Hole Refilling Technique

Many nonlinear interactions of molecules with light are relatively difficult to measure since the effects are small. It is important to be capable of measuring nonlinear effects such as two-photon absorption (TPA) or self-phase modulation (SPM) since these are fundamental properties of molecules. They also provide new constrast for imaging applications (see Henry link). Here we describe the “hole refilling” method that can extract TPA and SPM contrast simultaneously. These signatures are not limited to the resonant level structure of specific molecules, but also contain a dependence on local tissue anisotropy, chemical environment, or other structural properties.
Our approach is to shape the individual laser pulses in such a way that new frequency components are created by the nonlinear effects. As an illustration of a TPA measurement, consider a symmetric laser pulse, where with pulse shaping techniques a hole was created in the center of its frequency spectrum, as indicated in Fig. 1. In the time domain, such a pulse must consist of an intense and short part, balanced against a long and weak part. The areas of these two parts must be equal, if the pulse is to have zero amplitude at the center frequency. TPA tends to decrease the amplitude of the intense part more than the weak one, leading to an imbalance in the two parts, and thus refills the spectral hole.

SPM is another nonlinear process that can alter the pulse spectrum. SPM modulates the phase of the pulse by an amount proportional to the instantaneous pulse intensity. In the example of a spectrum with a hole, the phase modulation (or equivalently frequency modulation) caused by SPM also tends to refill the spectral hole. Simple measurements of the intensity in the spectral hole offer little contrast between the two nonlinear contributions. However, the two contributions can be clearly distinguished by the phase of the electric field generated by the non-linear polarizations. In order to detect this phase, a homodyne method can be employed by introducing a local oscillator (a few percent of the peak intensity) in the spectral hole, as indicated in Fig. 2. The local oscillator interferes with the nonlinear signal so that the amplitude of the signal in the spectral hole depends on the relative phase of the local oscillator and the nonlinear signal. Rotating the local oscillator phase allows the reconstruction of the phase of the nonlinear signal from the measured spectral intensity in the hole. With this technique TPA and SPM contributions can be extracted separately.

We first demonstrated the “hole-refilling” technique on various samples in solution. Figure 3 shows TPA and SPM measurements in Rhodamine 6G (R6G). To obtain this data the cuvette with the dye solution was scanned along the beam propagation direction. The shaded areas correspond to cuvette positions where the focal point was in the respective material. The dye sample shows both SPM and TPA whereas the glass walls only show SPM. We now have implemented this method in a microscopy setup, with which we simultaneously acquired 3D, high-resolution TPA and SPM images of mounted B16 melanoma cells. Figure 4 compares TPA and SPM images in a single slice through a cell. It is apparent that the SPM dynamic range is greater than that of the TPA images, and the contrast is different. The TPA contribution is predominantly caused by melanin within the cell. The mounting medium shows a strong SPM contribution, which is apparent in the large background in the SPM images. Melanin within the cell also exhibits strong linear absorption of the incident light. A shadow of the cell is therefore cast in the otherwise uniform SPM images acquired above and below the cell.