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Live Cell Imaging


to fill the reflector area of a Texas Instruments digital micro- mirror device (DMD), which consists of several hundred thousand micromirrors arranged in a rectangular array. Elec- tronic control of the light path through the DMD eliminates moving parts for better stability and improved image resolu- tion. Since each individual mirror can be rapidly tilted ± 12° electronically, the DMD can be directed to create a mirror pattern which can rotate the beam through 360° around the optic axis at an angle of 53°. As the beam rotates, the data required for reconstruction (48 images of 1k × 1k) is acquired in less than a second. Each data set from the sample is com- bined with the reference beam to create 2D interferograms from which the RI is calculated for each voxel in the image. When the resulting multiple 2D RI measurements are stitched together a 3D RI tomogram image is displayed. Image data are “super-resolved” with voxels of about 110 nm × 110 nm × 220 nm. Selected bands of RI can be pseudo-colored to highlight structures within the cell. Holotomography capabilities. The HT microscopy


method exploits the intrinsic optical properties of all mate- rials. It directly measures the optical phase delay intro- duced by RI differences between the sample and its medium directly. In doing so, it eliminates the need for stains or other labeling agents to quantitatively and non-invasively investigate biological cells and thin tissues. Because the RI values are measured quantitatively, HT microscopy provides quantitative data about the sample, including morphologi- cal and chemical information. The reconstructed 3D RI dis- tributions of biological material provide information about dry mass, cell volume, shapes of sub-cellular organelles, cytoplasmic density, surface area, and deformability. Fur- thermore, since the method requires only a low-power laser system, compared to laser scanning microscope systems, HT microscopy minimizes the potential for photobleaching and phototoxicity. Figure 2 shows an example of holotomo- graphic capture of 3D images of live cells without exogenous labeling agents, imaging cell membranes and organelles at a resolution of 110 nm. Large fields of view and long-term imaging. Another


aspect of HT offered by the HT microscope is the opportunity for safe, long-term study of live cells on a large scale. Tis is possible, in addition to minimal specimen damage, because of the instrument’s motorized stage and custom-designed stitch- ing soſtware developed in cooperation with the renowned Korea Advanced Institute of Science and Technology (KAIST). Aſter an image is acquired at one position, the specimen is moved along the X and Y axis at 70 μm per step via the com- puterized stage to acquire the next image. By stitching these images together, specimens up to 8 mm × 8 mm can be imaged in their entirety, with 2D and 3D image acquisition rates of 150 and 2.5 frames per second, respectively. Because the HT microscope only needs a low-power laser to image live cells and tissues in three dimensions without stains or fluorescent probes, these long-term studies can be conducted without the fear of photobleaching and phototoxicity impacting the results (Figure 2). Fluorescence, then holotomography. While the label-free and high-speed 3D imaging capabilities of holotomographic


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Figure 2: Label-free 3D image of C. elegans: (left) Single frame from a video shows the entire worm image of C. elegans, acquired without labeling using the Tomocube HT microscope equipped with motorized stage. (right) Repre- sentation of 3D image showing organelles in different colors. To download the image, please use this link: http://www.catalystforcontent.com/uploads/sim- plex/images/pr/images/Tomocube_C_elegans_stitch.jpg.


QPI are highly advantageous for long-term LCI, it would be useful to combine HT with standard fluorescence microscopy to obtain molecular specificity of the same specimen area. In such a multipurpose instrument, 3D QPI and fluorescence could be used in concert to create a correlative imaging part- nership. Tis is the idea behind the design of the Tomocube HT-2 (Figure 3). Te HT-2 microscope takes the existing HT setup and


adds a 3D fluorescence imaging capability. Te HT-2 is the first instrument of this type; it delivers HT and fluorescence correlative analysis in 2D, 3D, and 4D (including time). Te microscope’s 3D HT images and measurements enable the monitoring of cells and their structures with minimal stress, while highly detailed fluorescence images can be captured simultaneously to show the position of specific target organ- elles or molecules in the living cells. For long-term studies especially, the combination of HT


microscopy, fluorescence microscopy, computer-controlled motorized stage, and custom-designed stitching soſtware is especially advantageous. For these studies conducted on the HT-2, potentially damaging fluorescence observations can be restricted to mapping molecular specificity, while the gentler holotomographic QPI can be employed for the longer-term observations of the same cells.


Results Homotomography. Figure 4 shows the power of quan- titative studies using the Tomocube HT-1 microscope alone.


www.microscopy-today.com • 2020 January


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