Olympus Corporation

Mitsushiro Yamaguchi

Tetsuya Tanabe

Seiji Kondo

Takuya Hanashi

Kunio Hori

Hidetaka Nakata

Kazutaka Nishikawa

An optical analysis device includes a light source, a beam shaping unit, a relative movement unit, a photodetector, and a position detector. The light source unit generates a light beam. The beam shaping unit forms a flat beam portion. The relative movement unit is configured to cause the flat beam portion and a test sample including marker particles to relatively move in a minor axis direction of the flat beam portion. The photodetector is configured to detect a light intensity and a light emitting position in a plane orthogonal to the minor axis direction. The position detector is capable of detecting spatial positions of the marker particles on the basis of information on a relative movement amount of the flat beam portion, information on the light intensity and the light emitting position, and a change of the light intensity generated according to a relative movement of the flat beam portion.

Optical analysis device, optical analysis method, and recording medium

There is provided an optical analysis technique of detecting light of a light-emitting particle in a sample solution in the scanning molecule counting method using the light measurement with a confocal or multiphoton microscope, for suppressing the scattering in detected results of signals of light of light-emitting particles smaller and achieving the improvement of accuracy. The inventive technique comprises moving the position of a light detection region along a predetermined route for multiple circulation times by changing the optical path of the optical system; detecting light from the light detection region and generating time series light intensity data during the moving of the light detection region and detecting individually a signal indicating light from each light-emitting particle existing in the predetermined route using the time series light intensity data obtained in the circulating movements of the light detection region of multiple times.

Optical analysis device, optical analysis method and computer program for optical analysis using single light-emitting particle detection

There is provided a microscopic observation technique capable of detecting a light-emitting object or a light-emitting particle moving in a thick sample by the scanning molecule counting method. In the inventive technique, the light from a light detection region of is detected the optical system of a confocal or multiphoton microscope is detected with while moving the light detection region in each observed subregion obtained by dividing a region to be observed into plural regions; the signal of the light from a light-emitting particle is individually detected; and the position of the light-emitting particle corresponding to the detected signal is determined in the region to be observed. The moving of the position of the light detection region in each observed subregion is performed continuously in at least two directions or and/or continuously multiple times in each observed subregion.

Optical microscope device, microscopic observation method and computer program for microscopic observation using single light-emitting particle detection technique

An optical system of an optical analysis device is easily evaluated with high accuracy. There is provided a method of evaluating an optical analysis device including an optical system A capable of forming a confocal volume C at a focal position by condensing excitation light B, the method including the steps of: placing, at the focal position of the optical system A, a phantom sample in which two or more types of solid members having different fluorescent substance concentrations are arranged adjacent to each other; irradiating the phantom samplewith excitation light through the optical system A while relatively moving the confocal volume C formed by the optical system A and the phantom sample in an arrangement direction of the solid members; detecting fluorescent light generated in the solid members placed in the confocal volume C; and evaluating the optical system A based on the detected fluorescent light.

System for applying phantom sample to evaluate optical analysis device, storage device storing instructions, method and phantom sample

There is provided a structure to make the setting of a criterion for eliminating noises easy in the scanning molecule counting method. In the inventive optical analysis technique of detecting light of a light-emitting particle in a sample solution, time series light intensity data of light from a light detection region detected with moving the position of the light detection region in the sample solution is generated, and a signal of a light-emitting particle individually is detected in the time series light intensity data, wherein a signal having a light intensity in a light intensity range set based upon a signal generation frequency integrated value distribution which is a distribution, obtained by using as a variable an intensity of a signal, of integrated values of generation frequencies of signals having an intensity not lower than the variable is extracted as the signal of the light-emitting particle.

In the scanning molecule counting method detecting light of a light-emitting particle in a sample solution using a confocal or multiphoton microscope, there is provided an optical analysis technique enabling the scanning in a sample solution with moving a light detection region in a broader area or along a longer route while making the possibility of detecting the same light-emitting particle as different particles as low as possible and remaining the size or shape of the light detection region unchanged as far as possible. In the inventive optical analysis technique, there are performed detecting light from the light detection region and generating time series light intensity data during moving the light detection region along the second route whose position is moved along the first route, and thereby, the signal indicating light from each light-emitting particle in a predetermined route is individually detected using the time series light intensity data.

There is provided a way of enabling the discrimination or identification of the kind of a light-emitting particle corresponding to each pulse form signal in the scanning molecule counting method using the optical measurement by the confocal or multiphoton microscope. In the inventive technique, the position of a light detection region in a sample solution periodically along a predetermined route is moved in measuring the light intensity from the light detection region; and a signal of light from a light-emitting particle is detected individually. Then, an index value indicating a translational diffusional characteristic of one light-emitting particle in a plane perpendicular to the moving direction of the light detection region is determined based upon intensity values of signals of light of the same light-emitting particle for identifying a light-emitting particle.

The inventive technique of detecting and analyzing light from a light-emitting particle in accordance with the scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope is characterized by detecting intensities of components of two or more wavelength bands of light from a light detection region of an optical system with moving the position of the light detection region in a sample solution by changing the optical path of the optical system of the microscope; detecting individually signals of the light from each light-emitting particle in the intensities of the components of the two or more wavelength bands of the detected light; and identifying a kind of light-emitting particle based on the intensities of the components of the two or more wavelength bands of the signals of the light of the detected light-emitting particle.

Optical analysis device and optical analysis method using a wavelength characteristic of light of a single light-emitting particle

There is provided an optical analysis technique enabling identification of a kind of light-emitting particle corresponding to a signal on a time series light intensity data or identification of a signal corresponding to light-emitting particles other than a particle to be observed in an optical measurement using a confocal microscope or a multiphoton microscope. The inventive optical analysis technique measures simultaneously and separately intensities of lights of two or more wavelength bands from a light detection region in a sample solution containing light-emitting particles of two or more kinds to generate time series light intensity data of the respective wavelength bands; detects signals simultaneously generated on the time series light intensity data of at least two wavelength bands; and identifies the simultaneously generated signals as signals of a light-emitting particle of at least one specific kind.

Optical analysis method using measurement of light of two or more wavelength bands

There is provided an optical analysis technique which observes a polarization characteristic of a light-emitting particle using the scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope. In the inventive optical analysis technique, the light detection region is irradiated with excitation light consisting of predetermined polarized light component(s) and the intensity of at least one polarized light component of the light from the light detection region is detected with moving the position of the light detection region of the optical system in a sample solution; a signal of each light-emitting particle is detected individually in the intensity of at least one polarized light component; and based on the intensity of at least one polarized light component of the signal of the detected light-emitting particle, the polarization characteristic value of the light-emitting particle is computed.

Optical analysis device, optical analysis method and computer program for optical analysis for observing polarization characteristics of a single light-emitting particle

There is provided a scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope, enabling characterization of a light-emitting particle or identification of a light-emitting particle with emitted light intensity of a single light-emitting particle measured individually. In the inventive optical analysis technique, with reference to the ratio of the intensities of simultaneously generated signals of the lights of at least two light-emitting sites having mutually different emission wavelengths, possessed by a light-emitting particle contained in a sample solution, the intensities being measured with moving the position of the light detection region of an optical system by changing the optical path of the optical system, a single light-emitting particle corresponding to the signals is identified, and the kind, the size, etc. of the light-emitting particle is identified.

Optical analysis method using the light intensity of a single light-emitting particle

There is provided an optical analysis technique enabling the detection of the condition or characteristic of a particle to be observed contained at a low concentration or number density in a sample solution. The inventive optical analysis technique uses an optical system capable of detecting light from a micro region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, to detect the light from the light-emitting particle to be observed while moving the position of the micro region in the sample solution (while scanning the inside of the sample solution with the micro region), thereby detecting individually the light-emitting particle crossing the inside of the micro region to enable the counting of the light-emitting particle(s) or the acquisition of the information on the concentration or number density of the light-emitting particle.

Optical analysis device, optical analysis method and computer program for optical analysis

There is provided a method of measuring a diffusion characteristic value (for example, a diffusion constant) of a light-emitting particle using the scanning molecule counting method using the optical measurement with a confocal microscope or a multiphoton microscope. The inventive method of measuring a diffusion characteristic value of a light-emitting particle is characterized to measure light intensity from the light detection region with moving the position of the light detection region in the sample solution by changing an optical path of the optical system to generate light intensity data and to compute a diffusion characteristic value of the light-emitting particle based on a deviation time from a moving cycle time of the light detection region in an interval of generation times of two or more signals corresponding to a same light-emitting particle on the light intensity data.

Method of measuring a diffusion characteristic value of a particle

The inventive optical analysis technique uses an optical system capable of detecting light from a micro region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, to detect the light from the light-emitting particle to be observed while moving the position of the micro region in the sample solution (while scanning the inside of the sample solution with the micro region); generates time series light intensity data, computes a characteristic value of the light intensity indicating the presence or absence of the light from a single light-emitting particle in every time section of a predetermined width in the light intensity data; and detects the light-emitting particle crossing the inside of the micro region individually using the characteristic value, thereby enabling the counting of the light-emitting particle(s) or the acquisition of the information on the concentration or number density of the light-emitting particle.

The inventive optical analysis technique uses an optical system capable of detecting light from a micro region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, to detect the light from the light-emitting particle to be observed while moving the position of the micro region in the sample solution (while scanning the inside of the sample solution with the micro region); generates time series light intensity data, and after smoothing the time series light intensity data; and detects in the smoothed time series light intensity data the light-emitting particle crossing the inside of the micro region individually, thereby enabling the counting of the light-emitting particle(s) or the acquisition of the information on the concentration or number density of the light-emitting particle.

Growing community of inventors

Hachioji, Japan

Mitsushiro Yamaguchi