# Line-Shaped Illumination: A Promising Configuration for a Flexible Two-Photon Microscopy Setup
## 3 Overview of Applications
### Imaging of Biological Samples  
As far as imaging is concerned, the performance of the microscope setup has been tested with several biological samples. As a first example, we report results about murine macrophage-like RAW 264.7 cells incubated with lipoprotein(a), whose related morphological changes are studied. As lipoprotein(a) is well-known to be a genetic risk factor for atherosclerotic diseases, but its pathogenic mechanism is not completely clear, it is very interesting to highlight its internalization by cells. For this investigation, we have employed a 60\(\times\) objective with a 1.4 numerical aperture so that the field of view is 150 \(\times\) 112 \(\upmu\)m\({}^{2}\). The excitation wavelength was 830 nm and the average power impinging on the sample was 60 mW. The cells were stained with Nile red and they generated no fluorescence signal when they were not treated. On the other hand, a strong signal upon two-photon excitation could be easily detected following incubation, as the image in Figure 9 clearly shows. Interestingly, we observed that lipids tended to accumulate throughout the cells, and especially in the middle part.  
As a second example, we present images of mouse ovarian follicles stained with eosin and hematoxylin. Several samples have been sectioned from the same tissue and differently prepared to investigate the role of each dye and their mutual interactions. The experimental parameters were the same except for the average excitation power, which was set at 100 mW. In Figure 10, images from the following specimens are presented: eosin-stained (A), hematoxylin-stained (B), first eosin- and then hematoxylin-stained (C),  
Figure 8: Adapted from [14]. Measured two-photon absorption cross-section of (**a**) eosin in water solution at 1% (red), 0.5% (green), and 0.25% (blue) concentrations, and (**b**) hematoxylin in water solution at 0.1% (red), 0.05% (green), and 0.025% (blue) concentrations. Reproduced with permission from the authors, J. Microsc.; published by Wiley, 2018.  
Figure 9: Adapted from [17]. Green fire and blue coloring of Nile red-stained cells: the picture underlines high-density accumulation (fire-green zones) versus low-density accumulation of fluorescence.  
and first hematoxylin- and then eosin-stained (D). When drawing a comparison between the images of the two samples stained with either of the two dyes, it was clear that the eosin-stained sample provided a much higher two-photon fluorescence level than the hematoxylin-stained sample, as it was to be expected based on the different values of the cross-sections presented in the previous section. More interestingly, when the different regions from which the signal was generated were analyzed, it could be seen that eosin bound mostly to the nucleus and the follicle epithelium, while hematoxylin bound mostly to the zona pellucida.  
The two images of the counterstained specimens thus provided greater detail (see Figure 10). The reduced signal level with respect to the eosin-stained sample is an indication of quenching phenomena arising from the interaction of the two chromophores. The almost identical images suggest that the order in which the specimens are stained with the two dyes does not play any major role.  
The maximum speed of imaging that we could achieve was 96 frames/s, corresponding to the limit of the camera used in the described measurements. However, preliminary tests carried out with faster devices have demonstrated frame rates up to 350 frames/s. The improvement with respect to two-photon point-scanning microscopes which can acquire no more than a few images per second is clear, and appears to be very promising for the investigation of dynamic processes in biology and life sciences.