## 6 Full-Field OCT and Fluorescence
The first interest of combining OCT with fluorescence is to identify structures that are captured in OCT, in order to better understand and characterize OCT contrast [62]. The second interest is to use fluorescence to reveal some structures, proteins, or ultimately any structure that cannot be captured by OCT, therefore combining fluorescence molecular contrast with structural contrast from OCT. It can help to understand how a structure of interest integrates and evolves inside its micro environment, such as neurons in the intestine [149], or to find the location of stem cells in the cornea [12]. Figure 7 shows the example of imaging a tumorous intestine tissue, in which cell nuclei are labeled with fluorescence. This image shows the complementary contrast that can be obtained with both modalities. Additionally, with both sources of information, we can localize the cells on the contours of organized structures, named intestine will, indicating that this field of view has been acquired in a healthy region of the cancerous tissue. A final interest, that is often neglected, especially in the context of histology, is that fluorescence enables dynamic measurements that can probe the tissue biochemistry and its evolution, using specific probes. For examples, fluorescence measurements can follow the electrical activity of cells by quantitatively measuring their intracellular calcium concentration [150; 151], or directly measure the membrane potential [152]. It can be useful to assess the viability of a cortical or retinal explant from its spontaneous activity, or can assess the endocrine pancreatic function, as insulin production is triggered by calcium uptake. Fluorescence can also directly measure cell function by measuring the intracellular ATP concentration [153], and can specifically detect apoptotic cells, with the ClickIt TUNEL assay (ThermoFisher). This can be of major importance to study the efficiency of pharmaceuticals on a cancerous tissue, as the cellular recovery of apoptosis within the tumor is usually a promising sign of a treatment's success. Figure 8 shows the result of the ClickIt TUNEL assay (from ThermoFisher Scientific [154]) on a fresh liver tissue. The tissue is first purposely excised with a corner shape to elicit and follow the cell death propagation from the margin to the center of the tissue. After several minutes, the tissue is imaged with FF-OCT and D-FF-OCT, and an inactive region (dark region from the tissue margin) can be detected with D-FF-OCT (panels A to C). Directly after the imaging, the tissue is fixed, in order to start the TUNEL assay and detect the apoptotic regions of this tissue. Unfortunately, this assay requires fixed tissues, so we cannot overlay a D-FF-OCT, and a fluorescence image. However, we could  
Figure 7: Revealing cells in tumors with FF-OCT/SIM. A fixed tumorous tissue of intestine is imaged in FF-OCT (**A**); in fluorescence (**B**); and the overlay is displayed in **Panel C**. Fluorescence reveals cell nuclei in the tissue. These images show that the cells are located within a well defined structure captured by FF-OCT, indicating that we are imaging in a healthy part of the tissue. The scale bars represent 40 \(\upmu\)m.  
detect that almost all cells in a 200 \(\upmu\)m region from the tissue border were in apoptosis (panels D to F). The fluorescence signal started decreasing as we went further from the tissue margin, and is almost equal to zero at the center of the tissue. Figure 8 tends to show that apoptotic cells do not produce a dynamic contrast, at least for cells in apoptosis for a long time. Surprisingly, this indirect result is different from what was obtained with OCT measurements [155], where the authors showed a decrease of the speckle decorrelation time (i.e., an increase of the intracellular activity) in apoptotic cells. Explanations for this result may be the following: In contrast to [PERSON] et al. [155], we used a _brute force_ method to generate apoptosis, as we physically cut the sample, and imaged close to the tissue edge.