Method and device for determining a critical angle of an excitation light beam

An apparatus for determining a critical angle of total reflection at an interface of a sample, comprising:

• a light source operable to illuminate a sample with an angle of incidence;
• an image capturing sensor operable to capture fluorescence images of the sample, wherein each of the fluorescence images comprises a first image area including an object to be examined and a second image area outside the object;
• and
• a processor operable to determine a critical angle of total reflection at an interface of the sample based upon an analysis of the fluorescence images captured at a plurality of different angles of incidence, wherein said analysis comprises:
• forming for each fluorescence image an intensity ratio between a first fluorescence intensity in the first image area of one of the fluorescence images and a second fluorescence intensity in the second image area of said one of the fluorescence images, wherein said ratio being formed by dividing one of the first and second fluorescence intensities by the other of the first and second fluorescence intensities;
• and
• evaluating the intensity ratios for the plurality of angles of incidence.

A method for determining a critical angle of total reflection, comprising:

• illuminating a sample with an excitation light beam of a light source;
• using an image capturing sensor, capturing fluorescence images of at least part of the sample at a plurality of different angles of incidence of the excitation light beam and for at least a first image plane and a second image plane different from the first image plane, wherein each image plane comprises a first image area including an object to be examined and a second image area outside the object;
• and
• using a processor, determining a critical angle of total reflection at an interface of the sample based upon an analysis of the fluorescence images, wherein said analysis comprises:
• forming for each image plane an intensity ratio between a first fluorescence intensity in the first image area of one of the image planes and a second fluorescence intensity in the second image area of said one of the image planes, wherein said ratio being formed by dividing one of the first and second fluorescence intensities by the other of the first and second fluorescence intensities;
• and
• evaluating the intensity ratios for the plurality of angles of incidence.