Reusable Hepatic Imaging Tool: A Reusable 3D-Printed Tool for Hepatic Intravital Microscopy

The overall goal of this procedure is to showcase the Reusable Hepatic Imaging Tool, or RHIT, as a breakthrough tool for capturing high-quality, dynamic biological data, especially in liver research. Traditional scientific methods for studying mice in controlled experiments have long relied on behavioral observation, physiological monitoring, and genetic analysis. The complexity of these studies frequently results in inconsistent or limited data outputs.

Many experiments rely on data that is either incomplete or difficult to interpret, such as brief flashes of low-resolution images that quickly fade, making it hard to capture long-term trends or subtle changes. Introducing RHIT, enabling dynamic, high-resolution capture for liver research and beyond. The technique we demonstrate enables the fabrication of custom imaging tools using accessible materials and widely available 3D printing technology.

Designed for use in academic and resource-limited environments, this approach allows for the rapid development and deployment of devices to support high-resolution in vivo imaging. Our Reusable Hepatic Imaging Tool, or RHIT, was developed to improve tissue stability during intravital microscopy of the liver. RHIT's modular design accommodates mice of various sizes and supports extended imaging of dynamic metabolic processes with minimal physiological disruptions.

RHIT is fabricated in a single step using acrylonitrile, butadiene, styrene, ABS, a thermoplastic selected for its light weight, thermostability, and mechanical durability. By adjusting material density, RHIT effectively absorbs motion caused by respiration and heartbeat, thereby reducing image artifacts and enhancing clarity. ABS's chemical inertness and water insolubility ensure availability with biological tissue and enable efficient cleaning and reuse.

To recreate RHIT, use a 3D product design software. Autodesk Inventor was used for this design. Open Autodesk Inventor and create a new standard part.

Save the file with appropriate description. Create the sketch of RHIT on the front plane using the create 2D sketch feature. Click on the rectangle icon and place its bottom left corner at the origin of the sketch plane.

Draw a second rectangle inside the first one to represent the imaging window of RHIT. Add dimensions to both rectangles using the dimension feature, ensuring all lengths and widths are referenced to the origin and relative to each other. Adjust the RHIT dimensions according to the mouse size.

Add thickness to RHIT by selecting outer rectangle only and extrude the sketch using the extrude tool to leave an opening in the center. Select the bottom face of the extruded RHIT and start a new 2D sketch using the start 2D sketch feature. Draw a rectangle to define the pocket for the heat pad.

Define the dimensions of the rectangle based on the size and thickness of the heat pad that will be inserted into this slot. These measurements should match the physical heat pad to be used to ensure accurate temperature modeling of the mammal. Apply the required dimensions using the dimension feature.

Exit the 2D sketch and perform a material removal using the cut extrude tool to create a deep cavity for the heat pad. Zoom in as needed to precisely position the sketch. Start a new sketch for the temperature probe on the same face as the cavity.

Click on start a 2D sketch and select the circle icon. Place the circle on the plain surface and dimension accordingly. Exit the sketch mode.

Click on extrude and select the sketch of the circle. In extrusion mode, click on cut, extrude, and remove material. Place the circle on the right side of the face with the pocket insert.

It doesn't need to be centered yet. Add dimensions to align it with the center of the thickness of RHIT. Click extrude.

A popup will appear with an arrow showing the direction of the cut, along with the through all option. Select through all to cut through the entire material, then click okay. Exit extrusion mode.

Add fillets on the edges of window by clicking on fillet icon and select all the eight sharp edges. Add fillet size and exit fillet mode. Prepare the model for 3D printing by navigating to the environment tab and selecting 3D print tab.

To print the unmodified RHIT, import the STL file, either from figure 1A or the supplementary materials, into A 3D printing preparation software. Click import, select the file, and click prepare to slice the model into printable layers. Choose a compatible commercial 3D printer and click print.

Monitor the printing process. Once it's complete, carefully remove and clean the printed RHIT as needed. Wait 10 to 15 minutes after the ejections before beginning surgery and imaging.

Microscope preparation and utilizing RHIT. Prepare a conventional inverted confocal microscope for liver IBN, as shown in figure 2A. Insert the stage adapter to accommodate the sample.

For this protocol, a custom made metal insert was used with a 35 millimeter circular opening and a 40 millimeter cover glass. Thoroughly clean both sides of the cover glass before and after imaging, using 70%ethanol as necessary. Secure the appropriate RHIT based on mouse size with tape by aligning the RHIT aperture with objective lens, parallel to the imaging covering slip, as shown in figure 2G, on the metal insert with tape over the cover glass.

Select the objective for imaging based on an on-area size, spatial resolution, and imaging time, such as a 25X per 95 water objective. Level the objective close to the cover glass. Secure a strip of gauze across the wet window with tape.

This will help position the tissue and separate it from other abdominal organs. Prewarm RHIT using a heat pad inserted into the slot and secure the heat probe adjacent to the RHIT, shown in figure 2G through H.This will maintain the body temperature of the mouse during imaging. Shave the abdominal coat using a hair clipper and/or hair removal cream.

Move hair residue and disinfect the exposed skin with 70%ethanol. Depending on institutional IACUC requirements, an alternating scrub of Betadine and ethanol may be used to ensure appropriate surgical site preparation. Reassess the depth of anesthesia prior to surgery on the animal.

Expose the liver by making a horizontal crescent-shaped two to three centimeter incision under the ribcage with surgical scissors from the xiphoid, ending at the left lateral side of the upper abdomen. After the initial incision, carefully make a smaller secondary incision in the muscle layer to expose the left lobe of the liver. Cauterize as necessary to avoid bleeding.

Prevent heat damage to the liver while using the cauterizer by placing a saline-soaked gauze between the liver and muscle layer to absorb the emanating heat of the muscle. Avoid any excessive bleeding, surgical or mechanical trauma, or external compression. Once the liver is exposed, transfer the mouse to the microscope stage.

Positioning of the mouse on the microscope stage. Position the anesthetized mouse's abdomen facing down on top of the RHIT platform of the microscope. Adjust the nose cone so that the mouse remains under anesthesia.

Use a saline-soaked cotton-tipped applicator to gently guide the left lateral lobe of the liver against the cover slip while gently applying external pressure to the chest cavity. Take advantage of gravity to carefully maneuver the liver out and onto the cover glass. Use a gauze strip to gently separate the liver from the mouse's body.

Check the nose cone again to ensure the mouse remains under anesthesia during imaging. Prevent tissue dehydration by applying a water-based gel around the edges of the exposed cavity and avoiding direct contact with the heat pad. A microscope with an enclosed humidity chamber can also help maintain temperature and humidity.

Apply opthalmic ointment to the eyes to prevent corneal drying. Lastly, set up timelapse IVM to begin imaging. Open laser shutters, evaluate fluorescent labeling, and identify a stable region of interest with a notable blood flow.

Select appropriate image acquisitions, including format, speed, imaging intervals, and length of time, frame average, et cetera. Define upper and lower boundary for Z-stacks if required, and start timelapse recording. To validate our protocol, we quantified image stability using the structural similarity index, or SIM, shown in figure 3D.

SIM compares two images based on luminance, contrast, and structural patterns. A value of one indicates perfect similarity, while zero reflects distortion. Figure 3C shows that RHIT consistently yields the highest SIM values, up to 50 times greater than no insert, demonstrating superior image quality.

In images labeled A through C, we compared liver IVM without a stabilizer, with the cardboard inset, and with RHIT. RHIT maintains clear resolution of lipid droplets in magenta, nuclei in green, and vasculature in cyan. Black and white insets highlight image stability.

RHIT also enabled long-term IBM in obese mice for over eight hours, as shown in movie one. Together, these results confirmed that RHIT significantly improves imaging quality and reproducibility across conditions. Taken together, RHIT simplifies the complex setup traditionally required for hepatic IBM, improving both reproducibility and accessibility.

This approach anticipates a broader movement toward leveraging 3D printing for the standardization of advanced imaging protocols. Beyond liver imaging, RHIT's design principles may be extended to other applications, including organ tomography, material science, and engineering, where motion stability is critical for image fidelity.