Microscope and method for SPIM microscopy

A method for selective plane illumination microscopy (“SPIM”) with a microscope;

• wherein the microscope comprises:
• an illumination arrangement comprising
• an illumination light source;
• and
• an illumination beam path configured to illuminate a sample with a light sheet;
• a detection arrangement configured to detect light emitted by the sample with an objective;
• wherein the light sheet is substantially planar in a focus of the objective or in a defined plane in a vicinity of the focus of the objective, and the objective has an optical axis which intersects the plane of the light sheet at an angle different than zero;
• wherein the method comprises:
• displacing the sample through the light sheet in direction of the optical axis of the objective to detect different sample planes;
• illuminating the sample is under at least a first illumination angle and a second illumination angle;
• detecting a plurality of sample planes at each illumination angle, and storing the sample planes as at least a first image stack and a second image stack;
• and
• wherein the method further comprises:
• a step 1 of aligning the first and second image stacks relative to one another so that coordinate systems of all of the image stacks are aligned in a coordinate system of the first image stack;
• a step 2 of combining the first and second image stacks into a three-dimensional combined image stack;
• a step 3 of projecting the three-dimensional combined image stack into a two-dimensional rendering;
• a step 4 of aligning sample features captured from different illumination directions of the two-dimensional rendering relative to one another with respect to position;
• a step 5 of determining a coordinate transformation from coordinates of the aligned sample features;
• and
• a step 6 of applying the coordinate transformation for alignment to the combined image stack.