Imaging of Cellular Dynamics by Two-Photon Excitation Microscopy

Abstract

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.

Fluorescence correlation spectroscopy in vivo

“…6 illustrates such a measurement of either the labeled guide (left panel) or passenger strand (right panel). Using this ap- 61 proach, it could be shown that Ago2 is loaded with the guide strand in the cytoplasm and transported into the nucleus. It was reported that the nuclear import receptor Importin8 is responsible for the nuclear import of Ago proteins [59].…”

“…This implies that the region of fluorescence excitation is limited to a single focal plane, with virtually no out-of-focus photobleaching. Furthermore, nearinfrared light is typically less phototoxic in cells and is capable of penetrating deeper into thick biological specimen [61]. A drawback of TPE is that the large photon flux necessary to facilitate two-photon excitation can lead to strong higherorder photobleaching within the focal volume [62].…”

Video-Rate Scanning Two-Photon Excitation Fluorescence Microscopy and Ratio Imaging with Cameleons

“…This square dependence naturally confines the absorption to where the incident light is the strongest, such as the focal point of an objective lens, thus reducing photobleaching elsewhere in the specimen. A dramatic demonstration of this effect has been given by Piston et al (1995), who showed convinc-ingly that photobleaching is highly localized to a narrow layer in z, corresponding to the focal plane, in the case of TPEM, whereas nearly uniform bleaching occurs above and below the focal plane in the case of confocal (one-photon excitation) microscopy. The lack of bleaching outside the focal plane is particularly valuable when one is accumulating a stack of optical sections or viewing a diffusible fluorophore within a thick specimen.…”

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

“…The experiments with TPLSM were performed in a similar way as for microfluorometry (see above) with the perifusion chamber mounted on a Leica DMIRB microscope with a Leica TCS-NT confocal laser scanner (Leica Microsystems Heidelberg). We used a Ti:Sapphire laser (Tsunami; Spectra-Physics, Mountain View, CA) for ϳ100 fs excitation at ϳ82 MHz and two external PMT detectors (Hamamatsu R268; Hamamatsu Photonics, Shizuoka-Ken, Japan) with bandpass filters 480 Ϯ 15 nm (ECFP emission) and 535 Ϯ 13 nm (EYFP emission), arranged in-house for nondescanned fluorescence detection (20). The lens was Leica PL APO 100ϫ/1.40 oil, and average excitation power was below 10 mW at 790 nm.…”