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Surpassing the Diffraction Limit in Label-Free Optical Microscopy
ACS Photonics ( IF 6.5 ) Pub Date : 2024-08-27 , DOI: 10.1021/acsphotonics.4c00745 David Palounek 1, 2 , Milan Vala 1 , Łukasz Bujak 1 , Ivan Kopal 1, 2 , Kateřina Jiříková 1 , Yevhenii Shaidiuk 1 , Marek Piliarik 1
ACS Photonics ( IF 6.5 ) Pub Date : 2024-08-27 , DOI: 10.1021/acsphotonics.4c00745 David Palounek 1, 2 , Milan Vala 1 , Łukasz Bujak 1 , Ivan Kopal 1, 2 , Kateřina Jiříková 1 , Yevhenii Shaidiuk 1 , Marek Piliarik 1
Affiliation
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Super-resolution optical microscopy has enhanced our ability to visualize biological structures on the nanoscale. Fluorescence-based techniques are today irreplaceable in exploring the structure and dynamics of biological matter with high specificity and resolution. However, the fluorescence labeling concept narrows the range of observed interactions and fundamentally limits the spatiotemporal resolution. In contrast, emerging label-free imaging methods are not inherently limited by speed and have the potential to capture the entirety of complex biological processes and dynamics. While pushing a complex unlabeled microscopy image beyond the diffraction limit to single-molecule resolution and capturing dynamic processes at biomolecular time scales is widely regarded as unachievable, recent experimental strides suggest that elements of this vision might be already in place. These techniques derive signals directly from the sample using inherent optical phenomena, such as elastic and inelastic scattering, thereby enabling the measurement of additional properties, such as molecular mass, orientation, or chemical composition. This perspective aims to identify the cornerstones of future label-free super-resolution imaging techniques, discuss their practical applications and theoretical challenges, and explore directions that promise to enhance our understanding of complex biological systems through innovative optical advancements. Drawing on both traditional and emerging techniques, label-free super-resolution microscopy is evolving to offer detailed and dynamic imaging of living cells, surpassing the capabilities of conventional methods for visualizing biological complexities without the use of labels.
中文翻译：
超越无标记光学显微镜的衍射极限
超分辨率光学显微镜增强了我们在纳米尺度上可视化生物结构的能力。如今，基于荧光的技术在探索生物物质的结构和动力学方面具有不可替代性，具有很高的特异性和分辨率。然而，荧光标记概念缩小了观察到的相互作用的范围，并从根本上限制了时空分辨率。相比之下，新兴的无标记成像方法本身不受速度限制，并且有可能捕获整个复杂的生物过程和动力学。虽然将复杂的未标记显微镜图像超越衍射极限，达到单分子分辨率并在生物分子时间尺度上捕捉动态过程被广泛认为是无法实现的，但最近的实验进展表明，这种愿景的要素可能已经存在。这些技术使用固有的光学现象（如弹性和非弹性散射）直接从样品中获取信号，从而能够测量其他特性，如分子量、取向或化学成分。该观点旨在确定未来无标记超分辨率成像技术的基石，讨论其实际应用和理论挑战，并探索有望通过创新的光学进步增强我们对复杂生物系统的理解的方向。借鉴传统和新兴技术，无标记超分辨率显微镜正在不断发展，以提供活细胞的详细和动态成像，超越了传统方法在不使用标记的情况下可视化生物复杂性的能力。
更新日期：2024-08-27
中文翻译：
超越无标记光学显微镜的衍射极限
超分辨率光学显微镜增强了我们在纳米尺度上可视化生物结构的能力。如今，基于荧光的技术在探索生物物质的结构和动力学方面具有不可替代性，具有很高的特异性和分辨率。然而，荧光标记概念缩小了观察到的相互作用的范围，并从根本上限制了时空分辨率。相比之下，新兴的无标记成像方法本身不受速度限制，并且有可能捕获整个复杂的生物过程和动力学。虽然将复杂的未标记显微镜图像超越衍射极限，达到单分子分辨率并在生物分子时间尺度上捕捉动态过程被广泛认为是无法实现的，但最近的实验进展表明，这种愿景的要素可能已经存在。这些技术使用固有的光学现象（如弹性和非弹性散射）直接从样品中获取信号，从而能够测量其他特性，如分子量、取向或化学成分。该观点旨在确定未来无标记超分辨率成像技术的基石，讨论其实际应用和理论挑战，并探索有望通过创新的光学进步增强我们对复杂生物系统的理解的方向。借鉴传统和新兴技术，无标记超分辨率显微镜正在不断发展，以提供活细胞的详细和动态成像，超越了传统方法在不使用标记的情况下可视化生物复杂性的能力。
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