Apparatus and method of focusing a laser-scanning cytometer using the reflection of the laser

A method of focusing a scanning microscope on particles of a liquid sample, wherein the particles are arranged on an upper surface of an optically clear bottom of a sample vessel allowing the particles to be imaged through the bottom using an objective lens arranged below the sample vessel, the method comprising:

• scanning a light beam over the particles, using a scan mirror positioned between a light source and the objective lens, while moving the objective lens towards or away from the bottom of the sample vessel;
• for each of a plurality of positions of the objective lens, capturing an image of a spot formed by the light beam being reflected at the upper surface of the bottom;
• calculating a focus score of each of the images;
• determining, based on the focus score, a position of the upper surface of the bottom in relation to the objective lens;
• and
• determining a position of a sample plane, passing through a midpoint of the particles arranged on the upper surface, by adding half an average particle thickness to the position of the upper surface;
• followed by moving the objective lens such that a focal plane of the objective lens coincides with the sample plane.