of said pattern of contrasting light and dark areas as imaged within the fluid stream itself, wherein said dark areas mark the lateral edges of the stream as seen by said imaging means, and wherein said imaging means has an angle of light acceptance that captures a full width of the stream and light from said illumination field projecting externally and adjacent the lateral edges of the stream;
• and
• c) at least one information processing device for
• i. generating from multiple images of a stable sort having desirable characteristics an averaged numerical reference standard representative of said stable sort;
• ii. continuously collecting during the sorting multiple series of consecutive images of said portion of said stream and generating from said multiple series of consecutive images at least one numerical sample average representative of the sample sort of each collection of said multiple series of consecutive images;
• and
• iii. comparing each numerical sample average to said numerical reference standard and determining whether any of said sample averages exhibits a deviation from said reference standard requiring an adjustment to said sort, wherein said system continually corrects for any said deviation.

A flow cytometer for sorting particles in a fluid stream, comprising:

• a) an illumination field comprising
• i. a light that comprises Light emitting diodes;
• and
• ii. an opaque mask of a pre-determined geometric pattern of contrasting light and dark areas, wherein said mask consists of a single elongate slit defining said geometric pattern and extends substantially parallel to the fluid stream being imaged, said mask interposed between said light source and the fluid stream, said illumination field positioned relative to said fluid stream for illuminating the fluid stream from a side thereof, said illumination field providing a pattern of contrasting light and dark areas;
• and
• b) an image capturing device positioned to obtain an image of at least a portion of the fluid stream backlit by said illumination field such that said image of the fluid stream captured by said image capturing device includes said pattern of contrasting light and dark areas imaged within the stream acting as a lens, said dark areas marking the lateral edges of the fluid stream, wherein said image of said stream is improved in contrast, permitting more control of said sorting.

A flow cytometer for sorting particles in a fluid stream, comprising:

• a) an illumination field comprising
• i. a light source that comprises light emitting diodes;
• and
• ii. an opaque mask of a pre-determined geometric pattern of contrasting light and dark areas interposed between said light source and the fluid stream, wherein said geometric pattern includes at least one distinguishing mark that appears as a reference mark within said image of the fluid stream, and wherein said field is positioned relative to said fluid stream for illuminating the fluid stream from a side thereof;
• and
• b) an image capturing device positioned to obtain an image of at least a portion of the fluid stream backlit by said illumination field such that said image of the fluid stream captured by said image capturing device includes said pattern of contrasting light and dark areas imaged within the stream acting as a lens, wherein said image of said stream is improved in contrast, permitting more control of said sorting.