An apparatus for real-time optical imaging of a tissue specimen, the 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 range of wavelengths 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 being 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 a focal plane of the auxiliary imaging system; 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 generate a composite representation of the tissue specimen that includes a representation of cell nuclei in the 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, the apparatus being characterized in that: the primary imaging system and the auxiliary imaging system are both disposed on the same side of the specimen plane. 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 range of wavelengths 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 being 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 a focal plane of the auxiliary imaging system; 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 generate a composite representation of the tissue specimen that includes a representation of cell nuclei in the 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, the apparatus being characterized in that: the primary imaging system and the auxiliary imaging system are both disposed on the same side of the specimen plane.