Single-photon excitation and Two-photon excitation are two different mechanisms for exciting atoms or molecules to higher energy states. Single-photon excitation is the conventional method of fluorescence excitation. It involves the use of a single photon, which carries the energy required to excite a fluorophore. Two-photon excitation is a more advanced technique that allows for deeper tissue imaging and reduces photodamage compared to single-photon excitation. Two-photon excitation takes advantage of the nonlinear excitation process, where two photons of longer wavelengths combine their energies to excite a fluorophore. This technique relies on the principle of simultaneous absorption of two lower-energy photons that add up to the energy required for excitation.

From the figure above, in the single-photon excitation process, a sample is illuminated with a beam of light, typically from a laser or a lamp, at a wavelength (λ=400 nm) that matches the absorption spectrum of the fluorophore. When the fluorophore absorbs a single photon, it gains energy and undergoes an electronic transition to an excited state. Subsequently, the fluorophore emits a lower-energy photon (λ=500 nm) as it returns to its ground state, producing fluorescence that can be detected and visualized. In two-photon excitation, the combined energy of two photons (λ=800 nm) is used to excite the fluorophore to a higher energy level. The energy of each photon is typically half of the required excitation energy. When two photons with the appropriate wavelengths and energies coincide spatially and temporally, they can be absorbed simultaneously by the fluorophore. This simultaneous absorption results in the fluorophore undergoing a transition to the excited state, enabling fluorescence emission (λ=500 nm).

Single Photon Excitation

In single-photon excitation, a photon with sufficient energy is absorbed by an atom or molecule, causing an electron transition from a lower energy level to a higher energy level. The energy of the absorbed photon should be equal to or greater than the energy difference between the initial and final states of the system. The absorption of a single photon results in the excitation of a single electron. This process is commonly used in various applications, such as fluorescence spectroscopy, where molecules absorb photons and then emit lower-energy photons. It is also the principle behind the operation of many everyday electronic devices, including lasers, LEDs, and solar cells.

In the case of a resonant single-photon excitation, the energy of the absorbed photon precisely matches the energy difference between the initial and final states, leading to efficient and selective excitation of the system.

Advantages of Single Photon Excitation:

• Efficiency: A single photon with the exact or greater energy required allows for a direct absorption process. This means that the excitation can occur efficiently without any additional steps or intermediate states involved.
• Simplicity: The excitation with a single photon is conceptually simpler and requires fewer components in the experimental setup. It eliminates the need for precise timing control or complex pulse shaping associated with multi-photon processes.

Disadvantages of Single Photon Excitation:

• Photon availability: Generating a single photon with a specific energy can be challenging, especially if the desired energy is not readily available from common light sources. Specialized light sources or techniques, such as lasers or high-resolution monochromators, may be required, which can be costly and technically demanding.
• Background noise: When using a single photon source, it is possible to have additional background noise from other energy levels in the system or external sources. This can reduce the signal-to-noise ratio and make it more challenging to detect the desired excitation.

Two-Photon Excitation 

Two-photon excitation occurs when two lower-energy photons are simultaneously absorbed by an atom or molecule, leading to excitation. Unlike single-photon excitation, the combined energy of the two photons must be equal to or greater than the energy difference between the initial and final states. This phenomenon relies on a nonlinear optical effect called two-photon absorption. It occurs when the probability of absorbing two photons simultaneously is significantly higher than absorbing two individual photons sequentially. Two-photon excitation is more likely to occur when a sample is exposed to intense laser light with a high photon flux.

In resonant two-photon excitation, the combined energy of the two photons must be equal to the energy difference. This flexibility allows for efficient excitation of the system.

Advantages of Two-Photon Excitation:

• Photon availability: Two-photon excitation can be advantageous when the desired energy level cannot be directly accessed by a single photon source. The availability of photons at lower energies is generally more common than those at higher energies, making it easier to obtain the required photons.
• Background suppression: Two-photon excitation can offer improved background suppression compared to single-photon excitation. Since the two photons must interact simultaneously and in a specific manner, background noise from other energy levels or sources that do not satisfy the two-photon conditions can be significantly reduced.
• Enhanced penetration depth and reduced photobleaching: Two-photon excitation is advantageous for point measurements (such as FCS) conducted within limited volumes like cells. In this technique, only the confocal volume is susceptible to bleaching, minimizing undesired effects.
• Broad absorption spectra: The broad absorption spectra associated with two-photon excitation can be leveraged as an advantage in applications like tissue profiling through autofluorescence, enabling detailed examination of biological samples, cross-correlation measurements since the excitation volume is precisely determined by the same excitation beam, ensuring accurate and reliable results.

Disadvantages of Two-Photon Excitation:

• Efficiency: Two-photon processes generally have lower excitation probabilities compared to single-photon processes. The probability of two photons simultaneously interacting with the system decreases with increasing intensity, making the excitation less efficient.
• Complexity: Implementing two-photon excitation requires careful control of the relative timing and spatial overlap of the two photons. Precise synchronization and alignment of the light sources can be technically challenging, and the experimental setup may become more complex.
• Laser safety: Lasers employed in two-photon excitation typically fall under Laser Class IV, which signifies that they pose potential hazards to the eyes and skin both when directly viewed and through diffuse reflection.
• Low absorption: Compared to single-photon excitation, the absorption during two-photon excitation is significantly lower. However, this often results in higher intensities, which can pose a risk of cell destruction, potentially causing overheating or "boiling" of the cell.

The choice between the two strategies depends on the specific experimental constraints, including the availability of suitable light sources, technical feasibility, and the desired level of excitation efficiency.

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