Three-dimensional image obtaining device and processing apparatus using the same
An optical flux is radiated, which is focused at a measurement point in a specimen space, and a transmitted light amount is measured. A minute light absorption amount is measured from a transmitted light signal and a reference signal. While three-dimensionally scanning, a three-dimensional map in which light absorption amounts are represented by voxels (volume cells) is obtained. On this three-dimensional map, deconvolution processing with a light intensity distribution image in the vicinity of the measurement point being a convolution kernel is performed, so as to obtain a three-dimensional image of a specimen that is almost transparent in a non-dyed state.
1. A three-dimensional image obtaining device, in which a biological material that is almost transparent is mounted as a specimen, for obtaining a three-dimensional image from an attenuation amount of a transmitted light through the specimen, the device comprising:
a light source emitting an optical flux with light intensity being amplitude modulated by a predetermined frequency;
an objective lens focusing the optical flux at a measurement point in the specimen to generate a local intensity distribution in the vicinity of the measurement point;
a means for three-dimensionally scanning the specimen and the measurement point relatively;
a modulated low voltage signal processor calculating a difference between a reference signal voltage equivalent to a transmitted light amount of the optical flux of when there is no specimen and a specimen transmitted signal voltage equivalent to a transmitted light amount of the optical flux of when there is a specimen, and outputting a modulated signal voltage equivalent to a minute light attenuation amount of the optical flux corresponding to position coordinates of the measurement point to be scanned;
a means for making a three-dimensional map using the modulated signal voltage corresponding to the position coordinates of the measurement point; and
a means for making a three-dimensional image of the specimen from the three-dimensional map by either a deconvolution using a convolution kernel obtained from a light intensity distribution image in the vicinity of the measurement point or a blind deconvolution which does not initially set a convolution kernel.
2. The three-dimensional image obtaining device according to claim 1 , further comprising
a display controller comprising a means for displaying the three-dimensional image of the specimen on a display, correcting gamma of the three-dimensional image, and displaying the three-dimensional image again, and a means for changing a visual angle of the three-dimensional image and displaying the three-dimensional image again.
3. The three-dimensional image obtaining device according to claim 1 ,
wherein the deconvolution is performed by any one of a computer having three-dimensional deconvolution computer software, an electronic circuit structured including a digital signal processor (DSP), and an electronic circuit structured including a field programmable gate array (FPGA).
4. The three-dimensional image obtaining device according to claim 1 ,
wherein the three-dimensional scanning means is formed of an XYZ three-dimensional stage mounting the specimen.
5. The three-dimensional image obtaining device according to claim 1 ,
wherein the three-dimensional scanning means is formed of a one-dimensional stage of X axis mounting the specimen and a movable objective lens capable of scanning two-dimensionally in Y axis and Z axis.
6. The three-dimensional image obtaining device according to claim 1 ,
wherein the three-dimensional scanning means is an optical flux three-dimensional scanning means for scanning an optical flux three-dimensionally in XYZ axis.
7. The three-dimensional image obtaining device according to claim 1 ,
wherein the modulated low voltage signal processor divides a differential signal voltage between a specimen transmitted signal voltage and a reference signal voltage by the reference signal voltage to normalize the differential signal voltage.
8. The three-dimensional image obtaining device according to claim 1 ,
wherein the modulated low voltage signal processor is structured including a lock-in amplifier.
9. The three-dimensional image obtaining device according to claim 1 ,
wherein the reference signal voltage is a light detection output of an optical flux which is branched from the optical flux emitted from the light source and does not transmits through the specimen.
10. The three-dimensional image obtaining device according to claim 1 ,
wherein the light source is formed of a semiconductor laser, and the reference signal voltage is generated from a monitor diode mounted in the semiconductor laser.
11. A three-dimensional processing apparatus, comprising a three-dimensional image obtaining device, in which a biological material that is almost transparent is mounted as a specimen, for obtaining a three-dimensional image from an attenuation amount of a transmitted light through the specimen, specifying position coordinates of an arbitrary point in a specimen space of a biological specimen that is almost transparent, and optically stimulating and/or optically processing the arbitrary point,
wherein the three-dimensional image obtaining device comprises:
a light source emitting an optical flux with light intensity being amplitude modulated by a predetermined frequency;
an objective lens focusing the optical flux at a measurement point in the specimen to generate a local intensity distribution in the vicinity of the measurement point;
a means for three-dimensionally scanning the specimen and the measurement point relatively;
a modulated low voltage signal processor calculating a difference between a reference signal voltage equivalent to a transmitted light amount of the optical flux of when there is no specimen and a specimen transmitted signal voltage equivalent to a transmitted light amount of the optical flux of when there is a specimen, and outputting a modulated signal voltage equivalent to a minute light attenuation amount of the optical flux corresponding to position coordinates of the measurement point to be scanned;
a means for making a three-dimensional map using the modulated signal voltage corresponding to the position coordinates of the measurement point; and
a means for making a three-dimensional image of the specimen from the three-dimensional map by either a deconvolution using a convolution kernel obtained from a light intensity distribution image in the vicinity of the measurement point or a blind deconvolution which does not initially set a convolution kernel.