Pump–probe microscopy is a non-linear optical imaging modality used in femtochemistry to study chemical reactions. It is a kind of ultrafast laser spectroscopy. It generates high-contrast images from endogenous non-fluorescent targets. It has numerous applications, including materials science, medicine, and art restoration.
Advantages The classic method of nonlinear absorption used by microscopists is conventional two-photon fluorescence, in which two photons from a single source interact to excite a photoelectron. The electron then emits a photon as it transitions back to its ground state. This microscopy method has been revolutionary in biological sciences because of its inherent three-dimensional optical sectioning capabilities. Two-photon absorption is inherently a nonlinear process: fluorescent output intensity is proportional to the square of the excitation light intensity. This ensures that fluorescence is only generated within the focus of a laser beam, as the intensity outside of this plane is insufficient to excite a photoelectron. However, this microscope modality is inherently limited by the number of biological molecules that can undergo both two-photon excitation and fluorescence. Pump–probe microscopy circumvents this limitation by directly measuring excitation light. This expands the number of potential targets to any molecule capable of two-photon absorption, even if it does not fluoresce upon relaxation. The method modulates the amplitude of a pulsed laser beam, referred to as the pump, to bring the target molecule to an excited state. This will then affect the properties of a second coherent beam, referred to as the probe, based on the interaction of the two beams with the molecule. These properties are then measured by a detector to form an image.
Physics of pump–probe microscopy Because pump–probe microscopy does not rely on fluorescent targets, the modality takes advantage of multiple different types of multiphoton absorption.
Two-photon absorption Two-photon absorption (TPA) is a third-order process in which two photons are nearly simultaneously absorbed by the same molecule. If a second photon is absorbed by the same electron within the same quantum event, the electron enters an excited state. This is the same phenomenon used in two-photon microscopy (TPM), but there are two key features that distinguish pump–probe microscopy from TPM. First, since the molecule is not necessarily fluorescent, a photodetector measures the probe intensity. Therefore, the signal decreases as two-photon absorption occurs, the reverse of TPM. Second, pump–probe microscopy uses spectrally separated sources for each photon, whereas conventional TPM uses one source of a single wavelength. This is referred to as degenerate two-photon excitation.
Excited-state absorption Excited-state absorption (ESA) occurs when the pump beam sends an electron into an excited state, then the probe beam sends the electron into a higher excited state. This differs from TPA primarily in the timescale over which it occurs. Since an electron can remain in an excited state for a period of nanoseconds, thus requiring longer pulse durations than TPA.
Stimulated emission Pump–probe microscopy can also measure stimulated emission. In this case, the pump beam drives the electron to an excited state. Then the electron emits a photon when exposed to the probe beam. This interaction increases the probe signal at the detector site.
Ground-state depletion Ground-state depletion occurs when the pump beam sends the electron into an excited state. However, unlike in ESA, the probe beam cannot send an electron into a secondary excited state. Instead, it sends remaining electrons from the ground state to the first excited state. However, since the pump beam has decreased the number of electrons in the ground state, fewer probe photons are absorbed, and the probe signal increases at the detector site.
Cross-phase modulation Cross-phase modulation is caused by the Kerr effect, in which the refractive index of the specimen changes in the presence of a large electric field. In this case, the pump beam modulates the phase of the probe, which can then be measured through interferometric techniques. In certain cases, referred to as cross-phase modulation spectral shifting, this phase change induces a change to the pump spectrum that can be detected with a spectral filter.
Optical design
Excitation Measuring nonlinear optical interactions requires a high level of instantaneous power and very precise timing. In order to achieve the high number of photons needed to generate these interactions while avoiding damage of delicate specimens, these microscopes require a modelocked laser. These lasers can achieve very high photon counts on the femtosecond timescale and maintain a low average power. Most systems use a Ti:Sapphire gain medium due to the wide range of wavelengths that it can access. Typically, the same source is used to generate the pump and the probe. An optical parametric oscillator (OPO) is used to convert the probe beam to the desired wavelength. The probe wavelength can be tuned over a large range for spectroscopic applications. However, for certain types of two-photon interactions, it is possible to use separate pulsed sources. This is only possible with interactions such as excited-state absorption, in which the electrons remain in the excited state for several picoseconds. However, it is more common to use a single femtosecond source with two separate beam paths of different lengths to modulate timing between the pump and probe beams. The pump beam amplitude is modulated using an acousto-optic or electro-optic modulator on the order of 107 Hz. The pump and probe beams are then recombined using a dichroic beamsplitter and scanned using galvanometric mirrors for point-by-point image generation before being focused onto the sample.
Detection The signal generated by probe modulation is much smaller than the original pump beam, so the two are spectrally separated within the detection path using a dichroic mirror. The probe signal can be collected with many different types of photodetectors, typically a photodiode. Then, the modulated signal is amplified using a lock-in amplifier tuned to the pump modulation frequency.
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