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

Summary

This protocol introduces a cutting-edge, Reusable Hepatic Imaging Tool (RHIT) for intravital microscopy (IVM) of live mice, which enhances tissue stability for rapid and acute experiments. The RHIT's 3D-printed design offers cost-effective production, scalability, and versatility, enabling prolonged observation of metabolic processes in mice of various sizes.

Abstract

Hepatic function and dysfunction can be assessed by directly visualizing the dynamic processes of the liver using confocal-based intravital microscopy (IVM). In particular, excellent spatial and temporal precision can be achieved in the extraction of both qualitative and quantitative information. On the microscope stage, maintaining physiological tissue conditions while achieving stability and repeatability remains challenging. Increased weight and hepatic lipid content have a detrimental effect on this process in animal models of Type II diabetes (T2D) and non-alcoholic fatty liver disease (MAFLD). To overcome these constraints, we have created RHIT, a revolutionary imaging instrument design that improves tissue access and stability. RHIT is a reusable, scalable hepatic imaging device manufactured using 3D printing in an efficient one-step method. Its dimensions may be changed to fit mice of different sizes. Briefly, RHIT is a 3D-printed apparatus for positioning and stabilizing laboratory animals during intravital liver microscopy, used after a brief surgical preparation to minimize handling, stress, and variability. Its reusable, standardized design is easily produced and customized, supporting a range of sizes and potential experimental designs. Initial results demonstrate a notable improvement in the stability and reliability of hepatic intravital imaging, enabling the detailed visualization of subcellular structures, such as lipid droplets. This improved precision facilitates metabolic and physiological studies with improved clarity, reproducibility, and potential for longitudinal designs. The protocol offers a standardized, accessible approach that addresses long-standing technical challenges in hepatic IVM.

Introduction

Intravital microscopy is a powerful tool to visualize biological processes in vivo dynamically^1,2,3 in contrast to the traditional immunofluorescence histology with fixed tissue or live cell imaging and flow cytometry analysis of isolated cells. For liver inspection, IVM has been utilized to examine bile secretion and flow^4,5, glucose mobilization^6,7, nonparenchymal cells^8,9,

Protocol

All the animal studies were carried out at the animal facilities of the National Institutes of Health in compliance with guidelines and protocols authorized by the Ethics Committee for Animal Experiments (protocol No. LCMB-037). All personnel are required to be properly trained before proceeding with the following protocols. Animal handling was performed in compliance with institutional guidelines.

The RHIT protocol outlines the preparation of Reusable Hepatic Imaging Tools (RHITs) for use in intravital microscopy (IVM) experiments. The experimental procedures are performed in a sterilized microscope room, or hood, and involve a series of s....

Results

RHIT was designed using a CAD application (Figure 1). RHIT dimensions are scaled to accommodate small, medium, and heavy-sized mice (Table 1). RHIT design consists of a 107.50 mm long ABS flat material with an aperture positioned to accommodate liver imaging. IVM was performed under optimal (RHIT) and suboptimal (no insert or cardboard) conditions (see Figure 3A-C). This illustrates the range of possible outcomes and the extent .......

Discussion

Liver intravital microscopy has been extensively studied, however, previous methods often require highly invasive and unstandardized surgical procedures for stabilized terminal imaging^{5,6,7,9,31,32}. Motion is arguably the most challenging barrier preventing reproducible imaging in hepatic IVM. The most effective solution for .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
½-inch masking tape	3M General Purpose Masking Tape:	b40069428
40-mm coverslips	Bioptech	40-1313-03191
70% (v/v) ethanol	Thermo Fisher Scientific	BP2818-4	70% ethanol by volume and 30% water by volume
Anesthesia machine: AMD-3 Plus	Somni	AMD-17106
Aquasonic 100 ultrasound transmission gel	Parker Laboratories	01 08
BODIPY 665/67	Thermo Fisher Scientific (Invitrogen)	B3932	See Table 2
CAD software (Autodesk Inventor or equivalent)	Autodesk (representative CAD software)	n/a	Software
Cautery System	Gemini	GEM 5917
Clidox disinfectant	Pharmacal Research Laboratories Inc.	VWR, MSPP-96118F
Confocal microscope (Leica SP8 or equivalent)	Leica	SP8
Cotton-tip applicators	Puritan Medical Products	25-806 1WC
Custom-made aluminum stage insert (3 mm thick, 35-mm depression)	N/A	N/A
FORANE (isoflurane, USP) liquid for inhalation, 250 mL	Baxter	10019-360-60
Hair clipper (optional)	Onyx Fine Instruments	ASWRHC
Heat pad with rectal probe	ALA Scientific Instruments	HEATINGPAD-1/2
Heparin sodium, 1000 USP U/mL	Hikma	0641-0391-12
Hoechst 33342	Thermo Fisher Scientific (Invitrogen)	62249	See Table 2
Mouse heat pad	ALA Scientific Instruments	HEATINGPAD-1/2
Mus musculus	The Jackson Laboratory	C57BL/6	Laboratory mouse
Normal saline (0.9% sodium chloride)	Thermo Fisher Scientific	7210-16
RHIT (Reuseable Hepatic Imaging Tool) applicable size	Provided	N/A	See Table 1
Surgical forceps, curved tip	Fine Science Tools	11251-23
Surgical gauze	Mckesson	6939
Surgical scissors	Fine Science Tools	14001-14
Tetramethylrhodamine Dextran	Thermo Fisher Scientific (Invitrogen)	D7139	See Table 2
Ultra Fine U-100 Insulin Syringes, 31 G	Betty Mills	BD326730