Introduction
Two-photon excitation is the technique by which a fluorophore is stimulated by the uptake of two photons of minimum energy and greater spectrum compared to the single photon used for traditional excitation. This is the basis for two-photon microscopy.
In 1990, a method called two-photon microscopy was developed for capturing high-resolution, three-dimensional images of living cells and tissues. The technique relies on the specimen’s fluorescence, which is excited by the light and then emitted, creating a three-dimensional image.
Compared to conventional single-photon microscopy, the depth of observation is much improved when using two-photon microscopy to excite a fluorescent material. In order to excite the sample, a laser, often a femtosecond laser emits a pair of photons (Wang et al, 2010). This paves the way for 3D imaging of dense samples like living cells. Fluorescent molecules are excited by the two photons as they pass through the model, and this light is then identified and used to create an image.
In this traditional approach, excitation occurs when an electron in the fluorophore (a molecule that produces light when exposed to a photon) absorbs light of a shorter wavelength. This is because the fluorophore gets excited from its ground state to a higher energy excited state by the photon (a particle which transports light). The longer wavelength photon is emitted as the fluorophore relaxes to its ground state. Instead of using excitation wavelengths that are much longer than the emitted light, as is done in traditional fluorescence microscopy, two-photon microscopy can be used. This lessens the potential for damaging light effects, such as photobleaching and phototoxicity, which can hinder the study of living specimens. The approach is preferable because it more effectively identifies particular photons per excitation event.
Basic principles
In two-photon microscopy, infrared photons are employed to stimulate fluorescence in the focal region of the laser. The fluorophore may absorb both photons at once because their combined energy is larger than the difference in energy between the ground and excited states (Shaya et al., 2022). Most of the fluorescence signal originates from a zone about a millimetre thick surrounding the microscope’s focus point because the possibility of emission rises quadratically with excitation density.
Nonlinear excitation: To undergo two-photon fluorescence excitation, molecules must take in two photons with combined energy more extensive than the gap between their ground and excited states. The two photons’ combined power must be high enough to drive the molecule’s electrons into an excited state. Because it requires two photons for absorption instead of just one, this process is classified as nonlinear (Parodi et al., 2020). This indicates that a higher efficiency and better signal-to-noise ratio can be achieved in the measurement because fewer photons are required for molecule activation. In addition, two-photon fluorescence excitation is preferable to the more conventional single-photon excitation when imaging molecules in living cells. There is less collateral harm to the environment and greater efficiency in bringing molecules to an electrically excited state through the use of two-photon fluorescence excitation.
High resolution: Two-photon microscopy is a high-resolution imaging technology that studies living tissue in exquisite detail. Two-photon excitation, in which two photons of light simultaneously excite a molecule, yields a more detailed picture than single-photon excitation. Images obtained with two-photon microscopy are up to ten times clearer than those obtained with conventional single-photon microscopy (Zhu et al., 2017). Because of the improved resolution, scientists can examine cellular structures in considerable detail, which may lead to a deeper comprehension of biological processes. Two-photon microscopy is a powerful tool for imaging living tissue to depths of several millimetres and investigating dynamic processes. This method has been applied to imaging neuronal networks, cellular organelles, and other anatomical features of living tissues. For example, Stimulated emission depletion (STED) microscopy is an example of a laser-scanning super-resolution imaging technology that enhances two-photon excitation microscopy. This method uses doughnut-shaped beams to generate an excitation point spread function (PSF) that is restricted beyond the diffraction limit via the stimulated-emission effect.
Three-dimensional imaging: Using two photons of light to stimulate a fluorophore, two-photon microscopy (TPM) provides high-resolution images of biological material. It is used to examine many physiological systems at the levels of cells and molecules, and it generates high-resolution, high-contrast 3D images. Two-photon excitation, in which two light photons are absorbed concurrently by a molecule and then emitted, is the foundation of Two-Photon Microscopy. This phenomenon is utilized to photograph biological material with outstanding resolution and contrast and is highly sensitive to its immediate environment. This method is ideal for imaging at depth into tissue because it is less susceptible to light scattering by tissue features. Cell migration, cell division, protein transport, and gene expression are just some of the many biological processes that can be seen with TPM.
Low phototoxicity: Low phototoxicity is a fundamental tenet of two-photon microscopy, which uses near-infrared (NIR) light to excite a sample and produce a nonlinear optical response. When working with fragile samples and living specimens, this technique reduces photo bleaching and photo toxicity, two serious issues ( Choquet et al., 2021). Only a tiny portion of the material is ever exposed to light using two-photon microscopy since the released fluorescence is localized in space and time. This lowers the energy needed to photograph a sample and lowers the sample’s exposure to phototoxicity. Moreover, two-photon microscopy can see deeper into the sample than other imaging methods, which reduces the impact of phototoxicity even more.
Multiphoton imaging: Two-photon microscopy relies on multiphoton imaging. This imaging method requires a molecule or substance to absorb two or more photons simultaneously to achieve excitation. Equally important is that the photons have identical frequency and polarization. When the photons interact with the excited molecule or material in a way unique to it, the process is known as resonant excitation. When a molecule or substance is excited, it gives off a photon with less energy. An image is created by sensing this emitted photon and using it to create a picture. Multiple-photon imaging offers several benefits over its single-photon counterpart. Secondly, the photons can be concentrated into a tiny spot, which improves image quality because they have an identical frequency and polarization. Second, unlike single-photon imaging, photobleaching is less likely to occur when several photons are used. Finally, since the absorbed photons can enter tissue to a greater depth than single photons, using multiple photons enables the imaging of deeper structures.
Advantages of two-photon microscopy over one-photon fluorescence imaging
The main advantage of two-photon microscopy over one-photon fluorescence imaging is that the fluorescence is only produced in regions of the sample that are observed and photographed, two-photon excitation is termed “more efficient” since photo bleaching and photo toxicity are restricted to a much smaller volume of the sample than with conventional excitation ( Xu et al, 2022, pg 9(4)). Due to its extended excitation wavelength, two-photon microscopy causes minimal harm to specimens and may penetrate tissues five to twenty times deeper than ordinary fluorescent microscopes. There are several advantages associated with two-photon microscopy.
Improved Spatial Resolution: Two-photon microscopy has a superior spatial resolution to other methods. Unlike single-photon microscopy, two-photon microscopy can resolve features as small as a single molecule (Lee, M., & Serrels, A. 2016). The two-photon process needs the concurrent uptake of two photons with lower energy to create a single photon with more incredible energy. We can get a higher-resolution image by using this more powerful photon to excite fluorescent molecules in a sample. The image resolution is further enhanced because out-of-focus light less affects two-photon microscopy. Two-photon microscopy’s enhanced spatial resolution makes it well-suited for photographing living cells in their native habitat and examining minute features inside a sample.
Reduced Photo Bleaching: One of the main benefits of two-photon microscopy is that photobleaching is minimized. A decrease in signal is caused by photobleaching, which occurs when a fluorescent molecule loses its fluorescence after exposure to light. As the molecules in two-photon microscopy are only subjected to light for a fraction of a second, photobleaching is significantly mitigated ( Li et al., 2018). Because the sample does not have to be changed as often, long-term imaging of living cells and tissues is now possible. There is substantially less potential for sample destruction when using two-photon microscopy because it does not call for intense light. For example, in confocal microscopy, the specimen is irradiated with an excitation wavelength, and the emitted light is then transmitted through a small aperture to improve the quality of the resulting image. This setup blocks out most of the light from other focal planes before getting the detector.
Reduced Phototoxicity: There are several benefits to using two-photon microscopy instead of single-photon fluorescence microscopy. A lesser degree of phototoxicity is one of these benefits. Destruction of proteins, lipids, and other biological components can be caused by light, a phenomenon known as phototoxicity (Wu et al., 2021). This is a significant challenge for conventional single-photon fluorescence microscopy, which requires very bright lights to get a clear image. On the other hand, two-photon microscopy relies on the simultaneous absorption of two photons of light to excite fluorescence and may therefore function with much lower illumination intensity (Icha et al., 2017). This dramatically lessens phototoxicity and makes it possible to image living specimens over extended periods without harming them (Yuan et al., 2017).
Improved Imaging Depth: Compared to conventional single-photon microscopy, two-photon microscopy offers several benefits, including greater image depth. TPM’s capacity to use two- photons to form a signal, as opposed to the one photon used in conventional techniques, enables imaging at greater depths (Chen et al., 2016). This allows for more light penetration into deeper layers of tissue, resulting in images with greater depth resolution than those possible with single-photon microscopy. Visualizing structures in 3D is very helpful when working with thick tissue samples like brain tissue (Qian et al., 2022). More precise data on cellular processes as well as structures in deeper layers of tissue, are now possible thanks to this enhanced imaging depth, allowing researchers to gain insight into cellular processes that were previously unavailable.
Examples of Two-Photon in Vivo Imaging
Using two-photon microscopy, researchers have observed the activity of neurons in the living mouse brain ( Wei et al., 2022). Neuronal network dynamics and their functions in behaviours like learning and memory have been studied with this method.
It is possible to image calcium transients in the living heart using two-photon microscopy. Researchers have employed this method to analyze cardiac cell characteristics to learn more about the mechanisms of heart failure (Matsuura et al., 2018).
The development of blood arteries can be observed in vivo using two-photon microscopy. The mechanisms of angiogenesis and its part in wound healing and tumour growth have been investigated using this method (Miura et al, 2018).
The movement of leukocytes in vivo has been imaged using two-photon microscopy. Inflammation and its function in immune responses have been the subject of research using this method.
Two-photon in vivo imaging is a highly effective method for analyzing gene expression in real-time and in vivo (Park et al, 2015). Fluorescent molecules linked to a specific gene are imaged by a laser beam. The existence and activity of a gene can be detected by using a laser beam to excite fluorescent molecules, which then emit light. Two-photon in vivo imaging can be used to do more than detect gene expression; it can also be used to quantitatively assess the expression levels of multiple genes in real time (Piston, D. W. (2017). This method’s ability to identify gene expression changes in real-time makes it particularly well-suited to studies of gene expression in motion. Furthermore, two-photon in vivo imaging can examine human, mouse, and plant tissue gene expression. Because of this, it is a powerful method for investigating gene expression in different settings.
Conclusion
Two-photon microscopy is a game-changer in biological imaging, and this article has shown you why. Improved image depth, less photobleaching and phototoxicity, and higher resolution all make two-photon microscopy a powerful tool for studying cellular and tissue processes (Hazart et al,2022). Its nonlinear excitation and capacity to penetrate thicker materials have opened up new avenues of inquiry into the workings of the human body, including the mechanics of heart failure, the growth of blood vessels, and the in vivo migration of leukocytes. It has also made possible the in-vivo, real-time investigation of gene expression. Two-photon microscopy is a powerful tool for biologists thanks to its many benefits over single-photon fluorescence imaging.
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