Qin Miao

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

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 objectives 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 microm 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.

Multimodal 3D Imaging of Cells and Tissue, Bridging the Gap Between Clinical and Research Microscopy

Absorption dyes are widely used in traditional cytology and pathology clinical practice, while fluorophores and nanoparticles are more often used in biologic research. Optical projection tomographic microscopy (OPTM) is a platform technology that can image the same specimen in multiple modes in 3D, providing morphologic and molecular information concurrently and in exact co-registration. The depth-of-field of a high numerical aperture objective is extended by scanning the focal plane through the sample to generate an optical projection image. Samples of cells or tissue are brought into the OPTM instrument through a microcapillary tube filled with optical index-matching gel. Multiple optical projection images are taken from different perspectives by rotating the tube. Computed tomography (CT) algorithms are applied to these optical projection images to reconstruct 3D structure of the sample. Image segmentation and analysis based on these 3D images provide quantitative biosignatures for cancer diagnosis that represents a clear improvement over conventional 2D image analysis. In this article, we introduce the OPTM platform, optical Cell-CT, and Tissue-CT instruments, and some applications using these OPTM instruments.

Dual-mode optical projection tomography microscope using gold nanorods and hematoxylin-stained cancer cells

An optical projection tomography microscope (OPTM) can improve axial resolution by viewing a sample from different perspectives. Here, we report a dual-mode OPTM that can generate 3D images of single cancer cells in both absorption mode and polarization mode. Cancer cells were labeled with hematoxylin for absorption imaging and nanorods for polarization imaging. Absorption images can provide morphologic information, and polarization images can provide molecular information. The combination of molecular detection and 3D cytological cell analysis may help with early cancer diagnosis.

Simultaneous 3D imaging of morphology and nanoparticle distribution in single cells with the Cell-CT™ technology

The Cell-CT™ is an optical projection tomography microscope (OPTM) developed for high resolution 3D imaging of single cells based on absorption stains and brightfield microscopy. In this study we demonstrate the use of the Cell-CT™ in multi-color mode for simultaneous imaging of cellular 3D morphology and the 3D distribution of nanoparticle clusters in the cytoplasm. The ability to image cellular processes in relation to cellular compartments with a non-fluorescence 3D technology opens new perspectives for molecular research.

High resolution optical projection tomographic microscopy for 3D tissue imaging

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.

Dual-modal optical projection tomography microscopy for cancer diagnosis

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.

Multimodal three-dimensional imaging with isometric high resolution using optical projection tomography

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 objectives 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.

3D Imaging of Fine Needle Aspirates Using Optical Projection Tomographic Microscopy

Conventional Fine Needle Aspiration Biopsy (FNAB) requires minimal tissue sampling and causes minor tissue trauma. For these reasons, it is widely used to diagnose many types of cancers. However, the utility of FNA is limited, due to the lack of sensitivity and loss of structural information. This preliminary study introduces a new type of Three- Dimensional (3D) cytological imaging, Optical Projection Tomographic Microscopy (OPTM) that has the potential to reduce errors in FNAB analysis. We first demonstrate the functionality of OPTM, a microscopic imaging method that produces high resolution 3D images of single cell specimens in absorption mode, to perform multi-cellular imaging of cells stained with hematoxylin. Then, we use OPTM to image entire FNAB-like specimens in their three-dimensional form without experiencing errors from overlapping cells, choice of focal plane, and sampling. Our methods show success in generating volumetric data of large density of cells inside a cylindrical tube representative of FNA specimen within a 23-gauge needle. This 3D imaging technique may be applied to thin core needlebiopsyspecimens in the future, which may allow the preservation of tissue microstructure in FNAB specimens.

Tissue imaging using optical projection tomographic microscopy

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.

Dual Modal Three-Dimensional Imaging of Single Cell Using Optical Projection Tomography Microscope

The optical projection tomography microscope (OPTM) acquires three-dimensional images with isometric high resolution both in absorption and fluorescence modes, employing computed tomographic image reconstruction. The three-dimensional chromosome structure of a female muntjac cell is shown.