Introduction:
Two-photon excitation of fluorescence results in the absorption of two photons. The advantages of using two-photon fluorescence microscopy include reduced specimen photodamage and enhanced penetration depth, which shows higher contrast images and methods to trigger localized photochemical reactions. It is also used in increasing applications in biology and medicine. Two-photon microscopy is a better diagnostic tool used for developing optical microscopic instrumentation, and it is one of the revolutionary developments in biological imaging techniques due to its better capabilities. It helps significantly by reducing the photodamage and by allowing imaging of living specimens. In addition, two-photon microscopy allows high-sensitivity imaging by eliminating contamination of fluorescence signals by excitation of the light.
How Does It Work?
The two-photon absorption concept is the one that has two photons, which may be used at identical or different frequencies. It helps the molecule excite from one energy level or ground level to a higher energy state. Two photons microscopy, that is, two photons of light are used to double the wavelength that is used to excite the same or similar fluorescent dyes.
What Are the Basic Principles of Two-Photon Microscopy?
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Physical Basics of Two-Photon Excitation: Two-photon molecules are a nonlinear process involving combined energy sufficient to induce molecular transitions to an excited electronic state. Between one and two-photon absorption, conventional one-photon techniques use visible or UV (ultraviolet) to excite fluorescent molecules. Excitation occurs when the absorbed photon energy can match the energy gap between one ground and excited states. The same transition can be achieved by two fewer energy photons simultaneously absorbing.
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Optical Properties of Two-Photon Microscopy: This high numerical objective focuses the excitation source to a diffraction-limited spot.
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Two-Photon Laser Sources: One of the critical features of two-photon microscopy is the limitation of fluorescence excitation within the size of the focal volume.
How Are Two-Photon Microscopy Instruments Used?
Two-photon fluorescence microscopy is commercially available as that can be constructed from components or by modifying existing confocal microscopes. A typical form of two-photon microscopic features three essential components following:
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Excitation of the light source.
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High throughput screening fluorescence microscope.
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Detection system for high sensitivity.
Two-Photon Laser Sources: As two-photon is order process with small cross-section on the order of,
- Two-photon lasers must deliver high photon flux to the sample to generate efficient absorption.
- Typically achieved using ultrashort pulsed laser excitation.
Scanning Fluorescence Microscopy Optics: Typical two-photon microscopes generated by scanning mirrors of galvanometer-driven scanners. With appropriate beam power control and pulse width compensation, the excitation light enters from the microscope through a modified light path.
Fluorescence Detection System: Fluorescence emission is collected by imaging and transmitted through mirrors along the emission path. A barrier filter is needed to attenuate the further scattered excitation light because of the high excitation intensity used.
How Are Fluorescent Probes Used in Two-Photon Microscopy?
It is most important to examine fluorescent molecules' nonlinear form of absorption characteristics. Generally, most chromophores can be excited in two-photon mode as twice as the one-photon absorption. Therefore, spectroscopic properties of fluorophores under nonlinear excitation need to be quantified to optimize their use in two-photon absorption properties of some typical types, such as extrinsic and intrinsic fluorophores.
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Extrinsic and Endogenous Two-Photon Fluorophores: Extrinsic fluorophores are organic molecules designed for biological structures and measuring biochemical functions. Absorption spectra of many of the extrinsic fluorophores have been determined. Absorption properties of extrinsic fluorophores are present.
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Recent Effort Seen in Two-Photon Probe: Recent development in two-photon probe utilization has been identified in drug molecules excitable by light sources of two photons. Two-photon probe developments can achieve the ability to monitor drug delivery efficiency and develop therapeutic strategies.
How Is Molecular Level Application Used in Two-Photon Microscopy?
Many technological advances in light sources, optics, and detectors have led to tremendous improvement in the sensitivity of fluorescence methods.
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Single Molecule Detection in Solution: The first demonstration of single molecule detection is by two photons. The photon bursts from single molecules diffusing through the two-photon volumes. In these highly excited states reached by two photons.
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Imaging Single Molecules With Two-Photon Excitation: Ultra-sensitive two-photon fluorescence shows two-photon imaging molecules by cooled CCD (charge-coupled device) and a spectrograph. Discrete photobleaching of individual fluorescence peaks as a polarized emission for single molecule detection.
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Fluorescence Correlation Spectroscopy: It is a powerful method used for studying a large variety of experimental systems that includes:
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Measurement of diffusion.
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Chemical reactions.
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Molecular interactions.
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Number concentration.
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Hydrodynamic flow.
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Photophysical parameters.
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Photon Counting Histogram: Photon counting is an alternative approach for analyzing photon bursts to measure sample concentration and detect molecular interactions accurately. It is one of the powerful methods used for measuring molecular concentration and detecting and quantifying molecular interaction.
What Are the Cellular Level Applications of Two-Photon Microscopy?
Two photons can simultaneously monitor the cellular biochemical activity and structure at the subcellular level to understand essential processes. It produces 3D (three-dimensional) localized unchanging and photobleaching experiments.
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Two-Photon Multiple Color Imaging: There is apossibility of using two-photon microscopy to simultaneously as different color fluorophores for multiple image labeling. Used for imaging, four cellular structural components and interactions of various cellular organelles can be monitored over time.
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Three-Dimensional Localized Uncaging of Signaling Molecules: It is one of the essential abilities to initiate localized chemical reactions, such as uncaging molecule signaling.
What Are the Tissue Level Applications of Two-Photon Microscopy?
Two-photon imaging is used in tissue-level applications as it is used to study the physiology of the tissues.
Applying Two-Photon Microscopy to Study About Tissue Physiology: Two-photon microscopy is successfully applied to many tissue types, such as:
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To study the physiologies of many tissue types.
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To study the corneal structure of rabbits.
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Human and mouse dermal subcutaneous tissue structures.
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Toxins in the human intestinal mucosa.
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To study the neuron structures and functions seen in intact brain slices.
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Role of calcium signaling in dendritic functions of the spine.
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Associated cellular morphologic changes and neuronal plasticity.
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Rat hemodynamics.
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Embryo development.
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Calcium passage during sperm-egg fusion.
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It is applied in an increasing number of tissue systems.
Applying Two-Photon Excitations for Clinical Diagnosis and Treatment: Traditional tissue biopsy is a new imaging technique used in clinical diagnosis that requires:
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Removal.
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Fixation.
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Imaging of tissues.
However, this method is used to diagnose good pathological results and accuracy compared to traditional histology.
Conclusion:
This two-photon fluorescence microscopy is a powerful high-resolution imaging technique used in many ways, both in clinical and industrial applications. Tissue structural images and surgical margin determination are shown using these highly sensitive two photons appropriately can be helpful in many quantitative analyses.
