Leland Stanford Junior University

Axon Instruments, Inc.

Stephen J Smith

Kristina D Micheva

Yuri Vladimirovich Osipchuk

Yuriy Roll

Sasha Dromaretsky

James S Harris, Jr

Evan P Thrush

Ofer Levi

High-resolution three-dimensional imaging of a specimen is facilitated. According to an example embodiment of the present invention, a series of very thin slices from a specimen are serially and robustly arranged on an imaging device such as a microscope slide. The slices are imaged and the images are used to reconstruct a three-dimensional image having high resolution at depths into the specimen. The serial arrangement of the slices facilitates the proper ordering of images for reconstruction. Further, the robust nature of the slice arrangement facilitates treatment of the slices and, in some applications, multiple treatments with corresponding imaging sequences for each treatment. Various embodiments are directed to methods and arrangements for three-dimensional characterization of biological specimen and to data that is accessible and/or executable by a computer for linking different images together in order to characterize such biological specimen in three dimensions.

Arrangement and imaging of biological samples

Imaging specimen such as biological tissue is facilitated. According to an example embodiment of the present invention, a relatively thin, surface portion of a specimen is stained and imaged. The thin portion is removed, exposing a new portion of the specimen. The newly-exposed portion is stained and imaged. Subsequent new surface portions (newly exposed) are similarly stained and imaged, with multiple images obtained from the specimen in an automated fashion. Some applications are directed to the distinct imaging of specimen characteristics having relatively small vertical separation (e.g., less than about 40 nanometers), with an upper characteristic imaged, removed, and the lower characteristic subsequently imaged.

Optical imaging of molecular characteristics of biological specimen

A variety of types of molecules are detected and/or analyzed using an integrated micro-circuit arrangement. According to an example embodiment of the present invention, a micro-circuit arrangement detects excitable target markers in response to an excitation source. The excitation source emits a first electromagnetic radiation to excite one or more target markers into emitting a second electromagnetic radiation. The excitation source and detector combination can be optimized to detect a specific characteristic of a biological specimen. In this manner, an excitation source can be combined with several optical-detectors or detection channels, where each optical-detector is measuring or sensing the same or different characteristic of the biological specimen.

Excitable target marker detection

A fluorescent optical imaging system ( ) produces two separate spots (S and S ) on a sample ( ) by a pair of excitation laser beams (B and B ) that are generated by first and second lasers (L and L ). Excitation laser beams (B and B ) pass at slightly different angles, first through an aperture ( ) of a 45&deg; fold mirror ( ), and then through an objective element ( ). As a result, emission light beams ( ) are generated from each illuminated spot (S and S ) and are reflected and redirected by mirror ( ) through a secondary lens ( ) before reaching one of two detectors (PMT and PMT ). Emission beam ( ) reflects off a second 45&deg; mirror ( ) prior to reaching detector (PMT ), while emission beam ( ) travels directly to (PMT ). If desired, optical separation elements ( ), such as dichroic filters, prisms, or gratings, can be positioned in front of each detector (PMT and PMT ). Fluorescent optical imaging system ( ) may employ a scanning system ( ) for illuminating and imaging the entire area of sample ( ).

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Los Altos, CA, United States of America

Stephen J Smith