Two-photon excitation microscope

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],... 

Another important lesson from Table 8.1 is that all three of the methods tested yielded virtually the same FRET efficiencies for the same samples. The sRET method as implemented used two-photon excitation on a Zeiss 510 META/NLO microscope, as did the FLIM-FRET method, but FLIM-FRET used auxiliary time... 

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... 

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. 

S. HeU, E.H.K. Stelzer, Fundamental improvement of resolution with a 4Pi-confocal Huorescence microscope using two-photon excitation. Opt. Commun. 93, 277-282 (1992)... 

RE. Hanninen et al., Two-photon excitation 4Pi confocal microscope Enhanced axial resolution microscope for biological research. Appl. Phys. Lett. 66, 1698-1700 (1995)... 

Two-photon excitation by femtosecond NIR laser pulses can be used to obtain clear images of tissue layers as deep as 1 mm [132, 278, 279, 344, 462, 495, 534]. The efficiency of two-photon excitation depends on the square of the power density. It therefore works with noticeable efficiency only in the focus of the laser beam. With a microscope lens of high numerical aperture a lateral resolution around 300 nm and a longitudinal resolution of about 1 pm is obtained. Two-photon laser scanning microscopy has therefore become a standard technique of tissue microscopy. Two-photon laser scanning can be combined with... 

Fig. 5.71 Optical principle of a laser scanning microscope. Left. One-photon excitation. Right. Two-photon excitation...

Commercial laser scanning microscopes use the same microscope body and the same scan optics for one-photon and two-photon excitation. Most two-photon microscopes have lasers for one-photon excitation as well. They can switch between both modes, and between descanned and nondescanned detection. Moreover, in both the descanned and the nondescanned detection path, the light is split spectrally by additional dichroic mirrors or dispersion prisms and several detectors are used to record images in selectable wavelength ranges. The dichroic mirrors and filters are assembled on motor-driven wheels and are changed on command. The laser power... 

Fig. 5.72 Intensity distribution around the laser focus, seen from the surface of a thick sample. Left One-photon excitation. Fluorescence comes from the complete excitation light cone. The confocal microscope obtains a sharp image by detecting through a pinhole. Right Two-photon excitation. Fluorescence is excited only in the focal plane. Nevertheless, scattering in a thick sample blurs the image seen from the sample surface. The two photon microscope obtains a sharp image by assigning all photons to the pixel in the current scan position...

Fig. 5.96 Lifetime microscope with piezo-driven scan stage. One-photon excitation leji) and two-photon excitation (right)...

Optically driven photon correlation experiments normally require confining the detection or the excitation to an extremely small sample volume. This is achieved either by confocal detection or two-photon excitation in a microscope. The optical principles are the same as in confocal and two-photon laser scanning microscopes (see Sect. 5.7, page 129). However, most correlation experiments do not require scanning and can be performed in relatively simple microscopes. 

An FCS system with two-photon excitation is shown in Fig. 5.108, right [51, 457]. A femtosecond Ti Sapphire laser of high repetition rate is used to excite the sample. Because there is no appreciable excitation outside the focal plane of the microscope lens a small sample volume is achieved without a confocal pinhole. This makes the optical setup very simple. In terms of signal recording there is no difference between one-photon and two-photon FCS. 

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