Two-photon fluorescence microscope

Figure 11.13 Schematic representation of a two-photon fluorescence microscope system two-photon excitation of fluorophores within the sample is achieved using the output of a Ti sapphire laser. This is scanned in the X-Y plane to produce an image of a section of the sample. The insert shows a two-photon fluorescence image of pig kidney cells labelled with two-photon fluorophores (image provided courtesy of Dr Mireille Blanchard-Desce (CNRS UMR 6510 Rennes))...

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

Two-photon laser scanning microscopes and microscopes for two-photon single molecule experiments use femtosecond lasers. Two-photon fluorescence excitation with a picosecond diode laser cannot be expected to work with the same efficiency. The average intensity is only a few mW. Moreover, the pulse duration is of the order of 100 ps, so that the peak power is many orders of magnitude lower than for a fs Ti Sapphire laser. The relative excitation efficiency of different lasers can be estimated as follows ... 

Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. and Barry, N. (2003). Fluorescence lifetime imaging for the two-photon microscope time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381-90. 

The advantage of two-color excitation over two-photon excitation is not an improvement in imaging resolution, but the easier observation of microscopic objects through highly scattering media. In fact, in two-color excitation, scattering decreases the in-focus fluorescence but only minimally increases the unwanted fluorescence background, in contrast to two-photon excitation. 

As described above, two-photon excitation microscopy provides several advantages (reduced photobleaching, deeper penetration into the specimen). A fluorescence microscope combining two-photon excitation and fluorescence time-resolved... 

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 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... 

Two-photon excitation fluorescence is currently the most widely nsed nonlinear contrast mechanism for microscopic investigations. The first experimental demonstration of two-photon excitation fluorescence was provided in 1961 (Kaiser and Garrett 1961), even though the first theoretical description of two-photon excitation flnorescence stems back to 1931 (Goppert-Mayer 1931). Three-photon absorption was demonstrated a few years later by Singh and Bradley (1964). Two-photon absorption is a third-order nonlinear effect, whereas three-photon absorption is a fifth-order nonlinear effect. The transition rate for two-photon absorption, R, depends on the square of the intensity, /, as follows (see Boyd 1992) ... 

BariUe, R., Canioni, L., Rivet, S., Sarger, L., Vacher, R, and Ducret, T. 2001. Visualization of intracellular Ca + dynamics with simultaneous two-photon-excited fluorescence and third-harmonic generation microscopes. Appl. Phys. Lett. 79 4045M7. 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

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