Label-FreeHigh-ResolutionPhotothermalOpticalInfraredSpectroscopyfor Spatiotemporal Chemica lAnalysis in Fresh, Hydrated Living Tissues and Embryos

Label-free chemical imaging of living and functioning systems is the holy grail of biochemical research. However, existing techniques often require extensive sample preparation to remove interfering molecules such as water, rendering many molecular imaging techniques unsuitable for in situ structural studies. Here, we examined freshly extracted tissue biopsies and living small vertebrates at submicrometer resolution using optical photothermal infrared (O-PTIR) microspectroscopy and demonstrated the following major advances: (1) O-PTIR can be used for submicrometer structural analysis of unprocessed, fully hydrated tissue biopsies extracted from diverse organs, including living brain and lung tissues. (2) O-PTIR imaging can be performed on living organisms, such as salamander embryos, without compromising their further development. (3) Using O-PTIR, we tracked the structural changes of amyloids in functioning brain tissues over time, observing the appearance of newly formed amyloids for the first time. (4) Amyloid structures appeared altered following standard fixation and dehydration procedures. Thus, we demonstrate that O-PTIR enables time-resolved submicrometer in situ investigation of chemical and structural changes in diverse biomolecules in their native conditions, representing a technological breakthrough for in situ molecular imaging of biological samples.