Conventional Optical Projection Tomography (OPT) can image tissue samples both in absorption and fluorescence mode. Absorption image can show the anatomical structure of the sample, while fluorescence mode can determine specific molecular distribution. The depth of focus (DOF) of the lens in conventional OPT needs to transverse the whole sample. As a result, resolution will be poor due to the low numerical aperture (NA) needed to generate large DOF. In conventional pathology, the specimens are embedded in wax and sliced into thin slices so that high NA objective lens can be used to image the sections. In this case, the high resolution is obtained by using high NA objective lens, but 3D images can be only obtained by stitching different sections together. Here, we propose a new method that can image entire specimen without sectioning with the same high resolution as the conventional pathology. To produce high resolution that is isotropic, the original OPTM system scans the focal plane of the high NA objective through the entire specimen to produce one projection image. Then the specimen is rotated so that the subsequent projection is taken at different perspective. After all the projections are taken, 3D images are generated by the filtered back-projection method. However, the scanning rate is limited by scanning objective lens due to the large mass of the lens. Here we show a new OPTM system that scans the mirror in the conjugate image space of the object to produce projections.
Optical projection tomography (OPT) requires a large depth of field (DOF) of a low numerical aperture (NA) lens
resulting in low resolution. However, DOF of a high NA objective can be extended by scanning the focal plane through
the sample. This extended DOF image is called pseudoprojection, which is used by optical projection tomographic
microscope (OPTM) for tomographic reconstruction. The advantage of OPTM is the acquisition of relatively high
resolution and large depth of field concurrently. This method requires the working distance of the lens to be larger than
the size of the sample, so proper lens should be chosen for samples of different sizes. In this paper, we imaged
hematoxylin stained muntjac cells inside capillary tube with two different sizes. Two objective lenses with different NA
are used for these two tubes. Experimental results show that resolution improves over 10 times in OPTM compared to
conventional OPT, which make it possible for OPTM technique to resolve sub-cellular features for large samples.
Therefore, OPTM can be used for 3D histological analysis of hematoxylin & eosin (H&E) stained biopsy specimen with
sub-cellular resolution in the future.
A dual-modal optical projection tomography microscope (OPTM) is presented, which produces three-dimensional
images of single cells with isometric high resolution both in fluorescence and absorption mode. Depth of field of a high
numerical aperture objective is extended by scanning the focal plane through the sample in order to enable
reconstruction by back-projection method. Cells are fixed, stained, and mixed with optical gel and injected into the
capillary for imaging. Combining absorption and fluorescence mode allows us to image different aspects of the disease
process. Images of cells stained with both hematoxylin and fluorescence probes are shown. Registrations between two
modes are discussed.
The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 µm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.
The optical projection tomography microscope (OPTM) is an optical microscope that acquires focus-invariant images from multiple views of single cells. Although the depth of field of the objective is short, it can be extended by scanning the objective's focal plane. This extended depth of field image is similar to a projection in conventional X-ray CT. Samples flow through a microcapillary tube filled with optical gel. Optical distortion is minimized by matching refractive index of optical gel and tube. Multiple projection images are taken by rotating the microcapillary tube with sub-micron mechanical precision. After these pseudoprojection images are further aligned, computed tomography methods are then applied to the images to create a 3D reconstruction with isometric resolution of 0.35 microns. Three-dimensional reconstructed images of fluorescent microspheres and cells are shown.