Scanning microscopy, fluorescence detection, and laser beam positioning

A laser beam positioner having an optical path for a laser beam and a positioning apparatus that includes a mechanical portion having inertia adapted to move to a location having a specific address on a surface which is to receive a focused laser spot, in which the positioning apparatus includes an inertia-less deflector and a deflector control adapted to cause the inertia-less deflector to change the angle of the optical path within a limited range, a position sensor for the portion of the apparatus having inertia is arranged to provide an instantaneous motion signal for said deflector control during settling motions of said portion of said apparatus at said location, and said deflector control is adapted to change the angle of the laser beam in response thereto to move the spot instantaneously with negating motion opposite to the instantaneous settling motions of the portion of the apparatus having inertia, to cause the focused spot to be directed upon the respective location before said settling motions end.

A wide range microscope scanner or laser beam positioner comprising, in combination, a laser, a focusing lens, and a scanning or positioning apparatus comprising a wide range positioner and an inertia-less deflector, a control system which commands the positions of said wide range positioner and said inertia-less deflector, said combination constructed and arranged to position a focused laser spot on an extended surface, to hold said focused laser spot stationary relative to said surface for a time between 10 and 1000 microseconds, and to move said laser spot a few spot diameters from one specific location on said surface to another in less than ten percent of the interval during which said focused laser spot is stationary, the scanning or positioning apparatus constructed and arranged to address a sequence of at least twenty spot diameters in a scan direction.

A fluorescent scanner arranged to produce a laser beam that excites fluorescence at locations on a surface, the scanner having an acousto-optic deflector that controls fine changes in the angle of the laser beam incident upon the surface to prolong the duration of incidence.

A fluorescent scanner arranged to produce a laser beam that excites fluorescence at locations on a surface, the scanner having an electro-optic deflector that controls fine changes in the angle of the laser beam incident upon the surface to prolong the duration of incidence.

A laser beam positioner in which a mechanical component of a positioning apparatus causes a laser beam passing through a focusing lens to move generally continuously, preferably at approximately constant speed, in relation to a surface and an inertia-less deflector, preferably an acousto-optic or electro-optic deflector, responsive to a deflector control, is arranged to change the angle of the laser beam incident upon the surface to produce stepped motion of the beam over the surface.

A fluorescence scanner comprising scanning apparatus constructed and controlled to scan a laser beam across an extended surface to excite fluorophores associated with features on the surface and to detect fluorescence with respect to the features scanned, characterized in that the scanning apparatus includes at least one inertia-less deflector associated with a deflector control that changes the angle of the laser beam during scan of a feature to prolong the incidence of the laser spot on the region of the feature inwardly of its boundary during the course of the scan.

A computer program in machine readable form adapted to perform the steps of converting generally continuous high speed motion, for instance approximately constant speed, to stepped motion across a micro-array of spaced locations of density of at least 400 per square centimeter, preferably, of density of at least 1000 per square centimeter.

A medium carrying information configured to cause a microscope scanner or a laser beam positioner to progressively change the angle of a beam passing through it in accordance with generally continuous motion of a mechanical portion of the scanner or beam positioner through which the beam passes, to cause the beam to dwell on a location on an object in a field of at least 400 locations per square centimeter, preferably at least 1,000 locations per square centimeter, and to step to a next feature in the field, and dwell upon it, in a repetitive manner.

A medium carrying information configured to determine control signals applied to an inertia-less deflector of a microscope scanner or a laser beam positioner to change the angle of a beam passing through it in accordance with motion of a mechanical portion of the scanner or beam positioner through which the beam passes, to cause the beam to stabilize, i.e. stop or dwell, upon a selected location on an object despite the motion of the mechanical portion.

A medium carrying information configured to cause a machine to superimpose a dwell on motion of a beam that would otherwise be moving continuously, e.g. at approximately constant speed, across an array of closely-spaced locations.

A medium carrying information configured to cause a high speed fluorescence scanner to collect fluorescence data based upon periodic dwell of a fluorophore-exciting focused laser beam upon successive features of an array and assign detected data to respective locations on the array.

A method of reading fluorescence from features arrayed on a surface at density in excess of 400 features per square centimeter, preferably at density in excess of 1000 features per square centimeter, comprising exciting fluorophores of the features by scanning a laser spot over the surface and detecting fluorescent photons from respective features, wherein, while scanning proceeds, a laser spot is caused to dwell at a position inwardly from boundary regions of a feature and to step relatively quickly to the next feature.

A microscope scanner, or laser beam positioner comprising a scanning or positioning apparatus constructed and controlled to scan or position a focused spot (e.g. a laser spot) over locations on an extended surface addressed by the optical path from a focusing lens, the scanning apparatus including an inertia-less deflector and a deflector control, characterized in that said inertia-less deflector is associated with a first deflector lens which focuses through a hole in a mask, and a second deflector lens arranged such that said first and second deflector lenses create an image on the focusing lens at the same position on the lens over the range of angles of deflection of said deflector, thereby causing said optical path to rotate about said focusing lens during deflection of said deflector, said mask arranged to block light other than that deflected in first order by said deflector, the focal lengths and locations of said first and second deflector lenses, in conjunction with the initial diameter of a defined input beam and the focal length of said focusing lens determining the diameter of said focused spot on said surface.