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Proceedings Paper

Cell imaging by transient fluorescence detected infrared microscopy
Author(s): Tsutomu Ohmori; Makoto Sakai; Miya Ishihara; Makoto Kikuchi; Masaaki Fujii
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Paper Abstract

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.

Paper Details

Date Published: 20 February 2008
PDF: 7 pages
Proc. SPIE 6853, Biomedical Optical Spectroscopy, 685307 (20 February 2008);
Show Author Affiliations
Tsutomu Ohmori, National Defense Medical College (Japan)
Makoto Sakai, Tokyo Institute of Technology (Japan)
Miya Ishihara, National Defense Medical College (Japan)
Makoto Kikuchi, National Defense Medical College (Japan)
Masaaki Fujii, Tokyo Institute of Technology (Japan)

Published in SPIE Proceedings Vol. 6853:
Biomedical Optical Spectroscopy
Anita Mahadevan-Jansen; Robert R. Alfano; Alvin Katz; Wolfgang Petrich, Editor(s)

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