JP2015227940A5

JP2015227940A5 -

Info

Publication number: JP2015227940A5
Application number: JP2014113244A
Authority: JP
Filing date: 2014-05-30
Publication date: 2017-07-13
Anticipated expiration: 2034-05-30

Claims

A light source that irradiates a measurement object with excitation light that excites and emits a fluorescent indicator;
An enlarged optical system capable of obtaining an enlarged optical image of the fluorescent index excited by the excitation light;
An imaging device that obtains captured image data by capturing an optical image of the measurement object obtained by the magnification optical system;
An optical microscope having a focus control unit for adjusting a focus position of the magnifying optical system;
An analysis unit that selects cells to be measured from the captured image data, observes the fluorescence index for the selected cells to be measured, and measures the dynamics of one molecule in a living cell; Optical microscope system.

The optical microscope includes a shielding member that shields at least a part of the excitation light, which is disposed at a position that is a conjugate point with a focal position of the objective lens of the magnification optical system in the irradiation optical system that irradiates the excitation light. Prepared,
The optical microscope system according to claim 1, wherein the analysis unit determines a focus position of the objective lens based on an optical image of the shielding member, and an autofocus operation of the optical microscope is performed by the focus control unit.

The irradiation optical system can irradiate the measurement object by two irradiation methods of total reflection illumination and epi-illumination from the optical axis of the objective lens, and the optical microscope based on the light image of the shielding object The autofocus operation is performed under epi-illumination from the optical axis of the objective lens, and a captured image for dynamic measurement of one fluorescent index of the measurement object is obtained under total reflection illumination. 2. The optical microscope system according to 2.

The analysis unit performs binarization processing on the captured image to acquire binarization information of the captured image, and the enlargement is performed based on a change in the binarization information accompanying a focus adjustment operation by the focus control unit. The optical microscope system according to claim 2 or 3, wherein an autofocus operation is performed in the optical system.

The analysis unit uses the binarization information to digitize the contrast of the boundary portion of the shielding member in the captured image, and the position where the digitized contrast value is the largest is the focus position in the magnifying optical system. The optical microscope system according to claim 4, which is determined to be present.

The analysis unit obtains a position interval of the optical pattern within a predetermined range of the captured image from the captured image obtained by capturing an optical pattern having regularity in at least one direction arranged in the measurement visual field. The optical microscope system according to claim 1, wherein the imaging magnification information is acquired using an average value of the obtained position intervals.

The analysis unit identifies a two-dimensional coordinate position in the captured image from a position interval of the optical pattern within a predetermined range of the captured image, and extracts a plurality of captured images from the identified two-dimensional coordinate position. The optical microscope system according to claim 6, wherein captured images arranged side by side are acquired.

The optical microscope includes a plurality of imaging optical systems capable of acquiring captured images from each of a plurality of types of optical images having different wavelengths obtained by spectrally dividing by an optical spectroscopic unit and the optical spectroscopic unit,
The analysis unit uses the captured images of the plurality of imaging optical systems obtained by imaging an optical pattern having regularity in at least one direction arranged in the measurement visual field, and the imaging by the imaging optical systems. Get the correction value to correct the position information error between images,
The optical microscope system according to any one of claims 1 to 7, wherein an error of a captured image obtained by the plurality of imaging optical systems is corrected using the correction value.

The analysis unit creates a CV image that is a dispersion value / average value image for each pixel from a captured image obtained from a light image of the measurement object by transmitted light, and for the obtained CV image The optical microscope system according to claim 1, wherein a binarization process is performed to select cells to be measured.

The analysis unit, when acquiring the optical image of the measurement object by the transmitted light, controls the focus control unit to acquire a plurality of captured images including a captured image in a defocused state. The optical microscope system according to claim 9, wherein a cell to be measured is selected based on a composite image of the captured images.

The analysis unit acquires a captured image under total reflection illumination of the same measurement object and a captured image under epi-illumination from the optical axis of the objective lens, and is based on a correlation coefficient between these two captured images. The optical microscope system according to claim 1, wherein an expression state of the imaging object is determined.

The optical microscope system according to any one of claims 1 to 11, and
A well transfer means capable of transferring a well plate having a plurality of wells each capable of accommodating an object to be measured, and arranging each well in the objective lens field of the magnification optical system,
A screening control unit that controls the well transfer means and measures the dynamics of one biological cell molecule in a measurement object in each well;
A screening apparatus that enables automatic screening of one molecule of biological cells in a plurality of measurement objects located in each well.

An oil supply device for supplying oil immersion oil between the well and the objective lens of the magnification optical system;
The oil supply device has one oil supply system that supplies the oil immersion oil and two oil discharge systems that discharge the oil immersion oil, and at least one of the two oil discharge systems. The screening apparatus according to claim 12, which is an oil discharge system that sucks and discharges the oil-immersed oil.

An oil adapter disposed between the well and the objective lens of the magnification optical system;
The oil adapter is
An oil supply cable connected to the oil supply system for supplying the oil immersion oil to a gap between the oil adapter and the objective lens;
An oil suction cable connected to the oil discharge system for sucking and discharging the supplied oil immersion oil from the upper surface side of the oil adapter;
The screening apparatus according to claim 13, further comprising: an oil discharge cable formed on a bottom surface of the groove portion for discharging the oil oil in a groove portion formed in a peripheral portion of the oil adapter.

The screening control unit recognizes in advance center positions of a plurality of wells included in the well plate based on the captured image obtained from the optical microscope, acquires well center information, and based on the well center information The screening apparatus according to any one of claims 12 to 14, wherein the well transfer means is controlled.

The screening apparatus according to any one of claims 12 to 15, further comprising a measurement environment control unit capable of applying different stimuli to each of the measurement objects in the well at a predetermined timing.

An oil supply device that supplies oil immersion oil between a well and an objective lens of a magnifying optical system, the oil supply system supplying a single oil supply system, and two systems discharging the oil immersion oil An oil supply apparatus comprising an oil discharge system, wherein at least one of the two oil discharge systems is an oil discharge system that sucks and discharges the oil immersion oil.

An oil adapter disposed between the well and the objective lens of the magnifying optical system,
An oil supply cable connected to an oil supply system for supplying oil immersion oil to a gap between the oil adapter and the objective lens;
An oil suction cable connected to an oil discharge system for sucking and discharging the supplied oil immersion oil from the upper surface side of the oil adapter;
An oil adapter comprising: an oil discharge cable formed on a bottom surface of the groove portion for discharging the oil-immersed oil in a groove portion formed in a peripheral portion of the oil adapter.