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.
[3] The method modulates the amplitude of a pulsed laser beam, referred to as the pump, to bring the target molecule to an excited state.
Because pump–probe microscopy does not rely on fluorescent targets, the modality takes advantage of multiple different types of multiphoton absorption.
[3] 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.
[3] 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.
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.
[3] 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.
[3] Processing pump–probe data is challenging for several reasons – for example, the signals are bipolar (positive and negative), multi-exponential, and can be significantly altered by subtle changes in the chemical environment.
In melanoma studies, the principal components have shown good agreement with the signals obtained from the different forms of melanin.
However, the principal components do not necessarily reflect actual properties of the underlying chemical species, which are typically non-orthogonal.
[12] The development of high-speed and high-sensitivity pump–probe imaging techniques has enabled applications in several fields, such as materials science, biology, and art.
[3][7] Pump–probe imaging is ideal for the study and characterization of nanomaterials, such as graphene,[13] nanocubes,[14] nanowires,[15] and a variety of semiconductors,[16][17] due to their large susceptibilities but weak fluorescence.
In particular, single-walled carbon nanotubes have been extensively studied and imaged with submicrometer resolution,[18] providing details about carrier dynamics, photophysical, and photochemical properties.
Therefore, imaging the distribution of eumelanin and pheomelanin can help to distinguish benign lesions and melanoma with high sensitivity[24] Artwork consists of many pigments with a wide range of spectral absorption properties, which determine their color.