Bioimaging by molecular-vibration-sensitive quantitative phase microscopy based on wide-field mid-infrared photothermal excitation
Bioimaging by molecular-vibration-sensitive quantitative phase microscopy based on wide-field mid-infrared photothermal excitation
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DOI：
10.1117/12.2545728
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发表时间：
2020-02
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影响因子：
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通讯作者：
Miu Tamamitsu;Keiichiro Toda;Yu Nagashima;R. Horisaki;T. Ideguchi
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文献类型：
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作者：
Miu Tamamitsu;Keiichiro Toda;Yu Nagashima;R. Horisaki;T. Ideguchi
无标记光学成像对于研究脆弱的生物现象是有价值的，其中不希望与生物分子的外源标记相关的化学和/或光学损伤。分子振动（MVI）和定量相位成像（QPI）是两种最成熟的无标记成像方法，分别提供样品的生物化学和形态学信息。尽管这些方法在过去二十年中沿着其密集的技术发展已经开创了许多重要的生物分析，但其固有的局限性仍然没有得到解决。在这篇文章中，我们提出了一个统一的成像方案，弥合MVI和QPI之间的技术差距，实现同时和原位集成的两个互补的无标记对比度使用中红外（MIR）光热效应。我们的方法是一个超分辨率MIR成像，其中宽场MIR激发引起的振动共振和由此产生的光热RI变化被检测和本地化的空间分辨率由基于可见光的QPI系统确定。我们证明了这种方法的适用性，称为MV敏感的QPI（MV-QPI），活细胞成像。我们的MV-QPI方法可以允许以无标记和无损伤的方式定量绘制全球细胞形态内的亚细胞生物分子分布，提供复杂和脆弱的生物活性的更全面的图片。
Label-free optical imaging is valuable for studying fragile biological phenomena where chemical and/or optical damages associated with exogenous labelling of biomolecules are not wanted. Molecular vibrational (MVI) and quantitative phase imaging (QPI) are the two most-established label-free imaging methods that provide biochemical and morphological information of the sample, respectively. While these methods have pioneered numerous important biological analyses along their intensive technological development over the past twenty years, their inherent limitations are still left unresolved. In this contribution, we present a unified imaging scheme that bridges the technological gap between MVI and QPI, achieving simultaneous and in-situ integration of the two complementary label-free contrasts using the midinfrared (MIR) photothermal effect. Our method is a super-resolution MIR imaging where vibrational resonances induced by wide-field MIR excitation and the resulting photothermal RI changes are detected and localized with the spatial resolution determined by a visible-light-based QPI system. We demonstrate applicability of this method, termed MV-sensitive QPI (MV-QPI), to live-cell imaging. Our MV-QPI method could allow for quantitative mapping of subcellular biomolecular distributions within the global cellular morphology in a label-free and damage-less manner, providing more comprehensive pictures of complex and fragile biological activities.