Quantitative phase image generating method, quantitative phase image generating device, and program

Abstract

A quantitative phase image generating method for a microscope, includes: irradiating an object with illumination light; disposing a focal point of an objective lens at each of a plurality of positions that are mutually separated by gaps Δz along an optical axis of the objective lens, and detecting light from the object; generating sets of light intensity distribution data corresponding to each of the plurality of positions based upon the detected light; and generating a quantitative phase image based upon the light intensity distribution data; wherein the gap Δz is set based upon setting information of the microscope.

What is claimed is: 
     
         1 . A quantitative phase image generating device for a microscope, the device comprising:
 a light source that irradiates illumination light to an object;   a first detection unit that detects a first observation light, which is fluorescence from the object irradiated by the illumination light, via a first optical system;   a first image generation unit that generates a fluorescence observation image based on the detected first observation light;   a second detection unit that detects, with plural different conditions, a second observation light, which is light transmitted from the object irradiated by the same illumination light from the light source, via a second optical system, the second observation light being different from the first observation light; and   a second image generation unit that generates a quantitative phase image based on the detected second observation light.   
     
     
         2 . The quantitative phase image generating device according to  claim 1 , wherein:
 the second detection unit detects the second observation light from the object by disposing a focal point of the second optical system at each of positions that are mutually separated by gaps Δz along an optical axis of the second optical system of the second detection unit;   the second image generation unit acquires light intensity distribution data corresponding to each of the positions based on the second observation light and generates the quantitative phase image based on the light intensity distribution data;   the quantitative phase image is an image including phases obtained by multiplying change in thickness of the object by change of a refractive index of the object;   the gap Δz is set based upon setting information of the microscope; and   the setting information includes at least one of the following: a numerical aperture NA of an objective lens, a wavelength λ of the illumination light, and a refractive index between the objective lens and the object.   
     
     
         3 . The quantitative phase image generating device according to  claim 1 , wherein:
 a stage of the microscope on which the object is mounted is disposed between the first detection unit and the second detection unit.   
     
     
         4 . The quantitative phase image generating device according to  claim 1 , wherein:
 the first optical system includes an objective lens and a third imaging lens;   on an optical path of the first optical system, in order to detect the first observation light, the object, the objective lens, the third imaging lens, and the first detection unit are disposed in this order;   the second optical system includes a condenser lens and a fourth imaging lens; and   on an optical path of the second optical system, in order to detect the second observation light, the object, the condenser lens, the fourth imaging lens, and the second detection unit are disposed in this order.   
     
     
         5 . The quantitative phase image generating device according to  claim 1 , wherein
 the microscope is a scanning type electron microscope comprising a fluorescence observation unit which includes the first detection unit.   
     
     
         6 . A quantitative phase image generating device comprising:
 a processor; and   a memory encoded with instructions executed by the processor, the instructions causing the processor to perform operations comprising:
 causing an illumination light source to irradiate illumination light to an object; 
 causing to be detected a first observation light, which is fluorescence from the object irradiated by the illumination light via a first optical system; 
 generating a fluorescence observation image based on the detected first observation light; 
 causing to be detected, with plural different conditions, a second observation light, which is light transmitted from the object irradiated by the same illumination light from the light source, via a second optical system, the second observation light being different from the first observation light; and 
 generating a quantitative phase image based on the detected second observation light. 
   
     
     
         7 . The quantitative phase image generating device according to  claim 6 , wherein:
 the instructions cause the processor to perform further operations comprising:
 by controlling a driver, disposing a focal point of the second optical system at each of positions that are mutually separated by gaps Δz along an optical axis of the second optical system of the second detection unit; 
 causing to be detected the second observation light from the object; 
 acquiring light intensity distribution data corresponding to each of the positions based on the second observation light; and 
 generating the quantitative phase image based on the light intensity distribution data; 
   the quantitative phase image is an image including phases obtained by multiplying change in thickness of the object by change of a refractive index of the object;   the gap Δz is set based upon setting information of a microscope; and   the setting information includes at least one of the following: a numerical aperture NA of an objective lens, a wavelength λ of the illumination light, and a refractive index between the objective lens and the object.