Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes

Abstract

Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and lightsheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.

Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma

“…Next, lymph nodes were prepared using CUBIC-R1-based tissue optical clearing for imaging as described previously. 39 Briefly, lymph nodes were immersed in CUBIC-R1 solution for at least 1.5 days (with a longer, up to 7 days incubation time for the enlarged lymph nodes) and subjected to imaging using a Zeiss Lightsheet Z.1 equipped with two 5× objectives (N.A. 0.1) that project two independent co-axial lightsheets onto the lymph nodes from the left and right.…”

Imaging of fluorescent polymer dots in relation to channels and immune cells in the lymphatic system

“…Then, put the sample into 4′,6-diamidino-2-phenylindole (DAPI) solution (MA0128, meilunbio, China) for nuclear staining and DiD solution (5 ​μM, MB6190, meilunbio, China) for cytomembrane staining. Each staining period last for 5 ​h, and washing solution containing 0.2% vol/vol Triton X-100 was used after each staining period for 24 ​h [ 26 ]. Using a Leica TCS SP5 CLSM, three different channels were as follows: fluorescent Pdots (excitation 458 ​nm, emission 530–600 ​nm), DAPI (excitation 405 ​nm, emission 425–500 ​nm), and DiD (excitation 633 ​nm, emission 655–675 ​nm).…”

Perspective: Extending the Utility of Three-Dimensional Organoids by Tissue Clearing Technologies

“…As in the case of FP, it should be noted that retention of the stained signals is dependent on the clearing reagents, as some may remove a portion of the staining target, while others may alter the binding affinity or antigenicity (Lallemant et al, 2020;Matryba et al, 2020). For example, the intensive comparison by Li et al (2017) showed that the preservation degree of antigenicity toward major cell type markers in the lymph node is varied among tested methods.…”