Calibrated focus sensing

A focusing method, comprising

• (a) transmitting collimated radiation through a focusing apparatus to a primary beam splitter, then to a calibration beam splitter, wherein a first portion of the collimated radiation from the calibration beam splitter is transmitted by the primary beam splitter, to form a first image on a detector,
• wherein an astigmatism is generated in the collimated radiation prior to being transmitted to the calibration beam splitter;
• (b) transmitting a second portion of the collimated radiation from the calibration beam splitter through an objective to a vessel, wherein collimated radiation is reflected from the vessel;
• (c) transmitting the reflected radiation through the objective, then through the calibration beam splitter, then through the primary beam splitter to form a second image on a detector;
• (d) determining a calibration state for the focusing apparatus from the first image;
• (e) determining a focus correction from the second image and from the calibration state;
• and
• (f) adjusting a relative position of the objective and the vessel according to the focus correction.

An apparatus for evaluating focus, comprising

• (a) a stage configured to hold a specimen;
• and
• (b) an optical train comprising a radiation source, a calibration optic, an objective, and detector,
• the optical train forming a first path wherein radiation from the radiation source is directed to the calibration optic and then a first portion of the radiation is directed to the detector, thereby forming a first image on the detector,
• wherein a second portion of the radiation follows a second path from the calibration optic then through the objective to the specimen,
• wherein the optical train forms a third path wherein radiation reflected from the specimen is transmitted through the objective, then to the detector, thereby forming a second image on the detector, and
• wherein the radiation that forms the first image is astigmatic.

A focusing method, comprising

• (a) transmitting radiation through a focusing apparatus to a calibration optic that directs a first portion of the radiation to a detector, thereby forming a first image on the detector, wherein the radiation that forms the first image is astigmatic
• (b) transmitting a second portion of the radiation from the calibration optic through an objective to a vessel, wherein radiation is reflected from the vessel;
• (c) transmitting the reflected radiation through the objective, then to the detector, thereby forming a second image on the detector;
• (d) determining a calibration state for the focusing apparatus from the first image;
• (e) determining a focus correction from the second image and from the calibration state;
• and
• (f) adjusting a relative position of the objective and the vessel according to the focus correction.