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Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy
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View: Figures


Image of FIG. 1.
FIG. 1.

Schematic representation of nonlinear processes: (a) TPEF, (b) SHG, (c) THG, and (d) CARS. Wiggly lines represent incoming and radiated photons, dashed lines represent virtual states, and dashed arrows nonradiative relaxation processes.

Image of FIG. 2.
FIG. 2.

Schematic of a multicontrast microscope (not to scale). L: lenses; PH: pinholes; DM: dichroic mirrors; EO: excitation objective; F: optical filter; D: detector; CO: collection objective; DAQ: data acquisition card.

Image of FIG. 3.
FIG. 3.

System schematic for pulse measurement at the focus of high NA optic. By adding a simple Michelson interferometer at the input of the beam path to the microscope system, interferometric autocorrleation traces of the pulse can be made at the focus of the objective in the microscope. A second-order intensity autocorrelation trace is shown, prior to being optimized—the wings of the trace show uncompensated dispersion as a result of the scan optics, and microscope optics.

Image of FIG. 4.
FIG. 4.

Autofluorescence and expression of YFP-labeled protein in maize leaves. Panel (a), Guard cells (arrow) in the epidermis of a maize leaf show cell wall autofluorescence at excitation wavelength of 800 nm. Panel (b) A plant expressing a protein tagged with YFP (RAB2A::YFP) shows protein localization in the cell cytoplasm (arrow) and around the nuclei in cells (arrowheads) at excitation wavelength of 1040 nm. The localization of RAB2A::YFP shown in (b) is expected for the protein, which is involved in vesicle trafficking in maize cells.

Image of FIG. 5.
FIG. 5.

Schematic representation of the electronic (bold lines) and vibrational (light lines) energy levels within a fluorescent molecule. Nonradiative transitions such as vibrational relaxation are shown with wavy lines, while radiative transitions such as TPA and fluorescence are represented with straight arrows. There can also be a nonradiaive internal transition (not shown) of excited molecules down to the ground state.

Image of FIG. 6.
FIG. 6.

SHG imaging of potato starch granule. Different polarizations of the fundamental laser radiation were used as indicated in the bottom left corner of the panels, including linearly polarized light oriented horizontally (a) and vertically (b), as well as circularly polarized light (d) and (e). The polarization anisotropy image shown in (c) was calculated from images (a) and (b). Image (e) presents a pre-treated starch granule in water for 20 s and shows a reduction in SHG intensity due to the heat treatment. The scale bar in (a) is and for (e). A schematic model of a starch granule is shown in (f).

Image of FIG. 7.
FIG. 7.

A comparison between SHG imaged structures of unstretched (a) and stretched (b) muscle cells from Drosophila melanogaster larva. The arrows point to the same sarcomere which appears in its double-banded form in the nonstretched myocyte (a), and in its single banded structure for the stretched myocyte (b). A line profile of the indicated (arrow) sarcomeres is plotted in (c) showing that unstretched sarcomeres have a double peak and stretched sarcomeres have a single peaked -band. The anisotropic muscle regions, indicated by A, produce SHG; while the isotropic muscle regions, indicated by I, show no SHG. The position of the M-line is indicated by M. The scale bar is .

Image of FIG. 8.
FIG. 8.

Axial views of polystyrene beads of different sizes imaged with THG. The bead sizes are 10, 3, 1, and , for panels (a) through (d), respectively. The laser propagation direction is indicated by the arrow. The scale bar is .

Image of FIG. 9.
FIG. 9.

Baker’s yeast imaged with TPEF (a) and THG (b). The THG image clearly shows the cell wall and some internal organelles, predominately mitochondria. The scale bar is .

Image of FIG. 10.
FIG. 10.

2D projections of 3D rendered multicontrast microscopy images of TMRM labeled mitochondria in myocytes. TPEF from TMRM is shown in (a), THG in (b), two-channel correlation in (c), uncorrelated MPF in (d), and uncorrelated THG in (e). The scale bar is .

Image of FIG. 11.
FIG. 11.

Multicontrast microscopy of freshly isolated face aligned chloroplasts from pea leaves Pisum savitum. Chlorophyll fluorescence in the range of 650–720 nm is shown in (a), SHG in (b), THG in (c), and the combined correlated image in (d). Uncorrelated SHG is green, uncorrelated THG is blue, uncorrelated TPEF is red and correlated TPEF and THG is cyan. The scale bar is .


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy