Morizet Josephine, Chow Darren, Wijesinghe Philip, Schartner Erik, Dwapanyin George, Dubost Nicolas, Bruce Graham D, Anckaert Ellen, Dunning Kylie, Dholakia Kishan
SUPA, School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews Fife KY16, U.K.
Robinson Research Institute, School of Biomedicine, The University of Adelaide, Adelaide 5501, Australia.
ACS Photonics. 2023 Nov 13;10(12):4177-4187. doi: 10.1021/acsphotonics.3c00900. eCollection 2023 Dec 20.
Cellular metabolism is a key regulator of energetics, cell growth, regeneration, and homeostasis. Spatially mapping the heterogeneity of cellular metabolic activity is of great importance for unraveling the overall cell and tissue health. In this regard, imaging the endogenous metabolic cofactors, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), with subcellular resolution and in a noninvasive manner would be useful to determine tissue and cell viability in a clinical environment, but practical use is limited by current imaging techniques. In this paper, we demonstrate the use of phasor-based hyperspectral light-sheet (HS-LS) microscopy using a single UVA excitation wavelength as a route to mapping metabolism in three dimensions. We show that excitation solely at a UVA wavelength of 375 nm can simultaneously excite NAD(P)H and FAD autofluorescence, while their relative contributions can be readily quantified using a hardware-based spectral phasor analysis. We demonstrate the potential of our HS-LS system by capturing dynamic changes in metabolic activity during preimplantation embryo development. To validate our approach, we delineate metabolic changes during preimplantation embryo development from volumetric maps of metabolic activity. Importantly, our approach overcomes the need for multiple excitation wavelengths, two-photon imaging, or significant postprocessing of data, paving the way toward clinical translation, such as in situ, noninvasive assessment of embryo viability.
细胞代谢是能量学、细胞生长、再生和内环境稳态的关键调节因子。在空间上绘制细胞代谢活性的异质性对于揭示整体细胞和组织健康状况至关重要。在这方面，以亚细胞分辨率且以非侵入性方式对内源性代谢辅因子烟酰胺腺嘌呤二核苷酸（磷酸）（NAD(P)H）和黄素腺嘌呤二核苷酸（FAD）进行成像，将有助于在临床环境中确定组织和细胞活力，但实际应用受到当前成像技术的限制。在本文中，我们展示了使用基于相量的高光谱光片（HS-LS）显微镜，以单一紫外光激发波长作为在三维空间中绘制代谢图谱的途径。我们表明，仅在375 nm的紫外光波长下激发，就可以同时激发NAD(P)H和FAD的自发荧光，而它们的相对贡献可以使用基于硬件的光谱相量分析轻松量化。我们通过捕获植入前胚胎发育过程中代谢活性的动态变化，展示了我们的HS-LS系统的潜力。为了验证我们的方法，我们从代谢活性的体积图中描绘了植入前胚胎发育过程中的代谢变化。重要的是，我们的方法克服了对多个激发波长、双光子成像或大量数据后处理的需求，为临床转化铺平了道路，例如对胚胎活力进行原位、非侵入性评估。
