Method and apparatus for imaging unsectioned tissue specimens

A method of real-time optical imaging of a tissue specimen comprising the steps of:

• applying one or more fluorescent contrast agents to a tissue specimen, wherein at least one of the one or more fluorescent contrast agents is a nuclear contrast agent;
• providing an apparatus, comprising:
• a primary imaging system configured to use an illumination source to acquire images of a tissue specimen through one or more spectrally separated channels, at least one of the one or more spectrally separated channels is configured to detect a range of wavelengths distinct from the wavelength of the illumination source, the primary imaging system being an inverted microscope having a frame acquisition rate and configured to perform optical depth sectioning, the primary image system being configured to acquire a sequence of images;
• an auxiliary imaging system, wherein the auxiliary imaging system is configured to acquire an auxiliary image of the tissue specimen, wherein the area of the auxiliary image is greater than the area of each image of the sequence of images acquired by the primary imaging system;
• a specimen holder having a transparent window therewithin, the specimen holder disposed in a specimen plane at a focal plane of the primary imaging system, the specimen holder being configured to hold the tissue specimen on the transparent window, the specimen holder comprising one or more position sensors, wherein the specimen holder is configured to be translatable in the specimen plane, the one or more position sensors being configured to measure a specimen holder position, and wherein the specimen holder is configured to be translatable to the focal plane of the auxiliary imaging system;
• wherein the primary imaging system and the auxiliary imaging system are both disposed on the same side of the specimen plane, the apparatus further comprising:
• a user input device configured to accept user input, wherein the specimen holder is configured to translate in response to the user input in real-time;
• a processing unit in electrical communication with the primary imaging system, the auxiliary imaging system, and the position sensors, wherein the processing unit is configured to execute a sequence of instructions on the sequence of images acquired by the primary imaging system, the auxiliary image, and at least one specimen holder position to form a representation of cell nuclei and to generate a composite representation of the tissue specimen;
• and
• a display device in electrical communication with the processing unit, the display device being configured to display the composite representation of the tissue specimen in real-time,
• situating the tissue specimen in the specimen holder;
• positioning the specimen holder at the focal plane of the auxiliary imaging system;
• causing the auxiliary imaging system to acquire the auxiliary image;
• positioning the specimen holder at the focal plane of the primary imaging system;
• causing the primary imaging system to acquire the sequence of images;
• causing a processing unit to detect cell nuclei within the tissue specimen and to generate a composite representation of the tissue specimen;
• causing the display device to display the composite representation of the tissue specimen in real-time;
• and
• causing the specimen holder to translate in the specimen plane using the user input device.

A histological specimen cassette apparatus comprising:

• an open-end receptacle, including a bottom wall and a specimen retaining structure;
• a specimen cover, configured to close the open-end receptacle;
• a specimen cover connector, configured to connect the open-end receptacle and the specimen cover;
• a transparent window;
• a transparent window connector, configured to connect the bottom wall or the specimen cover and the transparent window;
• a plurality of perforations in one or more of the open-end receptacle, the transparent window or the specimen cover.