What is Multiphoton Microscopy?

Explain Two-Photon Microscopy in detail?

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- GoPhotonics

Jun 12, 2023

Multiphoton Microscopy is an advanced imaging technique used in biological and medical research that allows high-resolution imaging of living tissues and cells. It is a form of fluorescence microscopy that utilizes the nonlinear optical process known as multiphoton excitation

In this process, multiphoton excitation, which involves the simultaneous absorption of multiple lower-energy photons by a fluorophore (also known as a fluorochrome), is employed to excite the molecule. As a result of this absorption, the fluorophore undergoes an electronic transition to an excited state, leading to the generation of up-converted fluorescence. Up-converted fluorescence refers to the emission of light with a shorter wavelength than incident photons, and this unique phenomenon is harnessed to capture high-resolution images in various imaging techniques. 

The above figure depicts the energy diagram for one-photon and two-photon excitation. In one-photon excitation, a fluorophore absorbs a single photon of higher energy, promoting the molecule from its ground state S0 to an excited state S1. This energy level transition enables the fluorophore to emit fluorescence with a longer wavelength than the incident photon. On the other hand, in two-photon excitation, the fluorophore simultaneously absorbs two photons with the same or different energy. The combined energy of these photons is equivalent to the energy required for one-photon excitation. As a result, the fluorophore undergoes a transition from the ground state S0 to an excited state S1, leading to the emission of fluorescence with a shorter wavelength than the incident photons, occurring during the return from the excited state S1 to the ground state S0.

Both two-photon and three-photon absorption-induced up-converted fluorescence are commonly used in multiphoton microscopy. They involve the simultaneous absorption of multiple lower-energy photons by a fluorophore. In two-photon absorption, two photons are absorbed nearly simultaneously, while in multi-photon absorption, three or more photons are absorbed together. But, due to the demanding nature of three-photon absorption, which requires high peak power, two-photon microscopy has emerged as the dominant technique for bioimaging, offering practical advantages.

Two-Photon Microscopy

Two-Photon Microscopy is a specific type of multiphoton microscopy that uses two-photon absorption in which two photons are used to excite fluorescent molecules in a sample. Two-photon absorption refers to the phenomenon where a molecule can be excited from a lower energy state, typically the ground state, to a higher energy state through the simultaneous absorption of two photons with the same or different frequencies. This process involves the electron within the molecule transitioning from one atomic orbital to another, which is relatively less stable. 

The energy difference between these two states corresponds to the sum of energy of the two photons that were absorbed. i.e., in two-photon absorption, the energy of a single photon may be insufficient to excite the molecule to the higher energy state. However, the simultaneous absorption of two photons, whose combined energy matches the energy difference between the two states, allows for excitation to occur.

To facilitate this process, the two photons need to interact with the molecule within an extremely short timeframe of 1 femtosecond (10-15 seconds). For achieving this, a focused laser that produces fast light pulses, typically at a frequency of around 80 MHz delivering peak power levels from a few hundred milliwatts to a few watts is required.

The energy level of light is directly related to its wavelength. As the wavelength becomes shorter, the energy increases. This relationship follows a linear pattern, meaning that a photon with a wavelength of 400 nm has twice the energy of a photon with a wavelength of 800 nm.

Conventional fluorescence microscopy uses a single photon to stimulate fluorescent dyes primarily using visible excitation wavelengths ranging from 390 nm to 700 nm. Once excited, the electron undergoes a transition back to its stable state, emitting a photon of light with slightly less energy compared to the excitation photon. This energy loss occurs due to various processes such as vibrational relaxation, intersystem crossing, etc.

In two-photon microscopy, two photons of light, each with half the energy (or double the wavelength) of the single photon used in traditional fluorescence microscopy, are simultaneously absorbed by a fluorescent dye. This allows for excitation of the dye to a higher energy state. When the excited electron returns to a more stable orbital, it emits a single photon, which has the same wavelength as the corresponding one-photon fluorescence method. This emitted photon has a longer wavelength than half the excitation wavelength, as it corresponds to the energy difference between the excited state and the ground state of the fluorophore. The longer wavelength is due to the energy loss that occurs during the relaxation process of the excited state to the ground state.

The typical excitation spectra of widely used fluorescent dyes fall within the range of 400 to 500 nm. So, when employing two-photon excitation for these dyes, the corresponding wavelengths used are generally situated in the infrared spectrum, specifically between 800 nm and 1000 nm (approx.).

Two-Photon Excitation

Two-photon microscopy uses a short-pulse laser with red and near-infrared wavelengths, typically in the picosecond and femtosecond range, as the excitation source to generate fluorescence within the visible spectrum. A fluorophore, which absorbs ultraviolet light at a wavelength of around 350 nm, can also be stimulated by two near-infrared photons with a wavelength of approximately 700 nm. For this excitation to occur, both photons must simultaneously reach the fluorophore. Here, "simultaneously" refers to an interval of approximately 10 x 10-18 seconds. This capability allows for simultaneous multicolor imaging, effectively expanding the range of observable dynamic processes. The probability of this simultaneous two-photon absorption is directly proportional to the square of the instantaneous light intensity, necessitating the use of high-intensity laser pulses. 

To minimize any potential thermal damage to cells or biological specimens, it is advantageous to employ ultra-short laser pulses, such as those produced by mode-locked lasers, with pulse durations in the picosecond or femtosecond range. A popular choice for two-photon microscopy has been the Ti:Sapphire laser, which emits very short pulses (approximately 100 fs) of light around 800 nm, operating at a repetition rate of around 80 MHz, and delivering a significantly high peak power of 50 kW.

Working of Two-Photon Microscopy

A mode-locked Ti:Sapphire laser is used to emit ultrashort pulses of near-infrared light, essential for achieving two-photon excitation. These pulses are then directed across the sample using a scanning mirror, which scans the laser beam in a controlled pattern. A lens placed after the scanning mirror focuses the laser beam onto the sample, ensuring a tight spot for maximum intensity. After that, a dichroic mirror reflects the laser light towards the objective while allowing emitted fluorescence to pass through, separating the excitation light from the fluorescence. The objective lens, with its high numerical aperture, collects the emitted fluorescence from the sample, providing high-resolution imaging. 

A filter blocks any remaining excitation or scattered light, allowing only the emitted fluorescence to reach the detector. Another lens collects and focuses the emitted fluorescence onto a highly sensitive detector called a photomultiplier tube (PMT), which converts the photons into an electrical signal for further amplification and image formation.

Merits of Two-Photon Microscopy

In two-photon laser scanning microscopy (TPLSM), a tightly focused excitation beam is employed, resulting in a significantly reduced probability of excitation outside the focal region. This eliminates the considerable occurrence of "out-of-focus" fluorescence, which is often observed in one-photon excitation without the use of a confocal aperture. As a result, TPLSM inherently possesses optical sectioning capabilities without the need for a confocal aperture. Also, two-photon excitation offers a notable advantage in terms of minimizing photobleaching. It is because only the specific region at the focal point can be excited, reducing the overall photodamage. This unique characteristic arises from the quadratic dependence of two-photon excitation on intensity, ensuring a highly localized excitation precisely at the focal point where the intensity is at its maximum.

When compared to excitation in the UV-visible range, longer wavelength excitation using near-infrared (near-IR) light has the advantage of deeper tissue penetration due to reduced scattering and absorption at longer wavelengths. Also, the absence of UV light enhances cell and tissue viability, enabling more scans and facilitating the acquisition of improved 3-D images. 

In addition to the inherent optical sectioning ability, reduced photodamage, and improved tissue penetration, resolution is another important factor for evaluating the potential of two-photon laser scanning microscopy. The resolution of two-photon microscopy is determined by several factors, including the numerical aperture (NA) of the objective lens, the wavelength of the excitation light, and the quality of the imaging system. Also, it is influenced by factors such as the scattering and absorption properties of the sample. In thick or turbid samples, scattering can degrade the resolution and limit the penetration depth. 

In general, the resolution of two-photon microscopy is better than that of traditional widefield fluorescence microscopy and comparable to confocal microscopy. Techniques such as adaptive optics and image processing algorithms can further enhance the resolution of two-photon microscopy by compensating for aberrations and correcting for scattering effects, respectively.

Applications of Two-Photon and Multiphoton Microscopy

Two-photon and multiphoton microscopy have a wide range of applications in various fields of research. In neuroscience, these techniques are used to study neuronal activity, synaptic connections, and structural dynamics in live brain tissue, enabling insights into brain function and disorders. They also find applications in cellular biology, allowing the visualization of subcellular structures, organelles, and intracellular processes with high spatial resolution. 

In developmental biology, two-photon and multiphoton microscopy enable long-term imaging of embryogenesis and tissue morphogenesis in living organisms. These techniques have also been utilized in immunology, studying immune cell interactions and immune responses within complex tissues. Moreover, two-photon and multiphoton microscopy have proven valuable in investigating tumor biology, vascular imaging, drug delivery, and nanomaterial interactions, contributing to advancements in biomedical research and clinical diagnostics.