Towards a Field-Deployable Orthogonal View Microscope

Introduction:: Hydrodynamics and biomechanics of aquatic organisms could provide critical metrics for formulating effective conservation strategies. To this end, the Ortho-View Microscope was developed to study the hydrodynamics of aquatic organisms. Three considerations were essential in creating this imaging system. First, we wanted an imaging system to provide images or videos of freely moving organisms. The Ortho-view Microscope uses two orthogonal planes to visualize three-dimensional living aquatic organisms, eliminating the need to immobilize organisms before visualization. Second, the Ortho-View Microscope produces images of sufficient quality for analysis in the field. Other microscopes are difficult to calibrate once moved. Finally, The Ortho-view Microscope can be made with off-the-shelf components and 3D-printed parts. This imaging system can then be easily assembled and readily configurable to the differing needs of imaging studies.

Materials and Methods:: As demonstrated in Figure 1, the Ortho-view has two imaging paths. Each path included a camera; specifically, 20 MP (FLIR) and 12 MP (Arducam) CMOS cameras were used. Depending on the configuration, one or two computers were used to control the cameras. Each path included a tube lens (f=50 mm), a long-pass emission filter (500 nm), and an objective lens (4X, 10X, or 20X were used). These components were held via 3D-printed enclosures. Each pathline also included a guide rail to adjust the distance between the objective and the sample. Guide rails were printed with nylon composites for durability. Only bright field imaging was used for the results presented. To this end, 3W cool white LEDs were used. Wires were routed under the optical breadboard and secured to create a clutter-free working environment. Organisms were housed inside a glass cuvette (3.5 mL, 10 mm optical path length with 4 clear windows). A small PDMS block closed the cuvette (in place of the lid) to enable oxygen diffusion. The cuvette was placed on an XYZ micromanipulator for fine adjustments. Figure 2 demonstrates the system. LEDs can be swapped for fluorescence imaging. Ortho-view Microscope was characterized using a standard slide (3" x 1" positive 1951 USAF Wheel Pattern Test Target). Figure 3 demonstrates a sample image from the Ortho-view and a commercially available EVOS imaging system, both 4x magnifications. A 2-tailed t-test confirmed no difference between the measurements obtained from Ortho-view and the EVOS system.


Results, Conclusions, and Discussions:: Three case studies, as proof-of-concept investigations, were conducted to show the application of the Ortho-view in studying aquatic organisms. First, we used Ortho-view to examine the movement of Hydra Vulgaris. Figure 4 demonstrates a top view of Hydra (4X magnification). We confirmed that videos obtained from the Ortho-view were suitable for biomechanics analysis. To this end, DeepLabCut was used to quantify the movement of seven user-defined anatomical positions (e.g., tentacles). Figure 5 demonstrates the starting positions with a square and the final positions with a triangle; time is encoded via the change in the color of the line connecting the indicators. Second, we used Pacific sand dollar larvae (Dendraster excentricus). Marine larvae possess a three-dimensional structure and swim rapidly. Here, we used the top and side views to visualize organisms from two orthogonal views. Ortho-view provided an approach to explore the hydrodynamics of freely-swimming larvae in a 3D microenvironment (Figure 6). Finally, we used the Ortho-view to visualize sea anemones (Figure 7). We used the side view option here since sea anemones were attached to materials blocking light. Our results demonstrate the utility of the Ortho-view system in studying aquatic organisms. In future iterations, we will exchange the light source to enable fluorescent imaging.

Acknowledgements (Optional): : Authors are thankful to CSULB marine lab, Pace-Lab, Ahrar-Lab, Moses Villeda, Yvette Ralph, and Dr. Rob Steele.

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