Pham Dan L, Gillette Amani A, Riendeau Jeremiah, Wiech Kasia, Guzman Emmanuel Contreras, Datta Rupsa, Skala Melissa C
University of Wisconsin-Madison, Department of Biomedical Engineering, Madison, Wisconsin, United States.
Morgridge Institute for Research, Madison, Wisconsin, United States.
J Biomed Opt. 2025 Dec;29(Suppl 2):S22702. doi: 10.1117/1.JBO.29.S2.S22702. Epub 2024 Feb 29.
Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging).
This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs.
Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended.
Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.
无标记显微镜技术的进步能够提供实时、非侵入性成像，具备独特的对比度来源以及自动化的标准化分析，以表征异质性和动态生物学过程。这些工具将克服广泛使用的方法所面临的挑战，这些方法具有破坏性（例如组织学、流式细胞术）或缺乏细胞分辨率（例如基于平板的检测、全动物生物发光成像）。
本观点旨在（1）阐明在未受干扰的系统中，随着时间和空间追踪异质性细胞功能对无标记显微镜技术的需求，以及（2）针对仪器设备、图像分析和图像解读提出改进建议，以满足这些需求。
考虑了三个关键研究领域（癌症研究、自身免疫性疾病以及组织和细胞工程），以支持使用无标记显微镜技术来表征生物系统内的异质性和动态变化的需求。基于几种无标记显微镜技术的优势（例如多种分子对比度来源、非侵入性监测）和劣势（例如成像深度、图像解读），对未来成像系统提出了改进建议。
仪器设备的改进，包括提高分辨率和成像速度的策略、图像分析工具的标准化和集中化，以及可靠的数据验证和解读，将扩大无标记显微镜技术在研究异质性和动态生物系统方面的应用。
