최소 침습적 광학 투명 매체: 생체 외 및 생체 내 생세포 이미징

Abstract Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues. Similar content being viewed by others Main Live biological tissues are dynamic by nature. Thanks to various chemical and genetically encoded fluorescent biosensors, we can image and measure dynamic biological phenomena within the live tissues and organs using fluorescence microscopy. However, the imaging depth is often limited by tissue opacity. It has been a long-standing challenge to make live and healthy biological tissues transparent to facilitate live imaging. The opacity of the biological tissues is largely due to the inhomogeneity of refractive index within the samples. Two-photon microscopy uses a near-infrared excitation laser instead of visible light to reduce light scattering; however, the imaging depth is limited to a few hundred microns in mammalian tissues in vivo1. Adaptive optics uses a deformable mirror or spatial light modulator to correct aberrations caused by macroscopic refractive index distortions, but it is not effective for highly scattering samples2. For fixed tissues, optical clearing is a powerful approach: light refraction and scattering are minimized by removing high-index components (for example, lipids) and/or by immersing the sample in high-index solutions with refractive indices of 1.43–1.55 (refs. 3,4,5,6,7,8,9,10,11,12,13). However, most of the clearing agents developed for fixed tissues are toxic to live cells. Less toxic chemicals (for example, glycerol, dimethyl sulfoxide and sugars) have been tested for highly fibrous extracellular structures, such as skin and skull in vivo14,15,16,17,18,19; however, these chemicals interfere with cellular functions. A recent study claimed to have achieved optical clearing of the skin in live animals using a strongly absorbing dye, such as tartrazine18. However, the osmolality of the dye solutions used in the study was several-fold higher than the physiological osmolality condition, precluding its application to live imaging of normal physiological functions. Therefore, live mammalian cells and tissues have not yet been rendered transparent while maintaining intact cellular functions. Some chemicals have been proposed to be compatible with live cell imaging. Iodinated contrast agents were attractive candidates because of their low osmolarity. One of them, iodixanol, improves the transparency of bacteria and some multicellular organisms20,21. However, its toxicity to mammalian cells has not been fully evaluated. Another study attempted to improve transparency of the mouse brain by adding glycerol to drinking water22. However, it is unclear whether the marginal change in transparency was due to an increase in the refractive index in the brain, as glycerol should be easily metabolized once absorbed in the gut. Here we developed SeeDB-Live, a tissue-clearing medium for live mammalian cells and tissues. SeeDB-Live contains bovine serum albumin (BSA), which has exceptionally low osmolarity when dissolved in water and is minimally invasive to live cells. SeeDB-Live improved the imaging depth of spheroids, organoids, acute brain slices and the mouse brain in vivo. Results Strategies for minimally invasive optical clearing of live mammalian cells Light scattering in tissues is caused by refractive index mismatch between the light scatterer and the medium. Previously, simple immersion-based clearing agents (refractive index, 1.46–1.52) have been developed (for example, fructose, iohexol and tartrazine)8,9,18; however, osmolarity of these clearing agents is extremely high. To make live tissues transparent under isotonic conditions, we would have to use either (i) membrane-permeable or (ii) membrane-impermeable low-osmolarity (that is, high molecular weight) chemicals to reduce the refractive index mismatch (Fig. 1a). For (i) membrane-permeable chemicals, we do not need to change the concentration of the saline; however, when (ii) membrane-impermeable chemicals are added to the medium, we would need to