An automatic focus system for an optical microscope that facilitates faster focusing by using at least two offset focusing cameras. Each offset focusing camera can be positioned on a different side of an image forming conjugate plane so that their sharpness curves intersect at the image forming conjugate plane. Focus of a specimen can be adjusted by using sharpness values determined from images taken by the offset focusing cameras.

Techniques for acquiring focused images of a microscope slide are disclosed. During a calibration phase, a “base” focal plane is determined using non-synthetic and/or synthetic auto-focus techniques. Furthermore, offset planes are determined for color channels (or filter bands) and used to generate an auto-focus model. During subsequent scans, the auto-focus model can be used to quickly estimate the focal plane of interest for each color channel (or filter band) rather than re-employing the non-synthetic and/or synthetic auto-focus techniques.

Particles such as blood cells can be categorized and counted by a digital image processor. A digital microscope camera can be directed into a flowcell defining a symmetrically narrowing flowpath in which the sample stream flows in a ribbon flattened by flow and viscosity parameters between layers of sheath fluid. A contrast pattern for autofocusing is provided on the flowcell, for example at an edge of a rear illumination opening. The image processor assesses focus accuracy from pixel data contrast. A positioning motor moves the microscope and/or flowcell along the optical axis for autofocusing on the contrast pattern target. The processor then displaces microscope and flowcell by a known distance between the contrast pattern and the sample stream, thus focusing on the sample stream. Blood cell images are collected from that position until autofocus is reinitiated, periodically, by input signal, or when detecting temperature changes or focus inaccuracy in the image data.

A system for optimizing optics is provided. The system is configured to calibrate a position of a reference arm of an interferometric imaging system such that an image of a sample is visible when the sample is positioned at a working distance of an objective lens to provide an initial calibrated position. An image is obtained using the initial calibrated position. Image quality of the obtained image is assessed to determine if the obtained image is a valid image. A path length of the reference arm is adjusted if it is determined that the obtained image is not a valid image. A difference between the calibrated position of the reference arm and the adjusted position of the reference arm is calculated. System elements are adjusted based on the calculated difference such that the ample is visible when the sample is positioned at the working distance at the adjusted position.

The present invention provides a technology whereby relative positioning in the horizontal direction between a specimen observation area in a specimen container and an imaging field of view can be reliably performed, even prior to adjusting the focal position in the vertical direction using an auto-focus system. This specimen observation apparatus: obtains a luminance value for an image at a plurality of locations in the specimen container, prior to performing auto-focus; and uses the number of high-luminance regions and the width of those regions and identifies a central position, in the horizontal direction, in the specimen container or uses the number of low-luminance regions and the width of those regions and identifies the central position, in the horizontal direction, in the specimen container.

A microscope for computational microscopic layer separation may include an imaging device that includes a lens and an image sensor, an illumination system for illuminating a sample, and an actuator to adjust an axial position of a focal plane with respect to the sample. The microscope may also include a processor operatively coupled to the imaging device and the illumination system. The processor may be configured to measure, using the image sensor and the illumination system, optical aberrations of the imaging device at the axial position, and determine whether to adjust the focal plane with respect to the sample in response to the one or more optical aberrations. Various other systems and methods are also disclosed.

An apparatus for evaluating focus, including (a) a stage configured to hold a specimen; and (b) an optical train including a radiation source, calibration optic, 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.

An auto-focusing system is disclosed. The system includes an illumination source. The system includes an aperture. The system includes a projection mask. The system includes a detector assembly. The system includes a relay system, the relay system being configured to optically couple illumination transmitted through the projection mask to an imaging system. The relay system also being configured to project one or more patterns from the projection mask onto a specimen and transmit an image of the projection mask from the specimen to the detector assembly. The system includes a controller including one or more processors configured to execute a set of program instructions. The program instructions being configured to cause the one or more processors to: receive one or more images of the projection mask from the detector assembly and determine quality of the one or more images of the projection mask.

A rapid autofocus method for a stereo microscope includes steps of: calculating a disparity of each of stereo microscopic images in a stereo microscopic calibration image sequence; extracting a clear stereo microscopic image sequence from the stereo microscopic calibration image sequence; then, finding out a largest disparity and a smallest disparity among the disparities of all the stereo microscopic images in the clear stereo microscopic image sequence; at a chosen magnification, arbitrarily acquiring a stereo microscopic image; finally, determining a disparity range according to the disparity of the acquired stereo microscopic image, the largest disparity and the smallest disparity, and realizing an autofocus of a target object in the acquired stereo microscopic images. The disparity range is obtained via once off-line calibration at the same magnification, and applicable to the autofocus at an arbitrary timing.

This disclosure also teaches a system and method for scanning a specimen into a focus-stacked scan. In one embodiment, a method for scanning the specimen into a focus-stacked scan can comprise illuminating the specimen with a light. The specimen can comprise a topography. The depths of the topography can be variable along a z-axis. The method can also comprise dividing the specimen into a plurality of regions. Each of the regions can comprise a regional peak in the topography. Additionally, the method can comprise sampling each of the regions at a plurality of focal planes orthogonal to the z-axis by capturing, at each focal plane, an image of the region. The image can be focused on the focal plane. Lastly, the method can comprise focus-stacking, for each of the region the images within the region, into a focus-stacked image, and stitching together the focus-stacked images.