Nonlinear Optical Imaging — Introduction and Pharmaceutical Applications

Nonlinear optical imaging is an emerging technology with much potential in pharmaceutical analysis. The technique encompasses a range of optical phenomena, including coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and twophoton excited fluorescence (TPEF). The combined potential of these phenomena for pharmaceutical imaging includes chemical and solidstate specificity, high optical spatial and temporal resolution, nondestructive and non-contact analysis, no requirement for labels, and the compatibility with imaging in aqueous and biological environments. In this article, the theory and practical aspects of nonlinear imaging are briefly introduced and pharmaceutical and biopharmaceutical applications are considered. These include material and dosage form characterization, drug release, and drug and nanoparticle distribution in tissues and within live cells. The advantages and disadvantages of the technique in the context of these analyses are also discussed. Introduction to Nonlinear Optics Nonlinear optics deals with processes where two or more photons interact with the sample material simultaneously. This is only possible when a high enough number of photons is confined in a small volume, i.e. the intensity of the incident light has to be much higher when compared to linear effects (normal absorption, refractive index). In practice this is made possible using ultrashort laser pulses with durations from picoseconds to femtoseconds. The narrow temporal width limits the interaction time between laser pulses and the target medium. Therefore, they offer a way to precisely probe the medium with reduced risk of optically induced damage. Even more importantly, the energy confined within the narrow optical pulse gives rise to very high light intensities and, thus, ultrashort pulses constitute ideal means for provoking a variety of nonlinear optical phenomena. Since the signal is produced selectively at the focus where the intensity is highest, nonlinear methods offer intrinsic axial sectioning property wellsuited for three-dimensional imaging. In optical microscopy, the two most commonly exploited phenomena are TPEF and SHG. CARS microscopy is yet another emerging nonlinear optical technique which is suitable for imaging various biological and pharmaceutical samples. The energy level diagrams of these processes are illustrated in Fig. 1. Figure 1. Energy level diagrams of two-photon excited fluorescence (left), second harmonic generation (middle), and coherent anti-Stokes Raman scattering (right). Two-photon excitation occurs through the absorption of two photons via intermediate virtual energy levels (dashed line). In SHG, two photons are combined similarly, but the emission occurs directly from a short-lived virtual level without excitation to the higher electronic states. In the CARS process, two photons stimulate a selected vibrational mode. Probing of this level with the third excitation photon results in the CARS emission. In the TPEF process (Fig. 1, left) a fluorophore molecule is excited by the simultaneous absorption of two photons in the infrared spectral range. In the conventional one-photon fluorescence the same transition to the higher energy level requires more energetic photons in the ultraviolet or visible range. The same fluorophores can often be used with both techniques. However, due to the fundamentally different quantum-mechanical processes, the excitation efficiencies are typically not equivalent. The fluorescence emission spectra are essentially the same in both cases. In TPEF, the longer incident wavelength leads to improved depth penetration in tissues, with reduced potential for photolytic damage [1, 2]. This has led to its increasing popularity in the biomedical setting, and the technique is commonly referred to as multiphoton imaging (this is because it is the most established nonlinear optical imaging method, even though other nonlinear phenomena also involve multiple photons). SHG is a nonlinear process where two photons interact with the target medium so that the energy from the incident laser beam is transferred to a beam with precisely double the original frequency (half the wavelength). This process does not involve the absorption of the photons, but relies on so-called virtual energy levels (Fig. 1, middle). SHG can only occur in materials that exhibit non-centrosymmetric structure. In biology, one such example is collagen, and most pharmaceutical crystals are also examples (whereas the amorphous form is not). One benefit of the technique is that it is straightforward to spectrally separate the SHG signal from other emission sources such as autofluorescence. In CARS microscopy, the signal originates from vibrational motion of the molecules in the sample. The process is based on a nonlinear phenomenon called four-wave mixing. In this process, three beams are interacting with the target medium to produce the fourth at a different wavelength (Fig. 1, right). The frequency difference between two of the input beams is selected to match the frequency of a vibrational energy state of the target molecule. When this resonant condition is fulfilled, the stimulating beams enhance the selected vibrational mode (e.g. stretching of a C-H molecular bond). Using the third beam, this motion can be probed. The resulting CARS signal is generated at the blueshifted (antiStokes) spectral range when compared to the input beams. Stimulated Raman scattering (SRS) is another variant of coherent nonlinear processes used to selectively probe molecular vibrations. When the diff erence frequency between two input beams matches a vibrational resonance, a small fraction (about one millionth) of the incident energy is transferred from one beam to the other. In order to measure the subtle variation, one of the input beams is intensitymodulated at high frequency. This modulation is transferred to the other beam and can be measured using sensitive photodetection. The main benefit of SRS over CARS is the lack of non-resonant background. Like conventional Raman microscopy (based on spontaneous Raman scattering), CARS and SRS are mostly used for imaging different chemical components without the need for labels. The major advantage of CARS and SRS compared to conventional Raman microscopy is the much faster imaging speed (orders of magnitude). This allows video rate imaging of dynamic processes, not to mention dramatically increasing analytical throughput. On the other hand, the spontaneous Raman microscopy typically gives richer spectral information which facilitates analysis of complex mixtures. The development of coherent Raman imaging systems capable of rapidly collecting rich spectral information is gradually overcoming this limitation. As a result, it is entirely possible that Raman microscopy based on spontaneous Raman scattering will largely be replaced with coherent Raman imaging in the pharmaceutical setting in the coming years. Nonlinear optical microscopes capable of TPEF and SHG (multiphoton imaging microscopes) are relatively widespread. CARS and SRS microscopes, which are also capable of TPEF and SHG imaging, are technically more complex but capable of richer sample analysis, especially when multiple nonlinear phenomena are simultaneously used (multimodal imaging). To illustrate the value of nonlinear optical imaging in the pharmaceutical setting, various examples are presented below. These include imaging drugs and dosage forms during the lifecycle of the product, from manufacturing to their fate in the body. Only label-free imaging examples are presented. Imaging Drugs and Dosage Forms Nonlinear optical imaging is slowly gaining interest in the area of drug and formulation imaging (Table 1). Some of the earliest work focused on using CARS to image the composition of emulsions [3]. In this work, the authors utilized CARS microscopy to image based on the C-H stretch vibrational region (2800-3200 cm-1). Later work conducted by Day et al. [4] used oil-in-water emulsions and investigated lipid digestion with CARS microscopy. CARS microscopy allowed them to discriminate between undigested oil and lipolytic products without the need for labeling. Table 1. Some publications for nonlinear optical imaging of pharmaceutical formulations