Optical clearing based cellular-level 3D visualization of intact lymph node cortex

Abstract

Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellularlevel structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.

The morphological changes in the internal organs of laboratory animals after prolonged oral administration of gold nanoparticles

“…The detection and photothermal treatment of nonpigmented breast cancer cells in sentinel lymph nodes were achieved by previous labeling them with nanoparticles. In this study and in recent work,33 the enhancement of imaging depth in lymph nodes were demonstrated by using of optical clearing techniques.…”

Advances in Ex Situ Tissue Optical Clearing

“…Recent progress in TOC and imaging opens new avenues to study cells phenotype and distribution within transparent, entire intact lymph nodes, an approach called histocytometry. Until now FocusClear, CUBIC, SeeDB, CLARITY, BABB, and uDISCO were used for lymph nodes clearing, with one study describing a completely new method for their clearing, C e 3D . C e 3D is the first approach to utilize N ‐methylacetamide with histodenz for TOC, a solution selected from 32 tested possible OCAs.…”

In vivo cellular-level real-time pharmacokinetic imaging of free-form and liposomal indocyanine green in liver

“…For in vivo video-rate cellular-level monitoring of ICG pharmacokinetic dynamics, a custombuilt video-rate laser-scanning confocal microscopy system previously described [37][38][39][40][41][42] was modified for simultaneous imaging of green and near infrared ICG fluorescence imaging as shown in Fig. 1(a).…”