High Refractive Index Imaging Buffer for Dual-Color 3D SMLM Imaging of Thick Samples

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Analytical Chemistry Pub Date : 2024-09-19 DOI:10.1021/acs.analchem.4c02893

Lulu Zhou, Wei Shi, Shuang Fu, Mengfan Li, Jianwei Chen, Ke Fang, Yiming Li

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Abstract

The current limitations of single-molecule localization microscopy (SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations caused by refractive index (RI) mismatch, present a significant challenge in achieving high-resolution images at greater depths. To extend the imaging depth, we optimized the imaging buffer of SMLM with the RI matched to that of the objective immersion medium and systematically evaluated five different RI-matched buffers, focusing on their impact on the blinking behavior of red-absorbing dyes and the quality of reconstructed super-resolution images. Particularly, we found that clear unobstructed brain imaging cocktails-based imaging buffer could match the RI of oil and was able to clear the tissue samples. With the help of the RI-matched imaging buffers, we showed high-quality dual-color 3D SMLM images with imaging depths ranging from a few micrometers to tens of micrometers in both cultured cells and sectioned tissue samples. This advancement offers a practical and accessible method for high-resolution imaging at greater depths without the need for specialized optical equipment or expertise.

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用于厚样品双色 3D SMLM 成像的高折射率成像缓冲液

目前，单分子定位显微镜（SMLM）在深部组织成像中的局限性主要是由于折射率（RI）不匹配引起的深度畸变，这对在更深的深度获得高分辨率图像提出了巨大挑战。为了扩展成像深度，我们优化了 SMLM 的成像缓冲液，使其折射率与物镜浸泡介质的折射率相匹配，并系统评估了五种不同的折射率匹配缓冲液，重点关注它们对红色吸收染料闪烁行为和重建超分辨率图像质量的影响。我们特别发现，基于鸡尾酒的透明无阻脑成像缓冲液能与油的 RI 匹配，并能清除组织样本。在 RI 匹配成像缓冲液的帮助下，我们在培养细胞和切片组织样本中展示了成像深度从几微米到几十微米不等的高质量双色三维 SMLM 图像。这一进步为更深度的高分辨率成像提供了一种实用、便捷的方法，而无需专门的光学设备或专业知识。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.