## 6 Full-Field OCT and Fluorescence  
In principle, usual histology staining and fluorescence techniques are quite similar, except that fluorescence techniques use fluorescence probes instead of absorbing probes in histology. Some fluorescence techniques have been part of the histology toolkit for decades [136], such as immunofluorescence, which aims at detecting the interaction of a labeled antibody with a structure of interest [137], or fluorescence in situ hybridization (FISH) ([138; 139], Chapter 6), which aims at detecting the interaction of a labeled nucleotide sequence with its specific complementary sequence in a cell. More recently, confocal microscopy [140] and two photon microscopy [141] have been used in order to detect fluorescent probes in depth in tissues. Interestingly, thanks to the optical sectioning provided by these techniques, it has become possible to follow some fluorescent structures in ex vivo tissues, and in vivo [142; 143]. However, fluorescence techniques still require fluorescent labels, that need to be injected in the tissue. It can take a long time before the label diffuses in depth inside a tissue. Moreover, and similarly to classic histology stains, these labels only tag a few structures of interest, and it might be hard to understand the organization of these structures inside their microenvironment. Multiple labeling can be used to tag several structures, but it increases the complexity, and the cost of such experiments. We can also add that usually, confocal and two photon microscopy can only access shallow depth inside a tissue, down to a few hundred \(\upmu\)m at best. OCT techniques on the other hand can access deeper regions down to one millimeter, since the signal is often enhanced by the reference arm. Recently, clearing techniques [144; 145] with low tissue distortions have been developed in order to increase tissue transparency, and to image through the entire tissue. OCT can also benefit from these approaches [146], even if we would expect the intrinsic signal to be reduced in OCT, since clearing tends to remove lipid structures and to average the refractive index fluctuations. However, it can help to detect exogenous OCT contrast agent at important depths, such as metallic nanoparticles. Here again, clearing processes are often long, and can induce tissue distortions. Fluorescence and OCT techniques are quite complementary, and multimodal systems combining the two can help with diagnosis, as we will illustrate throughout this section. Many such multimodal setups have been developed with both scanning OCT approaches [5; 147] and full-field approaches, using either a flip mirror to switch from one modality to the other [18; 148], or a simultaneous approach [41; 42]. If all these multimodal approaches share a common interest, scanning OCT approaches are more easily combined with scanning fluorescence approaches, such as confocal or two photon microscopies, whereas FF-OCT systems have been developed in combination with widefield fluorescence techniques, such as structured illumination microscopy (SIM). SIM acquisition speed intrinsically depends on the fluorescence camera frame rate divided by the number of grid modulation steps. The SIM acquisition rate can go up to 100 frames per second in our measurements, even though the usual frame rate limit comes from the fluorophores' brightness, and from the minimal exposure time required to produce a decent image. Interestingly, simultaneous combination of FF-OCT and SIM [41; 42] enable the parallel acquisition of both modalities, so images with both modalities can be acquired at more than 100 frames per second. However, SIM is not very robust to aberrations and multiple scattering, and can hardly penetrate inside scattering samples after 30 to 50 \(\upmu\)m. Nevertheless, in mostly transparent samples, such as cornea or retina, with only a few structures labeled, we could perform multimodal FF-OCT and SIM images through the entire sample.