Fluorescence up-conversion microscope

FIGURE 3.3 Schematic diagram of the femtosecond fluorescence up-conversion microscope. (From Fnjino, T. and Tahara, T. J. Phys. Chem. B 107 5120-5122, 2003. Used with permission)... 

FIGURE 3.4 Performance of the fluorescence up-conversion microscope, (a) Evaluation of the time-resolution with the 100 x objective lens , up-converted fluorescence -F-, the first derivative. By the fitting analysis, the time-resolution of the microscope was evaluated as 520 fs. (b) Evaluation of the transverse (XY) spatial resolution with the 100 x objective lens. A CCD image of the excitation pulses (inset) and the beam profile along the lateral (X) direction. By the fitting analysis, the transverse resolution was evaluated as 0.34 pm. (c d) Evaluation of the axial (Z) spatial resolution with the 100 x objective lens , up-converted fluorescence -I-, the first derivative. By fitting analysis on the first derivative coefficient, the axial resolution was evaluated as 1.1 pm with the 50 pm pinhole (c) and 5.3 pm without pinhole (d). (Rhodamine B, 2 x 10" mol dm in methanol, 600 nm.) (Erom Eujino, T. and Tahara, T., Appl Phys. B 79 145-151, 2004. Used with permission.)... 

Two-photon excitation can be used for the fluorescence up-conversion microscope, and high axial resolution was achieved without a pinhole in this case. Figure 3.5 shows the up-converted fluorescence from a coumarin 522B solution at a fluorescence wavelength of 520 nm observed in the same manner of Figure 3.4d without pinhole. In this measurement, a fundamental laser pulse at 800 nm was used for excitation. The axial resolution with two-photon excitation was evaluated to be 0.97 pm (FWHM) by fitting for the first derivative of the obtained data. This result indicates... 

The second example of the application of fluorescence up-conversion microscope is imaging of organic microcrystals based on ultrafast fluorescence dynamics (femtosecond fluorescence dynamics imaging) (Fujino et al. 2005a). In this measurement, the site-specific energy transfer rate in a tetracene-doped anthracene microcrystal was measured, and the crystal was visualized based on the observed local ultrafast dynamics. 

As described in the previous section, the femtosecond fluorescence up-conversion microscope enabled us to visualize microscopic samples based on position-depen-dent ultrafast fluorescence dynamics. However, in the imaging measurements using the fluorescence up-conversion microscope, XY scanning was necessary as when using FLIM systems. To achieve non-scanning measurements of time-resolved fluorescence images, we developed another time-resolved fluorescence microscope. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

Here we first describe the ultrafast fluorescence microscope, which nses the fluorescence up-conversion method. This microscope simnltaneonsly achieves femtosecond time resolution and snbmicron space resolution (Fnjino and Tahara 2003, 2004). 

In recent years, we have developed a two-color super-resolution laser scanning fluorescence microscope based on the up-conversion fluorescence depletion... 

Watanabe, T., Iketaki, Y., Omatsu, T., Yamamoto, K., Sakai, M. and Fujii, M. (2003) Two-point-separation in superresolution fluorescence microscope based on up-conversion fluorescence depletion technique. Opt. Express, 11, 3271-3276. 

Fig. 16 a Different confocal images of a splitter-type channel waveguide polymerized inside a polymer gel by two-photon-induced up-conversion fluorescence, b Vertical cross-section of the channel waveguide imaged using both the fluorescence and reflection modes in a confocal microscope... 

Since a molecule or a quantum dot can emit no more than one photon per excitation, the optimum source for ultrafast fluorescence microscopy has a high repetition rate (10 -10 Hz) and a pulse energy sufficient to pump an excitation NOPA (noncollinear optical parametric amplifier) and to drive the Kerr or up-conversion gate. The current configuration of the Rutgers microscope relies on a cryogenically cooled Ti sapphire regenerative amplifier which produces 50 gJ... 

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