Olympus Corporation

Hidetaka Nakata

Tetsuya Tanabe

Takuya Hanashi

Kazutaka Nishikawa

Mitsushiro Yamaguchi

Kunio Hori

In the scanning molecule counting method of measuring light intensity from a light detection region while moving the position of the light detection region of a confocal or multiphoton microscope in a sample solution containing light-emitting particles, generating time series light intensity data and detecting each of signals of the light-emitting particles individually in the data, wherein the light-emitting particles are formed by binding to a particle to be observed a light-emitting probe which emits light through binding to the particle to be observed and in which a stochastic transition between a non-light-emitting state and a light-emitting state occurs in the unbound state, the moving speed of the position of the light detection region is adjusted to make the time during which the unbound probe is encompassed by the moving light detection region longer than an average lifetime during which the probe is in the light-emitting state.

Optical analysis method and optical analysis device using single light-emitting particle detection

There is provided optical analysis techniques in the scanning molecule counting method using the light measurement with a confocal or multiphoton microscope in which the measuring unit time in the light measurement is set to an appropriate value in order to surely detect an approximately bell shape profile of the signal of a light-emitting particle and avoid excessive increase data volume of time series light intensity data. The inventive optical analysis technique of detecting light of a light-emitting particle in a sample solution generates time series light intensity data of light from a light detection region detected during moving the position of the light detection region of a microscope in the sample solution and detects in the data a signal indicating light from each light-emitting particle individually. The measuring unit time is determined based on the size and the moving speed of the light detection region.

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

The present invention provides a method for indirectly and with high sensitivity detecting a particle dispersed and moving randomly in a solution using a luminescent probe. In the present invention, (a) a solution is prepared that contains a target particle and one or more types of a luminescent probe that directly or indirectly binds to the target particle, (b) a complex is formed that contains the target particle and the luminescent probe in the solution, (c) the complex is recovered by separating luminescent probe not bound to the target particle from the solution containing the complex, followed by (d) dissociating the luminescent probe from the recovered complex and mutually separating and separately recovering the free luminescent probe and target particle, (e) again binding a luminescent probe to the recovered target particle followed by dissociating the luminescent probe and mutually separating and recovering free luminescent probe and the target particle are repeated, followed by preparing a single measurement sample solution containing the total amount of the recovered free luminescent probe, and (f) the number of molecules of the luminescent probes in the measurement sample solution is calculated.

Method for detecting a target 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 using a light-emitting probe. 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 probe having bound to a 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 particle crossing the inside of the micro region to enable the counting of the particle(s) or the acquisition of the information on the concentration or number density of the particle.

Method of detecting sparse particles in a solution using a light-emitting probe

The method of the present invention includes: preparing a sample solution containing the target particles and luminescent probes to be bound to the target particles, and binding these in the sample solution; moving a position of a light detection region of the optical system in the sample solution using a confocal microscope or a multiphoton microscope, and detecting light signal emitted from the luminescent probe in the light detection region while moving the position of the light detection region, and individually detecting the target particles directly or indirectly; and counting the number of the detected target particles, and calculating the concentration of the target particles in the sample solution from the number of the counted target particles on the basis of a calibration curve that approximates the correlation between the concentration or quantity of the target particles in the sample solution and the number of the target particles.

Quantitative determination method for target particles, photometric analysis device, and computer program for photometric analysis

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

The present invention provides a method for identifying polymorphism of nucleic acids in a sample solution in which the concentration or number density of the observed nucleic acids is lower than that of conventional photometric analysis technologies. Namely, the present invention relates to a method for identifying polymorphism of nucleic acid molecules, in which is compared the result of hybridization between a target nucleic acid molecule and a first nucleic acid probe labeled with a fluorescent substance; and the result of hybridization between the target nucleic acid molecule and a second nucleic acid probe having a sequence different from the sequence of the first nucleic acid probe, and being labeled with a fluorescent substance. Each of the hybridization is conducted in a separate sample solution and the detection of the conjugate is done by counting the molecule of the conjugate using a scanning molecule counting method.

Method for identifying polymorphism of nucleic acid molecules

The present invention provides a method for identifying polymorphism of nucleic acids in a sample solution in which the concentration or number density of the observed nucleic acids is lower than that of conventional photometric analysis technologies. It includes: preparing a sample solution comprising a first nucleic acid probe, which specifically hybridizes with a single-stranded nucleic acid molecule including a first type of base sequence, and a target nucleic acid molecule; forming a hybrid of the nucleic acid molecules in the sample solution; calculating a number of molecules of the hybrid including the first nucleic acid probe in the sample solution by the scanning molecule counting method; and identifying polymorphism of the target nucleic acid molecule based on the calculating result. The sample solution includes an oligonucleotide having a base sequence complementary to a base sequence different from the first type of base sequence in the polymorphic sequence.

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Hachioji, Japan

Hidetaka Nakata