We built a photoacoustic tomographic (PAT) imaging system by scanning a single detector (φ 3.5 mm) made of
piezoelectric copolymer poly(vinylidene
difluoride-trifluoroethylene), P(VDF-TrFE), which had been fabricated for
diagnostic photoacoustic measurement of cartilage tissues in our group. The PAT images of a phantom were obtained at
two excitation wavelength of 687.5 nm and 795 nm. The phantom was made of agar including a black hair and agarose
gels dissolving indocyanine green (ICG) and methylene blue (MB). Laser pulses (685-900 nm) were generated from a
Ti:Sappire tunable laser to excite ICG and MB molecules. The PAT image at 687.5 nm shows signals due to all
absorption sources. This is good agreement with dimension of the phantom. The PAT image at 795 nm shows a strong
signal due to the ICG-dyed gel and almost no signal due to the
MB-dyed gel. This result indicated that absorption
sources were extracted by excitation wavelength according to their absorption spectra. The signal/noise ratio of the PAT
images were compared between the P(VDF-TrFE) transducer in our group and a PZT transducer (Parametrics V309, 5
MHz, φ 12.7 mm) which is commercially available. The P(VDF-TrFE) transducer was more sensitive by 9 times (120
times per area) than the PZT transducer. By using this imaging system with a P(VDF-TrFE) transducer which is highly
sensitive in a wide frequency range, we will achieve frequency analysis of the PAT images to associate photoacoustic
waveforms with physical properties of sample tissues.
We demonstrated cell imaging without any stain by far-field 2-color infrared (IR) super-resolution microscopy,
combining laser fluorescence microscope and picosecond transient fluorescence detected IR (TFD-IR) spectroscopy.
TFD-IR spectroscopy detects IR absorption by monitoring fluorescence due to an electronic transition from a vibrational
excited level by an additional visible light. By using the IR microscopy based on TFD-IR spectroscopy, the spatial
resolution of the image can be increased to the visible diffraction limit of sub-μm, i.e., the IR is super-resolved. Cell
auto-fluorescence due to flavin molecules was monitored for label-free detection of the cellular components. The
fluorescence image of an A549 cell was obtained by introducing both an IR light at 3300 nm and a visible light at 560
nm. The spatial resolution of the image was estimated to be 1.6 μm. This is about 2.5-times higher resolution than the
diffraction limit of IR light. The fluorescence intensity of the images at 3448 nm was smaller than that at 3300 nm,
corresponding to the smaller IR absorption. Therefore, IR spectral imaging of a single cell was achieved with superresolution.
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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