A revolution in specific staining was initiated with the introduction of antibody-mediated immunohistochemical stainings and DNA hybridization-based stainings. These allow a single protein or DNA sequence to be highlighted within a huge background of chemically similar molecules that could not be differentiated with the classical chemical dyes. The latest important progress in this context has been made with fluorescent proteins. These are introduced to stain structures, molecules and cells or are used in sensors for metabolites, ions and other species in living cells and organisms to provide a window for the observation of the machinery of life – in live mode. Nevertheless, all these staining procedures involve more or less invasive interventions, and require dead or fixed preparations in many cases. Additionally, the fluorescent protein method depends on the expression of foreign proteins in the target cells that often significantly interferes with the sensitively regulated homeostasis in cells. Therefore, methods are explored that allow imaging of bulky samples without any staining.
Fig. 1: Left: Overlay image of two recordings from sandwich sauce: Lipids are shown in red, water in green, 25x objective, scale bar 25 μm. Right: Study of localization and grade of accumulation of lipids in different types of macrophages: Lipids are displayed in red, actin in green, 63x objective. Courtesy: Alba Alfonso Garcia and Jeff Suhalim, Potma Lab, University of California, USA. Cells: Dr. Adelheid Kratzer, Department of Medicine Division of Pulmonary and Critial Care, University of Colorado.
Light can interact with matter not only by absorbing photons followed by the molecule assuming a higher excited state, but also by assuming what is called a "virtual state". In that case, the photon does not have the quantum of energy that would fit any of the state differences in the given molecule. For that reason, the released photon is not Stokes-shifted, as there is no thermal substate series in a virtual state which could cause the emission shifted to the red (in fluorescence, the excited state will first rapidly relax to the lowest vibrational substate of the excited state, releasing thermal energy which leaves the emitted photon with less energy – a redder tone). Under normal scattering conditions, the incident and the scattered photon therefore have the same energy, which is the same color, and cannot carry any specific information from the interacting molecule. This standard case is called “Rayleigh scattering” (Figure 2: Y).
Nonetheless, there is a chance that the emission does not cover the full span to the lowest vibrational state of the molecule, but, for example, the energy only returns to the first thermal state. In that case, the emitted photon only lacks the energy difference of that thermal state to the ground state. The emission is red-shifted. This phenomenon is known as Raman scattering (here, Stokes scattering, see below for an alternative, Figure 2: S). As the energy differences of these substates are very specific for molecular structures, the energy difference between incident and released photons reveals details of the compound that was interacting with the light. Raman spectroscopy has therefore become an extremely useful tool in analytical chemistry. Alternatively, the molecule might just happen to reside in the vibrational state (which to some extent happens even at ambient temperature). In this case, the virtual state upon illumination is higher compared to the example discussed above. As the molecule will typically assume the lowest energy after emission, the scattered photon has a higher energy than the incident photon. This is called Anti-Stokes scattering (Figure 2: A). Both processes occur very rarely; the Stokes process some 10–3-10–4 times less than the Rayleigh process, and the Anti-Stokes process even one or two orders below that. For imaging, the efficiency of spontaneous Raman scattering is by far too low to generate a sufficient signal within a reasonable time.
In order to generate useful intensities of Raman photons, it is necessary to artificially increase the population of the first vibrational state. When this is achieved, many photons will emit at Anti-Stokes wavelength, and a measurable signal is generated.
To achieve this goal, an intense infrared beam (pump beam) is initially focused into the sample by using a standard pulsed high power