Two-photon Excitation
Two-photon Excitation - Related Videos
04:32
Two-Photon In Vivo Imaging of the Mouse Retina
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2025
Source: Wang, Z., et al. Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp. (2021).This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed,...
07:04
A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)
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Cited by 2 •
2014
Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.
03:22
Deep-Tissue Imaging of a Zebrafish Brain Using a Three-Photon Fluorescence Microscope
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2025
Source: Hontani, Y., et al. Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain. J. Vis. Exp. (2022)This video demonstrates the setup and execution of three-photon imaging in an anesthetized zebrafish brain. The protocol enables deep-tissue visualization of fluorescently labeled neurons, revealing real-time neuronal activity and structures for neuroscience research applications.
07:00
Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
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Cited by 4 •
2010
In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.
02:52
Two-Photon Microscopy for Imaging Neuronal Morphology in a Mouse Pup
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2025
Source: Mizuno, H., et al. In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice. J. Vis. Exp. (2018)This video showcases two-photon microscopy being used to image neurons in the brain of an anesthetized transgenic mouse pup. The two-photon laser allows for deeper imaging with high spatial resolution and minimal light scattering, enabling the detailed capture of neuronal morphology, including dendritic projections.
FAQs
The sharp dependence of absorption probability on photon density confines excitation to the microscope’s focal point. Away from that location, photon density is insufficient for the two-photon event to occur frequently, so much less fluorescence is generated. This spatial restriction allows researchers to distinguish signals from selected regions within a biological specimen.
Pulsed near-infrared light supplies the lower-energy photons needed for the process while concentrating photons in time. Their absorption must occur nearly simultaneously, making photon density especially important. This combination supports localized excitation rather than broadly distributed fluorescence, which is central to obtaining focused signals from biological samples.
Compared with conventional fluorescence microscopy, localized excitation produces less out-of-focus fluorescence because regions outside the focal point are not excited as strongly. It can also limit photodamage in surrounding tissue. These differences make two-photon imaging useful when preserving neighboring biological structures and obtaining a cleaner signal are important.
A basic setup combines a microscope with a pulsed near-infrared laser and a biological specimen positioned for focal-point imaging. The microscope’s focal point determines where excitation is concentrated, so the resulting fluorescence represents a selected region rather than the entire optical field. This arrangement supports localized observation within cells, tissues, or organisms.
Researchers apply this approach to living cells, tissues, and organisms when they need high-resolution information from structures within biological specimens. Its reduced out-of-focus fluorescence is particularly relevant in scattering samples, where signals from surrounding regions could complicate interpretation. The method therefore supports localized optical imaging in living biological contexts.
The principal readouts are localized fluorescence and preserved temporal information about biological dynamics. Because excitation is concentrated mainly at the focal point, images can emphasize structures or regions at selected locations while limiting signal from elsewhere. This makes the technique suitable for following changing processes in living material, not only for recording static structural detail.