Department of Physics, Portland State University, Portland, Oregon, USA.
Astrobiology. 2024 Mar;24(3):300-317. doi: 10.1089/ast.2023.0020.
We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock.
我们报告了一种简单、廉价的荧光显微镜设计，使用发光二极管（LED）激发，用于检测矿物基质中标记和未标记的微生物。使用深紫外（DUV）激发可见发射不需要专门的光学器件或载玻片，可以使用倾斜照明几何结构轻松且廉价地实现。为了避免矿物自发荧光，DUV 激发（<280nm）优于近紫外（365nm）。当用 DUV 激发时，无色素的细菌显示两个发射峰：一个在近紫外区域约 320nm，对应于蛋白质，另一个在蓝绿色范围，对应于黄素和/或还原型烟酰胺腺嘌呤二核苷酸（NADH）。许多常用的染料在 DUV 中也显示出次级激发峰，其发射光谱和量子产率与主峰相同。然而，DUV 无法激发关键的生物特征分子，特别是蓝细菌中的叶绿素。可见激发（紫色到蓝色）也比近紫外产生的矿物自发荧光少，这里看到的矿物中的大多数自发荧光是绿色的，因此红色染料和叶绿素和卟啉的红色自发荧光很容易区分。DUV 和近紫外或可见激发的配对，以及整个可见光范围内的发射，代表了在土壤和岩石中检测标记和未标记细菌的最彻底方法。
