Cytometer with automatic continuous alignment correction
First Claim
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1. A flow cytometry system operable to examine a fluid stream having particles suspended therein, the cytometry system comprising:
- a light source focused on a sensing region in the fluid stream;
an objective positioned to receive light from the sensing region;
a lens positioned to receive light from the objective, wherein the lens and objective define an optical axis;
a field stop having an aperture;
a beam splitter that is positioned between the lens and the field stop that intersects the optical axis at an angle;
an imaging device positioned to generate images from light received from the beam splitter, the beam splitter and imaging device being oriented such that the generated images are along the optical axis;
an XY stage defining a support surface that is precisely controllable in at least two orthogonal directions, wherein the XY stage supports an optical element that determines the alignment of the light source with respect to the sensing region;
a computing device that receives the images generated by the imaging device and calculates from the images at least one parameter that characterizes the alignment of the light source with the sensing region, wherein the at least one calculated parameter is used to generate control signals that are transmitted to the XY stage such that a desired alignment of the light source with the sensing region is maintained.
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Abstract
A system and method for performing flow cytometry is disclosed wherein the alignment of the light source with respect to the sensing region of the fluid stream is continuously controlled using a feedback control loop. An imaging apparatus is provided that images the system along the optical axis, between the field stop and the sensing region. In an embodiment, the cytometer includes a field stop having an H-shaped aperture and light diverters over the ends of the aperture that divert a portion of the light to peripheral detectors.
71 Citations
23 Claims
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1. A flow cytometry system operable to examine a fluid stream having particles suspended therein, the cytometry system comprising:
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a light source focused on a sensing region in the fluid stream; an objective positioned to receive light from the sensing region; a lens positioned to receive light from the objective, wherein the lens and objective define an optical axis; a field stop having an aperture; a beam splitter that is positioned between the lens and the field stop that intersects the optical axis at an angle; an imaging device positioned to generate images from light received from the beam splitter, the beam splitter and imaging device being oriented such that the generated images are along the optical axis; an XY stage defining a support surface that is precisely controllable in at least two orthogonal directions, wherein the XY stage supports an optical element that determines the alignment of the light source with respect to the sensing region; a computing device that receives the images generated by the imaging device and calculates from the images at least one parameter that characterizes the alignment of the light source with the sensing region, wherein the at least one calculated parameter is used to generate control signals that are transmitted to the XY stage such that a desired alignment of the light source with the sensing region is maintained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 22)
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13. A method for performing flow cytometry of a fluid stream having a plurality of particles suspended therein with a cytometer, the method comprising:
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focusing a light source on a sensing region of the fluid stream; using an optical system, receiving light from the sensing region of the fluid stream and focusing the received light along an optical axis that intersects a field stop having an aperture; providing a beam splitter between the optical system and the field stop that is positioned to intersect the optical axis at an angle; with the cytometer operating, aligning the cytometer; imaging the beam splitter with an imaging device wherein the beam splitter and camera are aligned to produce an image along the optical axis; providing an XY stage defining a support surface that is controllable in at least two orthogonal directions, wherein the XY stage supports an optical element that determines the alignment of the light source with respect to the sensing region; and calculating from the image along the optical axis a parameter that characterizes the alignment of the light source with the sensing region for the aligned system; during continuous operation of the cytometer, imaging the beam splitter with the imaging device to generate a plurality of images along the optical axis; calculating from the plurality of images the parameter that characterizes the alignment of the light source with the imaging region for the continuously operated cytometer; comparing the parameter calculated for the aligned system with the parameter calculated for the continuously operated cytometer to determine control signals for the XY stage; and sending the control signals to the XY stage such that the optical element that determines the alignment of the light source is maintained in an aligned position. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 23)
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Specification