# Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching
## 1 Introduction  
Nonlinear optical microscopy has been proven to be a well-established technique based on nonlinear interactions between light and matter [1; 2; 3; 4]. In a nonlinear optical process, the emission intensity has a quadratic or high-order dependence on the excitation intensity, which ensures that the signal collected out of the focal volume reaches its minimum. Thus, nonlinear optical microscopy has submicron three-dimensional spatial resolution with higher signal-to-noise ratio and less out-of-focus photobleaching, compared with traditional confocal microscopy [5; 6; 7; 8; 9]. In addition, using near infrared (NIR) excitation wavelengths also increases the penetration depth, which is particularly significant for biomedical imaging [10; 11; 12]. Multiphoton microscopy (MPM) and coherent Raman scattering (CRS) microscopy are the most frequently applied nonlinear imaging technologies for biomedical microscopy [13; 14; 15; 16; 17; 18]. The former mainly includes two-photon fluorescence (TPF), three-photon fluorescence (ThPF), second-harmonic generation (SHG), and third-harmonic generation (THG) microscopy. The latter mainly consists of coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy. TPF microscopy has been the most preferred nonlinear microscopy for biological imaging since 1990 [13], thanks to its simpler setup compared to CRS microscopy and its superior ability to image a wider range of specimens compared to other multiphoton microscopy techniques. Intrinsic fluorescent biomolecules in biological tissues can be applied for imaging without the need of exogenous biomarkers, which ensures that TPF microscopy is a label-free technique. These biomolecules, called endogenous fluorophores, exist at both intracellular scale and in the extracellular matrix of biological tissues. The most widely used intracellular fluorophores are metabolic coenzymes, which include reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Investigations of physiological processes, e.g., cell differentiation and cancer development, have been performed by using autofluorescence derived from NADH and FAD [19]. Concerning the extracellular matrix, visualization of autofluorescence proteins, e.g., elastin, collagen, and keratin, has been used to diagnose diseases [20] such as asthma and pulmonary fibrosis. Even though TPF is the most widely used nonlinear process for biological microscopy, the first nonlinear label-free microscope is based on SHG [11]. Photons are scattered by molecules in the process of SHG, rather than absorbed in the process of TPF. SHG emitters are required to be spatially organized in non-centrosymmetric structures to avoid cancellation. In biological materials (e.g., collagen and myosin) with such structures, the signal of SHG could be very intense. The structures of collagen containing tissues, such as skin, tendon, and lung, have been revealed by SHG microscopy [20]. Meanwhile, pathological processes, such as liver fibrosis and hypertrophic scar development, have been investigated by SHG imaging [21]. TPF and SHG collect physiological and structural information from biological samples, respectively. Combining the two modalities will obtain more informative and comprehensive images. Simultaneous label-free TPF and SHG microscopy has been developed based on this concept over the last decade. Several groups have demonstrated simultaneous label-free acquisition of TPF from NADH and SHG from collagen using different excitation wavelengths [19; 20; 21]. Although these platforms provide high-quality images with sufficient biological information, only one depth can be imaged at the same time. Multi focal-plane microscopy and point-spread function (PSF) engineering are two typical methods to image fluorescent particles at different focal planes simultaneously, and several implementations have been established [22; 23; 24; 25].