Multiphoton processes are finding applications within a broad range of scientific disciplines. These include bioimaging, diagnosis of diseases and their monitoring. Thanks to the use of longer wavelengths (800–1200 nm) in the multiphoton processes, multiphoton microscopy (MPM) reduces sample’s optical damage and provides deeper penetration (250–500 µm) into the specimen. MPM is regarded as the method of choice for non-invasive imaging of living, intact biological tissues on length scales from the molecular level through the whole organism. The technology finds its success in skin imaging, neuroscience, oncology, immunology, embryology, deep-tissue imaging, functional and molecular imaging, intravital imaging, virology applications at hospitals, clinical laboratories, and research institutes.
The underlying reason for the utility of multiphoton processes is the quadratic (for two-photon excitation) or higher power dependence on the excitation probability as function of the intensity of the excitation light, allowing for spatially confined excitations in 3D. The resolution of microscopy scales with the wavelength of excitation/emission light, and the wavelengths used in multiphoton experiments are longer than those for the corresponding one-photon application by factors 2, 3, 4 etc. This implies that two-photon excitation typically uses light of 800 nm rather than 400 nm and, given everything else equal, the lateral and axial resolution of the two-photon experiment is no better than confocal imaging with corresponding one-photon excitation. While the last two decades have seen the rise of better than diffraction limited one-photon fluorescence microscopy, so called super-resolution microscopy, improved resolution in two-photon microscopy remains difficult to reach. The power dependencies that are typical for higher-order non-linear microscopy suggest that when implementing three- or four-photon excitations (at the same wavelength as for two-photon), a substantial resolution gain can be obtained to resolve structures down to 150 nm. However, the gain in the spatial resolution of three- or four-photon microscopy comes at the cost of reduced cross-sections and wavelengths that are not necessarily compatible with life cell imaging (800 nm in four-photon microscopy corresponds to excitation at 200 nm in a one-photon process). For these reasons, the more sophisticated three- and four-photon techniques so far have been merely of academic interest, and two-photon microscopy is by far the most commonly applied technique in the industry, making up 94.2% of the market share. As the current paradigm is that two-photon microscopy allows no better resolution than confocal microscopy, this is what sets the limit for the vast majority of today’s users of multiphoton microscopy.
What if a technique existed offering the same spatial resolution as four-photon microscopy, but relying on excitation by two-photon absorption? This would combine the upsides of two-photon microscopy (relatively low excitation energies, standard lasers at around 800 nm could be used, excitation light in the middle of the optical window where the penetration depth in tissue is maximal) with those of four-photon microscopy (spatial resolution of 150 nm or below, a substantial improvement as compared to one- or two-photon excitation schemes using similar hardware) implying a true paradigm shift in multiphoton microscopy. Such a technique is presented here. Based on preliminary experiments we will describe the development of the concept in order to reach the ultimate objective for this project: Deliver proof-of-principle for a technology where conventional two-photon microscopy can achieve a spatial resolution equivalent to that of four-photon microscopy. This will allow all users of multiphoton microscopy to enjoy the superior resolution offered by four-photon microscopy.
