Compact microscope auto-focus assembly

An auto-focus system for a microscope, comprising:

• a light source generating a laser beam;
• a set of mirrors oriented to direct the laser beam to and from an optical train of the microscope;
• a beamsplitting device configured to split the laser beam into a pair of beams, including a primary beam and a secondary beam, wherein the secondary beam is a mirror image of the primary beam;
• a controller, including executable instructions stored in non-transitory memory that, when executed, cause the controller to:
• apply a common mode rejection to define a centroid of an optical signal generated from reflections of the pair of beams at a sample;
• estimate a location of a target focal plane based on the centroid;
• align a focus of the microscope with the estimated location of the target focal plane;
• and
• maintain the focus of the microscope at the target focal plane during scanning and imaging of the sample.

A method for auto-focusing a microscope, comprising:

• generating coordinates of a first centroid based on a common mode rejection, the first centroid estimated based on a first pair of spectral intensity peaks corresponding to a pair of laser beams reflected from a first interface of a sample;
• inferring coordinates of a second interface of the sample, opposite of the first interface, based on a calibrated displacement of the second interface from the first centroid;
• and
• aligning a focus of the microscope with the second interface and maintaining the focus aligned with the second interface during scanning and imaging of the sample;
• wherein the pair of laser beams are mirror images of one another and split from a parent laser beam by a beamsplitting device.