Method and scanning fluorescence microscope for multi-dimensional high-resolution imaging a structure or a path of a particle in a sample

A method of multi-dimensional high-resolution imaging a path of a particle in a sample, the particle being marked with a fluorescence marker, the method comprising:

• focusing fluorescence enabling light into the sample, the focused fluorescence enabling light illuminating a measurement area in the sample;
• subjecting a partial area of the measurement area to fluorescence inhibiting light, wherein the partial area omits a center of the measurement area in that an intensity distribution of the fluorescence inhibiting light comprises a line-shaped or plane-shaped intensity minimum extending across the center of the measurement area, and wherein a minimal extension of the intensity minimum in a direction through the center of the measurement area is by a factor k≧2 smaller than a diameter of the measurement area in said direction;
• measuring fluorescence light emitted from the sample out of the measurement area without spatial resolution within the measurement area, wherein the fluorescence light emitted out of the measurement area is measured at a temporal resolution for a plurality of consecutive angle positions of the intensity minimum about the center, wherein the temporal resolution resolves the angle positions of the intensity minimum in the measured fluorescence light, and wherein the measurement area, for each angle position of the intensity minimum, is subjected to the fluorescence enabling light;
• measuring the fluorescence light emitted from the sample out of the measurement area without spatial resolution with the measurement area, wherein the fluorescence light emitted out of the measurement area is measured with spatial resolution for a plurality of consecutive angle positions of the line-shaped or plane-shaped intensity minimum about the center, wherein the temporal resolution resolves the angle positions of the intensity minimum in the measured fluorescence light, and wherein the measurement area is subjected to the fluorescence enabling light for each of the angle positions of the intensity minimum;
• and
• updating a position of the center of the measurement area in the sample such that a predetermined temporal course of intensities of the fluorescence light measured over the plurality of angle positions of the line-shaped or plane shaped intensity minimum is maintained;
• wherein the steps of focusing, subjecting, measuring and updating are repeated for a plurality of measurement areas in the sample.

A scanning fluorescence light microscope for multi-dimensional high-resolution imaging a structure of a sample, which is marked with fluorescence markers, the scanning fluorescence light microscope comprising

• a fluorescence enabling light source providing fluorescence enabling light and configured to illuminate a measurement area in the sample with the focused fluorescence enabling light;
• a fluorescence inhibiting light source providing fluorescence inhibiting light and configured to subject a partial area of the measurement area to the fluorescence inhibiting light, wherein the partial area omits a center of the measurement area in that an intensity distribution of the fluorescence inhibiting light comprises a line-shaped or plane-shaped intensity minimum extending across the center of the measurement area, and wherein a minimal extension of the intensity minimum in a direction through the center of the measurement area is by a factor k≧2 smaller than a diameter of the measurement area in said direction;
• a detector configured to measure fluorescence light emitted from the sample out of the measurement area without spatial resolution within the measurement area;
• a coding device configured to assign a value of the measured fluorescence light to a location defined by the position of the center of the measurement area in the sample;
• and
• a scanning device configured to scan a spatial area of interest of the sample with the center of the measurement area,
• wherein the fluorescence inhibiting light source has an angle adjusting device configured to adjust a plurality of consecutive angle positions of the intensity minimum about the center of the measurement area, wherein the fluorescence enabling light source is configured to subject the measurement area to the fluorescence enabling light for each angle position of the intensity minimum, and
• wherein the detector is configured to measure the fluorescence light emitted out of the measurement area for the plurality of consecutive angle positions of the intensity minimum about the center of the measurement area.

A scanning fluorescence light microscope for multi-dimensional high-resolution imaging a path of a particle in a sample, the particle being marked with a fluorescence marker, the scanning fluorescence light microscope comprising:

• a fluorescence enabling light source providing fluorescence enabling light and configured to illuminate a measurement area in the sample with the focused fluorescence enabling light;
• a fluorescence inhibiting light source providing fluorescence inhibiting light and configured to subject a partial area of the measurement area to the fluorescence inhibiting light, wherein the partial area omits a center of the measurement area in that an intensity distribution of the fluorescence inhibiting light comprises a line-shaped or plane-shaped intensity minimum extending across the center of the measurement area, and wherein a minimal extension of the intensity minimum in a direction through the center of the measurement area is by a factor k≧2 smaller than a diameter of the measurement area in said direction;
• a detector configured to measure fluorescence light emitted from the sample out of the measurement area without spatial resolution within the measurement area;
• and
• a tracking device configured to update a position of the center of the measurement area in the sample;
• wherein the fluorescence inhibiting light source has an angle adjusting device configured to adjust a plurality of consecutive angle positions of the intensity minimum about the center of the measurement area, wherein the fluorescence enabling light source is configured to subject the measurement area to the fluorescence enabling light for each angle position of the intensity minimum,
• wherein the detector is configured to measure the fluorescence light emitted out of the measurement area for the plurality of consecutive angle positions of the intensity minimum about the center of the measurement area at a temporal resolution, and
• wherein the tracking device is configured to update a position of the center of the measurement area in the sample such that a predetermined temporal course of intensities of the fluorescence light measured over the plurality of angle positions of the line-shaped or plane shaped intensity minimum is maintained.