Microscope and Method for SPIM Microscopy

摘要

A method for SPIM microscopy with a microscope winch includes (1) an illumination arrangement for illuminating a sample with a substantially planar light sheet, and (2) a detection arrangement for detecting light emitted by the sample with an objective. The sample is displaced through the light sheet in direction of the objective's optical axis, and the sample is illuminated under a first illumination angle and a second illumination angle. A plurality of sample planes are then detected at each illumination angle and stored as at least a first image stack and a second image stack. The image stacks are aligned relative to one another, and are combined in one image stack. A the three-dimensional image stack is projected into a two-dimensional rendering, sample features are aligned, a coordinate transformation is determined, and the coordinate transformation for alignment is applied to the combined image stack.

主权项

1. A method for SPIM microscopy with a microscope;
wherein the microscope comprises:
an illumination. arrangement comprising
an illumination light source; andan 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 carried 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 are into a combined image stack;a step 3 of projecting the three-dimensional image stack into a two-dimensional rendering;a step 4 of aligning sample features captured from different illumination directions relative to one another with respect to position;a step 5 of determining a coordinate transformation from coordinates of the alignment; anda step 6 of applying the coordinate transformation for alignment to the combined image stack.