Image processing apparatus, fluorescence microscope apparatus, and image processing program
A three-dimensional image without luminance irregularity is generated while achieving good contrast. An image processing apparatus is provided, including an image combining portion that generates combined images by combining, for each depth position in a specimen, a plurality of fluorescence images captured with differing exposure levels at each of different depth positions of the specimen; a smoothed-luminance calculating portion that calculates a representative luminance from the individual combined images and that calculates a smoothed luminance for the individual combined images by smoothing the calculated representative luminance in the depth direction; a luminance correcting portion that generates corrected images by correcting the luminance of the individual combined images on the basis of differences between the smoothed luminance and the representative luminance calculated; and a three-dimensional image generating portion that generates a three-dimensional image of the specimen from the plurality of corrected images.
1. An image processing apparatus comprising:
an image combining portion that generates a plurality of combined images, each of the plurality of combined images corresponding to one of a plurality of different depth positions in a specimen, by combining, for each of the depth positions, a plurality of fluorescence images captured with different exposure levels at the depth position, the plurality of fluorescence images being generated by capturing fluorescence coming from the specimen at each of the different depth positions;
a smoothed-luminance calculating portion that calculates a representative luminance for each of the plurality of combined images generated by the image combining portion and that calculates a smoothed luminance for each of the plurality of combined images by smoothing the calculated representative luminance along the depth direction;
a luminance correcting portion that generates a corrected image for each of the depth positions by correcting a luminance of a corresponding one of the plurality of combined images based on differences between the smoothed luminance and the representative luminance calculated by the smoothed-luminance calculating portion; and
a three-dimensional image generating portion that generates a three-dimensional image of the specimen from the corrected images each of which is generated for a respective one of the depth positions by the luminance correcting portion.
2. A fluorescence microscope apparatus comprising:
a light source that radiates excitation light onto a specimen;
an objective optical system that collects fluorescence generated at the specimen due to irradiation with the excitation light;
an image capturing portion that captures the fluorescence collected by the objective optical system;
a focal-position controller that causes the image capturing portion to capture images of the specimen at a plurality of focal positions by moving a focal position of the objective optical system in an optical axis direction of the objective optical system with respect to the specimen;
an exposure-level controller that causes the image capturing portion to capture the fluorescence multiple times at each of the plurality of focal positions with different exposure levels;
an image combining portion that generates a plurality of combined images, each of the plurality of combined images corresponding to one of the plurality of focal positions, by combining, for each of the plurality of focal positions, a plurality of fluorescence images having different exposure levels acquired by the image capturing portion at the focal position, in accordance with the control by the focal-position controller and the exposure-level controller;
a smoothed-luminance calculating portion that calculates a representative luminance for each of the plurality of combined images generated by the image combining portion and that calculates a smoothed luminance for each of the plurality of combined images by smoothing the calculated representative luminance along the optical axis direction;
a luminance correcting portion that generates a corrected image for each of the plurality of focal positions by correcting a luminance of a corresponding one of the plurality of combined images based on differences between the smoothed luminance and the representative luminance calculated by the smoothed-luminance calculating portion; and
a three-dimensional image generating portion that generates a three-dimensional image of the specimen from the corrected images each of which is generated for a respective one of the plurality of focal position by the luminance correcting portion.
3. The fluorescence microscope apparatus according to claim 2 , wherein the exposure-level controller controls the exposure level by adjusting at least one of an intensity of the excitation light from the light source, a fluorescence detection sensitivity of the image capturing portion, and an exposure time of the image capturing portion.
4. The fluorescence microscope apparatus according to claim 2 , further comprising:
an image-capturing position controller that, by moving relative positions of the specimen and the objective optical system in directions that intersect with the optical axis direction, causes the image capturing portion to acquire the plurality of fluorescence images with different exposure levels at a plurality of adjacent positions in the intersecting directions at individual focal positions,
wherein the exposure-level controller sets, for each of the plurality of adjacent positions in the intersecting directions, exposure conditions of the image capturing portion for the fluorescence.
5. A three-dimensional fluorescence image generating method for generating a three-dimensional image of a specimen from a plurality of fluorescence images acquired with varying exposure levels at each of a plurality of different depth positions of the specimen in an optical axis direction using a fluorescence microscope apparatus, the fluorescence microscope apparatus comprising a light source that radiates excitation light onto the specimen, an objective optical system that collects fluorescence generated from the specimen due to irradiation with the excitation light, a detector that detects the fluorescence collected by the objective optical system, and a focal-position moving mechanism that moves a focal position of the objective optical system in the optical axis direction of the objective optical system with respect to the specimen, and the three-dimensional fluorescence image generating method comprising:
generating a plurality of combined images each corresponding to a respective one of the different depth positions of the specimen by combining, for each of the depth positions, the plurality of fluorescence images acquired with different exposure levels at the depth position;
calculating an average luminance for each of the plurality of combined images generated and calculating a smoothed luminance for each of the plurality of combined images by smoothing the calculated average luminance along the optical axis direction;
generating a corrected image for each depth position by correcting a luminance of a corresponding one of the plurality of combined images based on differences between the calculated smoothed luminance and the calculated average luminance; and
generating the three-dimensional image of the specimen from the corrected images each of which is generated for a respective one of the depth positions.
6. The three-dimensional fluorescence image generating method according to claim 5 , wherein at least one of an intensity of the excitation light, a fluorescence detection sensitivity of the detector, and an exposure time of the detector is adjusted to vary the exposure levels.
7. The three-dimensional fluorescence image generating method according to claim 5 , further comprising:
acquiring, by moving relative positions of the specimen and the objective optical system in directions that intersect with the optical axis direction, the plurality of fluorescence images with the different exposure levels at a plurality of adjacent positions in the intersecting directions, at the different depth positions,
wherein variation in the exposure levels is performed to be set for each of the plurality of adjacent positions in the intersecting directions.
8. A fluorescence microscope apparatus comprising:
a light source that radiates excitation light onto a specimen;
an objective optical system that collects fluorescence generated at the specimen due to irradiation with the excitation light;
a detector that detects the fluorescence collected by the objective optical system;
a focal-position moving mechanism that moves a focal position of the objective optical system in an optical axis direction of the objective optical system with respect to the specimen;
a controller that controls at least one of the light source and the detector, and that controls the focal-position moving mechanism; and
an image processing device that processes an image acquired based on a detection signal from the detector,
wherein the controller is configured to perform:
a control in which a depth position at which an image acquisition is performed is varied in the optical axis direction by the focal-position moving mechanism, and
a control in which an exposure level of fluorescence detected by the detector is varied by gradually increasing or decreasing at least one of an intensity of the excitation light, a fluorescence detection sensitivity of the detector, and an exposure time of the detector, and
wherein the image processing device is configured to perform:
generating a plurality of combined images, each of the plurality of combined images corresponding to a respective one of a plurality of focal positions, by combining a plurality of fluorescence images with different exposure levels that are acquired at the respective one of the plurality of focal positions in accordance with the control by the controller;
calculating a representative luminance for each of the plurality of combined images generated and calculating a smoothed luminance for each of the plurality of combined images by smoothing the calculated representative luminance along the optical axis direction;
generating a corrected image for each of the plurality of focal positions by correcting the luminance of each of the plurality of combined images based on differences between the calculated smoothed luminance and the calculated representative luminance; and
generating a three-dimensional image of the specimen from the corrected images each of which is generated for a respective one of the plurality of focal positions.