Abstract
The absorption spectrum of light is known to be a “molecular fingerprint” that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 μm, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells.
Highlights
Our quantitative understanding of cellular function would be aided considerably by the accurate determination of the amounts of molecular components present in living cells
Because the absorption is proportional to the concentration and optical path length, as predicted by the Lambert-Beer law, conventional absorption spectroscopy requires large sample volumes and concentrations
cavity-reflection-enhanced absorption microscopy (CREAM) uses a light focused on a ~15-μm spot in an optical cavity (S1 Fig), with which we can measure the O.D. of a Venus fluorescent protein at an optical path length of 10-μm (Fig 2), to a concentration of approximately 5 μM
Summary
Our quantitative understanding of cellular function would be aided considerably by the accurate determination of the amounts of molecular components (such as nucleic acids, proteins, and lipid) present in living cells. One method for such quantitation is the measurement of light absorption, which reveals both the molecular amount and type, as in the commonly used assays for protein [1] and nucleic acid [2] concentration. We wondered if it would be possible to apply the same molecular fingerprinting approach to a living cell at a subcellular resolution to quantitatively estimate the molecular concentration and distribution, as this may reveal differences between cells that bulk biochemistry overlooked.
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