There are provided a microscope apparatus, an observation method, and a microscope apparatus-control program that can more efficiently perform auto-focus control and can shorten an imaging time in a case where a culture vessel is to be scanned by an image forming optical system and the auto-focus control is to be performed at each observation position. Focus information of a culture vessel is detected by a first displacement sensor and a second displacement sensor while a stage is moved to a scanning measurement position from an initial set position, and an auto-focus control unit performs auto-focus control at every observation position on the basis of the focus information in a case where the stage has been moved to the scanning measurement position.

A method is provided for determining the distance between two optical boundary surfaces spaced apart from each other in a first direction. A first image is ascertained wherein the plane into which the pattern acquired coincides with a first of two optical boundary surfaces or has the smallest distance to the first optical boundary surface in a first direction. A position of the first image in the first direction is determined. A second image is ascertained wherein the plane into which the pattern acquired coincides with a second of two optical boundary surfaces or has the smallest distance to the second optical boundary surface in the first direction. The position of the second image in the first direction is determined. The distance is calculated by means of determined positions of the first and second image.

A method for performing microscopy-based imaging of samples comprises: loading a sample holder (100) onto a support (50) configured to receive the sample holder (100); moving the sample holder (100) in a first direction, from a starting position on a first strip of the sample holder (100), to move the sample holder (100) relative to an imaging line of a line camera (10), to capture an image of the first strip of the sample holder (100); monitoring a focal plane using an autofocus system (15) as the sample holder (100) is moved in the first direction; in response to a signal from the autofocus system (15), moving an objective lens (25) along the optical axis to adjust the focal plane; and moving the sample holder (100) in a second direction, to align the imaging line of the line camera (10) with a position on a second strip of the sample holder (100).

A cell observation apparatus includes an imaging device capable of imaging a vessel containing cells while varying a focal position, an illuminating device for irradiating the vessel with illuminating light; and a controller for controlling the imaging device. The controller includes: a z-stack imaging controller for causing the imaging device to take a plurality of z-stack images while varying the focal position; a variance value calculation part for calculating a variance value of pixels values for each of the z-stack images; an edge index value calculation part for calculating an edge index value indicative of edge strength for each of the z-stack images; a focus evaluation value calculation part for calculating a focus evaluation value having a minimum value in an in-focus position, based on the variance value and the edge index value; and an in-focus position estimation part for calculating the focal position where the focus evaluation value has a minimum value to estimate the in-focus position. This achieves the estimation of the in-focus position with high precision while suppressing the increase in the number of images taken for z-stack imaging.

A focus detection unit to adjust a focal point of an image, of an object, formed by an optical system includes a first output section, a second output section, and a projection optical system. The first output section includes a first light modulation element configured to generate a first pattern image based on incident light and is configured to output the generated first pattern image. The second output section includes a second light modulation element configured to generate a second pattern image based on incident light and is configured to output the generated second pattern image. The projection optical system is configured to project the output first pattern image and the output second pattern image such that the output first pattern image and the output second pattern image have a predetermined positional relationship at an in-focus position of the optical system.

A variable focal length (VFL) lens system is utilized to determine surface Z-height measurements of imaged surfaces. A controller of the system is configured to control a VFL lens (e.g., a tunable acoustic gradient index of refraction lens) to periodically modulate its optical power and thereby periodically modulate a focus position at a first operating frequency, wherein the periodically modulated VFL lens optical power defines a first periodic modulation phase. A phase timing signal is synchronized with a periodic signal in the controller that has the first operating frequency and that has a second periodic modulation phase that has a phase offset relative to the first periodic modulation phase. A phase offset compensating portion is configured to perform a phase offset compensating process that provides Z-height measurements, wherein at least one of Z-height errors or Z-height variations that are related to a phase offset contribution are at least partially eliminated.

Regarding a microscope system, a technique capable of suitably achieving a focusing on a surface of a sample is provided. The microscope system includes an irradiation optical system (laser light source 101 or the like) that irradiates a surface of a sample 3 on a stage 104 with light from an oblique direction, an observation optical system (camera 112 or the like) that forms an image of scattered light from the surface of the sample 3, a focus mechanism (piezo stage 106 or the like) that changes a height position of focus with respect to the surface of the sample 3, and a computer system 100 that acquires an image from the observation optical system. Regarding the sample 3, the computer system acquires a first image in a first focus state and a second image in a second focus state, in which the first image and the second image have different focus heights, calculates an amount of change between a position of a first spot pattern in the first image and a position of a second spot pattern in the second image, calculates an amount of change in height of the sample 3 based on an incident angle in the oblique direction and the amount of change in position of spot pattern, and adjusts the height position of the focus by using the amount of change in sample height so as to focus on the surface of the sample 3.

The invention relates to a method for inspecting a sample with an assembly comprising a scanning electron microscope (SEM) and a light microscope (LM). The assembly comprises a sample holder for holding the sample. The sample holder is arranged for inspecting the sample with both the SEM and the LM, preferably at the same time. The method comprising the steps of: capturing a LM image of the sample in its position for imaging with the SEM; determining a position and dimensions of a region of interest in or on the sample using the LM image; determining values to which the SEM parameters need to be set to image the sample at a desired resolution; and capturing a SEM image of the region of interest, preferably using the first electron beam exposure of said region of interest.

The disclosure features methods and systems that include positioning a surface of a coverslip overlying a sample relative to an object plane of a microscope system, projecting a two-dimensional pattern of light onto the surface, where a focal plane of the two-dimensional pattern at a position of the surface is rotated by an angle β relative to the object plane, obtaining a two-dimensional image of the pattern of light reflected from the surface using a detector that includes an imaging sensor oriented perpendicular to a direction of propagation of the reflected pattern of light at the sensor, analyzing the image to determine a line of best focus of the pattern within the image, determining an offset of the line of best focus from an expected position of the line of best focus within the image, and determining a position adjustment of the surface based on the offset.

A method and system are provided for focusing an imaging device on a liquid sample flowing through a field of view of the imaging device. Objects are segmented in the captured frames and used to account for the fact that the sample is flowing. Object velocities are calculated and used in selecting an appropriate focus value. The calculation of a focus measure takes account of the number of objects in captured frames in order to ensure a consistent calculation of the focus measure.