## 7 Comparison with Other Popular Novel Microscopies  
As previously pointed out, although fixation and labeling protocols have been greatly improved these past years in histology, this field could still benefit from the important technological developments that optical imaging has known over the past twenty years. So far, we compared FF-OCT and its multimodal versions to standard histology procedures. However, to be fair, we should also compare them to other recently developed optical techniques, such as multiphoton microscopies, and coherent anti-Stokes Raman scattering (CARS) microscopy, that also are label-free and enable optical sectioning. Similarly to OCT, these techniques rely on the intrinsic properties of tissues to generate contrast. They both can provide optical sectioning, since they rely on the combination of two or three photons,  
Figure 8: Imaging apoptosis with multimodal FF-OCT systems. This figure presents the imaging of the edge of an ex vivo liver. Here, the liver has been excised with a triangular edge to illustrate the propagation of cellular death from the excised edge. First, a large field FF-OCT and D-FF-OCT images have been acquired with the commercial LLTech system (**A**, **B** and overlay in **C**) 30 min after the excision. Then, the tissue has been fixed, and labeled with ClickIt Plus TUNEL Alexa 488 assay (ThermoFisher) to reveal apoptotic cells; **Panels D**, **E** and **F** respectively show the FF-OCT and fluorescence signals (and the overlay) acquired from the part of the tissue in the yellow box in panel A. In this region close to the excised edge, almost all cells are apoptotic, as suggested by the low level of dynamic signal in panel B. Scale bars for panels A, B, and C represent 125 \(\upmu\)m, and scale bars in D to F represent 40 \(\upmu\)m.  
that can statistically only happen at the focal plane where the power density is high enough. Second harmonic generation (SHG) is measured in dense non-centrosymmetric structures, such as microtubule bundles, and collagen I fibers [157]. Third harmonic generation (THG) is measured at interfaces displaying high index mismatch, such as cell or nuclear contours, and lipid droplets [158]. Both techniques, especially when combined, are therefore useful to study 3D tissue organization, e.g., in the cornea [159], or in developing embryos [160]. The association of the two techniques gives a contrast similar to FF-OCT, but non-linear microscopes are a little more sensitive and specific than the FF-OCT signal, and can be naturally combined with two-photon fluorescence microscopy [161]. The transverse resolution is often higher than FF-OCT, as high NA objectives are required to generate a high power density at the focal point. We can add that SHG has already been extensively used to assess tissue disorganization, especially in the context of liver fibrosis [162]. A commercial SHG microscope for histology is already available at HistoIndex [163]. CARS [164], and its hyperspectral version [165], are other promising non-linear techniques for histology, which reveal a high density of either a single or multiple molecular vibrations inside a sample. Interestingly, CARS is one of the only techniques that is both label-free, and specific at the molecular level, as vibration spectra of given structures can be measured, and computationally extracted [166]. It is therefore one of the most promising label-free techniques today for quantification of tissue structure, and molecular content. Unfortunately, to our knowledge, there is no commercial CARS system available, and CARS systems often require careful and time consuming alignment steps. Finally, we can add that all these techniques are based on non-linear processes that require similar kinds of sources, i.e., femtosecond lasers, and can be ingeniously combined all together from a single laser source [167]. However, such non-linear processes are quite weak. They typically display a low signal-to-noise ratio, especially in dense tissues, and often require long exposure times to generate a measurable signal.