Principle of Non-linear Optical Imaging
Non-linear optical imaging involves irradiation of a sample with laser light (at one or two wavelengths) through an optical microscope and detection of scattered light at a different frequency. Non-linear optical imaging is also sometimes referred to as multi-photon imaging since the non-linear processes involve several photons (Figure 1). The technique encompasses a range of non-linear optical phenomena including second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (TPF). In SHG, the energy of two photons is combined to emit light at half the laser wavelength. This process depends on the structural symmetry of the sample, and can be used to resolve crystalline and amorphous materials and some different polymorphic forms. In CARS, three photons at two or three wavelengths interact to efficiently generate light at a shorter wavelength (anti-Stokes Raman scattering). The technique is related to normal (spontaneous) Raman imaging, and is also used for label-free chemically-selective imaging. However CARS imaging is orders of magnitude faster, the spatial resolution is usually better and interference from fluorescence may be avoided. TPF is related to normal (one-photon) fluorescence, but it involves the energy of two incident photons instead of one with the advantage of being inherently confocal. Some materials (e.g. indomethacin, doxorubicin) generate TPF and so can be imaged with this technique without the requirement for labels. Vibrational energy level diagrams representing the SHG, CARS and TPF processes are shown in Figure 1.
Since the non-linear optical phenomena have different advantages and specificities, it is often very helpful to collect a combination of these signals at the same time with the same imaging setup. This is known as "multi-modal" imaging.
Imaging Solid Drugs and Dosage Forms
It is becoming widely recognised that critical solid dosage form properties, such as drug dissolution and release, are dependent not only on the formulation composition but also the component and solid state form distribution. Non-linear optical imaging is well suited to imaging a range of dosage forms. It is capable of rapidly imaging different chemical components and solid forms with high resolution (micron or sub-micron) in three dimensions. In general the data for the images may be collected in a few seconds or less. The technique may also be used to image changes in dosage forms in situ during drug release/dissolution and storage .
Distributions of components in tablets may be imaged in 2D or 3D, as shown in Figures 2 and 3. Both drug and excipient distributions may be imaged.
As mentioned above, the technique is suited for real time imaging of drug release/dissolution. In collaboration with the Optical Sciences Group, University of Twente, The Netherlands and Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University, Duesseldorf, Germany we are currently working on imaging drug and dosage form changes in a flow-through cell while simultaneously analysing drug concentration in solution.
Non-linear optical imaging is also well suited to real-time imaging of cells and tissues. We are currently working on imaging delivery of poorly water soluble drugs in various types of formulations in vitro and in vivo. If feasible, this approach will facilitate bringing together the analysis of drug release/dissolution and permeability, and should help lead to better understanding of absorption of these drugs.
 Strachan, C., Windbergs, M., & Offerhaus, H. (2011). Pharmaceutical applications of non-linear imaging International Journal of Pharmaceutics, 417 (1-2), 163-172 DOI: 10.1016/j.ijpharm.2010.12.017
 Windbergs, M., Jurna, M., Offerhaus, H., Herek, J., Kleinebudde, P., & Strachan, C. (2009). Chemical Imaging of Oral Solid Dosage Forms and Changes upon Dissolution Using Coherent Anti-Stokes Raman Scattering Microscopy Analytical Chemistry, 81 (6), 2085-2091 DOI: 10.1021/ac8020856
Asst. Prof. Clare Strachan (Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki) has several years' experience in non-linear optical imaging of a range of solid dosage forms. A fully integrated commercial non-linear optical microscope (Leica TCS SP8 CARS microscope) is available at the University of Helsinki. This is the first commercially available fully integrated CARS microscope in the world. The microscope uses a picosecond solid-state-laser light source to excite single Raman lines within a range of 1250 cm-1 to 3200 cm-1 for CARS imaging. It gives access to molecular specific contrast based on a variety of Raman-active vibrations relevant to pharmaceutical applications. Second harmonic generation (SHG) and two-photon fluorescence (TPF) are also possible with the setup. The microscope is also capable of one-photon fluorescent imaging in the UV and visible wavelengths. All non-linear and fluorescence phenomena can be imaged on exactly the same sample with the same microscope, and therefore a direct comparison of the imaging approaches can be made.