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Transient fluorescence detected infrared (TFD-IR) microscopy was developed to overcome the diffraction limit of
infrared (IR) light without a near-field system. This microscopic technique is based on TFD-IR spectroscopy, which
converts information on IR absorption to fluorescence intensity by further electronic excitation of vibrationally excited
molecules by a probing UV/visible light. Roots of Arabidopsis thaliana and living A549 cells with fluorescent dyes
were chosen as samples. In the measurements using the TFD-IR microscope, tunable IR picosecond laser pulses were
used in the wavelength range from 2700 to 3700 nm, corresponding to CH, NH, and OH stretching modes. Fluorescence
images of the root cells of A. thaliana by the TFD-IR scheme were obtained with super-resolution compared with the
resolution of conventional IR microscopy. The resolution is estimated to be less than 2.6 μm by fitting of a gaussian
function. However, the TFD-IR images were dominated mainly by the fluorescent dyes because they were almost the
same as a conventional fluorescence image. To investigate other contributions hidden by that of fluorescent dyes, we
plotted the fluorescence intensity in several 5 μm squares at various IR wavelengths, called a TFD-IR spectrum. For root
cells of A. thaliana, the TFD-IR spectra show shapes similar to those of a conventional IR absorption spectrum of the
fluorescent dye. Therefore, the TFD-IR images are not due to the cellular components. For an A549 cell, the TFD-IR
spectra were different from a conventional IR absorption spectrum of fluorescent dyes in the wavelength region shorter
than 3100 nm. We speculate that the spectral difference is due to the cellular components, possibly assigned to the
combination band related to amino groups of cellular components bonded covalently to the fluorescent dyes.
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