Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging

Summary

Optical clearing techniques, such as absorbing molecules solutions, reduce light scattering temporarily to enhance biological imaging and offer biocompatible, reversible in vivo imaging strategies for deeply embedded organs. This protocol presents detailed experimental procedures as well as advanced image processing to achieve high-quality, dynamic imaging for real-time dynamics in living animals.

Abstract

Dynamic imaging is a foundational tool in biology and medicine, but it is limited by light scattering in live tissues caused by refractive index mismatches, which reduces penetration depth and resolution in biological tissues. Previous work introduced a novel approach to achieve optical transparency in live animals by utilizing strongly absorbing molecules. When these absorbing molecules, such as Tartrazine, an FDA-approved dye commonly used in foods, dissolve in water, they modify the refractive index of the aqueous medium through Kramers-Kronig relations. This modulation allows reduction of the refractive index mismatch between separate components in tissue with distinct refractive indices, thus mitigating scattering. This article reports the detailed methodology of the tissue clearing technique using a Tartrazine-based aqueous solution, applied to both ex vivo and in vivo samples. This approach reversibly renders live mouse bodies transparent in the visible spectrum, enabling clearer and deeper imaging of internal processes and structures. An advanced image processing algorithm was then used to improve the contrast and segmentation of organs of interest. Additionally, high transparency in ex vivo samples, such as chicken breast, treated with Tartrazine-based solution, is shown. This method offers a versatile and accessible approach to enhance imaging capabilities across various modalities and paves the way for advancement in the understanding of complex biological systems.

Introduction

Biological imaging has long been challenged by the scattering of light, which obscures structures of interest¹. Optical clearing techniques aim to reduce light scattering and enhance visual clarity, enabling the visualization of previously hidden structures². The development of these techniques has led to a diverse array of methods designed to improve optical imaging of ex vivo biological tissues, even whole animals, without physical dissection of the samples³. However, the development of similar techniques for in vivo applications has been challenging. Techniques such as CLARITY....

Protocol

All animal experiments conducted were approved by The University of Texas at Dallas' Institutional Animal Care and Use Committee (IACUC) in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The reagents and the equipment used are listed in the Table of Materials. Image processing code is available at Github: https://github.com/ou-group-utd/JoVE-2025-Image-Processing

1. Tartrazine solution preparation

1. For ex vivo samples
   1. To prepare dye solutions, calculate the required mass of dye powder (Tartrazine) to prepare the desired volume of the soluti....

Results

Demonstration of optical transparency in ex vivo chicken breast tissue
To investigate light propagation through ex vivo tissue, we used sliced pieces of raw boneless chicken breast with thicknesses of 1.5 mm, 2.5 mm, and 3.5 mm (Figure 2B). The tissues were cut parallel to collagen fibers, ensuring consistent muscle fiber orientation. Images were taken using both a mesh grid pattern and .......

Discussion

This work presents a comprehensive and reproducible protocol method to achieve optical transparency in biological tissues utilizing absorbing molecules, specifically FDA-approved food dye Tartrazine (approved for use in food products, not in medical applications). This method effectively minimizes intrinsic scattering in tissue by altering the refractive index within the tissue's microenvironment, which enhances optical transparency. This study demonstrated the effectiveness of this method through both ex vivo a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Analytical Balance	HZK	FA220S
Animal anesthesia Machine	RWD life science co	TAIJI-IE	Inhalation Anesthetics
Atipamezole HCL injection 5 mg/mL	Covertus	82124	Injectable anesthesia
CMOS camera	Thor labs	CS165CU/M, Zelux
Dexmedetomidine HCL injection 5 mg/10 mL	Covertus	72960	Injectable anesthesia
Fiber Light Illuminator	RWD life science co	76301	Light source for top illumination
Glass slides	CravitySci	0303-2122	Super grade microscope slides
Homeothermic Blanket	RWD life science co	69027	Animal temperature controller
Image Processing	OpenCV	Python	Software
Insulin Syringe 0.5 mL 29 G	Comfort point	26028	Injectable anesthesia
Isoflurane	Covertus	SKU 029405	Inhalation Anesthetics
Ketmine HCL injection 100 mg/mL	Dechra	20042240	Injectable anesthesia
LED light source	GGE corp	TLB-01	Light source for top illumination
Lens	Thor labs	MVL4WA	3.5 mm focal length and f/1.4 aperture
Nair	Church & Dwight	L 1224	Hair removal Cream
PBS ph 7.4 (1x)	Gibco	2902869	Injectable anesthesia
Q-Tip	Puritan medical products company	806-wc hospital
SeaPlaque Agarose	Lonza	50101	Chemical
Tartrazine	MilliporeSigma	CAS number: 1934-21-0	Chemical
Transfer pipette	Fisherbrand	13-711-9AMMD
UV-VIS-NIR Spectrophotometer	Shimadzu	SolidSpec-3700i	Instrument
Vials	Qorpak	242633
Volumetric pipettes	Eppendorf research plus	3123000063	100-1000µL
Water Purification System	Avidity science	SOLO S	Instrument