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.

Described herein are systems and methods for improving light-sheet microscopy with cost-effective and simplified components. Such cost-effective and simplified components can be implemented in a light focusing system, a light generation system, and/or in imaging system. The light focusing system can be improved by attaching a voice coil motor to a focusing lens to increase the imagable field of view. The light generation system can be improved with a multimode laser diode to increase the uniformity of the beam profile and to increase the usable optical power. The imaging system can be improved by using a fluid chamber with positive cylindrical optical window for minimizing spherical aberrations.

This disclosure is directed to an apparatus for securely holding a substrate, such as a microscope slide. A holder includes a frame including at least three walls, such as a base, a first arm, and a second arm. Each wall includes a platform or a portion of a platform to support the substrate. The first arm includes at least one securing block and a second arm opposite the first arm includes a secure bar with a securing block. The securing blocks include a ramp to guide the substrate off of the platforms and a stopper to set a maximum lift distance and to constrain the substrate.

This disclosure is directed to an apparatus for securely holding a substrate, such as a microscope slide. A holder includes a frame including at least three walls, such as a base, a first arm, and a second arm. Each wall includes a platform or a portion of a platform to support the substrate. The first arm includes at least one securing block and a second arm opposite the first arm includes a secure bar with a securing block. The securing blocks include a ramp to guide the substrate off of the platforms and a stopper to set a maximum lift distance and to constrain the substrate.

This disclosure is directed to optical microscope calibration devices that can be used with optical microscopes to adjust the microscope imaging parameters so that images of samples can be obtained below the diffraction limit. The microscope calibration devices include at least one calibration target. Each calibration target includes a number of features with dimensions below the diffraction limit of a microscope objective. Separate color component diffraction limited images of one of the calibration targets are obtained for a particular magnification. The color component images can be combined and image processed to obtain a focused and non-distorted image of the calibration target. The parameters used to obtain the focused and non-distorted image of the calibration target can be used to obtain focused and non-distorted images of a sample for the same magnification by using the same parameters.

This disclosure is directed to optical microscope calibration devices that can be used with optical microscopes to adjust the microscope imaging parameters so that images of samples can be obtained below the diffraction limit. The microscope calibration devices include at least one calibration target. Each calibration target includes a number of features with dimensions below the diffraction limit of a microscope objective. Separate color component diffraction limited images of one of the calibration targets are obtained for a particular magnification. The color component images can be combined and image processed to obtain a focused and non-distorted image of the calibration target. The parameters used to obtain the focused and non-distorted image of the calibration target can be used to obtain focused and non-distorted images of a sample for the same magnification by using the same parameters.

At least some embodiments of the technology are directed to an automated slide processing apparatus configured to apply at least one reagent to a specimen carried by a microscope slide. The slide processing station can include a support element with a support surface, at least one vacuum port, and a sealing member having a non-round shape. In an uncompressed state, the scaling member can extend upwardly beyond the support surface. In a compressed state, the scaling member can be configured to maintain an airtight seal with a backside of the microscope slide as the microscope slide is pulled against the support surface by a vacuum drawn via the at least one vacuum port.

Imaging systems and methods using an ancillary image detector for sample location. An exemplary system may comprise a light source to irradiate a sample on an irradiation axis, a first image detector on an optical axis oblique to the irradiation axis, and a stage. The system also may comprise a second image detector disposed on an imaging axis, and a drive mechanism configured to move the stage and the imaging axis relative to one another. The system further may comprise a processor configured to (a) receive an image of the sample detected by the first image detector, (b) determine a physical location for a region of contrast produced by the sample within the image, and (c) send a signal to the drive mechanism based on the physical location, to dispose at least part of the sample in a field of view of the second image detector.

A cassette for retaining a specimen of surgically exposed tissue from a patient in an orientation that facilitates optical sectioning of the tissue by a confocal microscopic or other optical imaging microscope. The cassette includes a base member having a rigid optically transparent window upon which a tissue specimen is situated, a pliable membrane locatable over a substantial portion of the base member including the window, and an upper member, having an aperture therethrough, which can cover the base member to provide an enclosed cavity between the membrane and the window sealing the tissue specimen therein. The edges of the tissue specimen may be positioned planar against the window and retained in that position by bonds formed between the membrane and window at multiple points or locations around the tissue specimen. The specimen retained in the cavity is imagable by a microscope through the window of the base member.

A cassette for retaining a specimen of surgically exposed tissue from a patient in an orientation that facilitates optical sectioning of the tissue by a confocal microscopic or other optical imaging microscope. The cassette includes a base member having a rigid optically transparent window upon which a tissue specimen is situated, a pliable membrane locatable over a substantial portion of the base member including the window, and an upper member, having an aperture therethrough, which can cover the base member to provide an enclosed cavity between the membrane and the window sealing the tissue specimen therein. The edges of the tissue specimen may be positioned planar against the window and retained in that position by bonds formed between the membrane and window at multiple points or locations around the tissue specimen. The specimen retained in the cavity is imagable by a microscope through the window of the base member.