**4. Two-Photon Fluorescence**

Two-Photon Fluorescence (TPF) is a validated technique for imaging and detection of labeled biological material such as peptides [81] and steroids [82,83]. TPF has numerous advantages over conventional one photon fluorescence (OPF), being the most important one the large Stokes shift between emission and excitation. The large energy gap between the fields lowers the background noise. Other advantages are the reduced static photo-bleaching of dyes due to the absorption's quadratic dependence on the intensity; and the wide range of fluorescent dyes with high quantum yields and molar extinction in the visible. However, TPF requires high photon density flux, reached by tightly focusing the laser light. To avoid tight focusing and achieve the needed intensities, much research has resorted to enhancing photonic platforms such as resonators [84]. MBR are the most suitable WGMR for TPF, being hollow they can be filled with liquids. For the TPF demonstration Pastell et al. [17] filled MBR with a 10−<sup>3</sup> and 10−<sup>4</sup> solution of fluorescein and 10−<sup>6</sup> solution of Rhodamine 6G.

The authors used a modified confocal microscope for coupling the light into the microbubble resonator [85], an inverted light microscope (Nikon Eclipse TE2000U) up-graded to a multi-modal imaging system even though it can be used either as a bright-field microscope or as a phase contrast microscope. The microscope was pumped by a Mira 900-f Ti: Sapphire (repetition rate 76 MHz and 150fs pulse duration, Coherent) with an average power of 1.2 W. It has an average power of 1.2 W and it can be tuned over a wavelength range of 690–950 nm within which falls the two-photon absorption spectra of many fluorophores [86]. The wavelength in the experiments was set to 800 nm. The raster scan of the beam was stopped and it was coupled into the MBR by focusing the laser beam tangential to the bubble wall with two different dry objectives, namely 4X and 10X and 0.5 NA. The excitation light was filtered by a dichroic mirror (FF720-SDi01, Semrock) and a BG39 Schott filter. We tested first the bubble with the fluorescein filling that was imaged with a 4X dry objective using a CCD camera to see the complete WGM at the equator. Figure 13 shows the TPF band around the equator and the TPF partially coupled back to the MBR wall. In this case, the MBR was filled with fluorescein and imaged with a 4X dry objective using a CCD camera to see the complete WGM at the equator.

**Figure 13.** Fluorescence image of the MBR filled with 10−<sup>3</sup> solution of fluorescein, showing the TPF band and lobes, and the fluorescence coupled back into the MBR wall . Reproduced with modifications from [17].

The two-photon nature of the emitted signal was validated by checking its dependence on the excitation laser power. Figure 14 shows an optical image of a MBR for an incident power of about 190 mW (the power at the focal plane is just the 30 of the incident power), its intensity plot (center); and a logarithmic representation of the TPEF signal from a Rhodamine 6G filled microbubble versus incident laser power at the focal plane. A linear fit to the data has slope close to 2, ensuring the quadratic dependence of the obtained signal. The two lobes that correspond to the WGM are clearly seen.

**Figure 14.** Fluorescence image of the MBR filled with 10−<sup>6</sup> solution of Rhodamine 6G, showing the TPF lobes, the corresponding intensity plot (center) and the TPF signal versus the pump laser power in log-log scale. The red line is the linear fit with slope close to 2. The probe wavelength is *λpump* = 800 nm. Reproduced with modifications from [17].
