Ultrafast Lasers for SHG Microscopy / Second Harmonic Imaging Microscopy
Incoherent nonlinear imaging techniques like 2-photon fluorescence microscopy have become a standard tool in cell biology for imaging of thick samples with high resolution. Comparable resolutions can also be obtained with coherent nonlinear techniques, based on photon scattering, such as second- or third harmonic generation (SHG or THG). In a SHG process the interaction of two photons with a nonlinear material creates a new photon having twice the energy and therefore half the wavelength of the incident light. This process was first observed on crystalline quartz in 1961, only years later was it also applied to investigate biological specimens. Since then second harmonic imaging microscopy (SHIM) has evolved to an established method to visualize structure and function of cells and intact tissue.
Analogous to 2-photon microscopy, the SHG process depends on the square of the intensity of the illumination light. Only in the very focus, the light intensity is high enough for these processes to occur. Therefore these nonlinear imaging techniques allow for intrinsic three-dimensional resolution without the need of a confining confocal pinhole. On the other hand, the probability of 2-photon excitation or second harmonic generation to occur is extremely small, therefore modelocked ultrafast lasers are required in order to create fs- to ps light pulses at sufficiently high intensities. The excitation light for both techniques is in the near infrared range (typically 700nm – 1000nm), which allows for a large penetration depth in the tissue due to reduced scattering at higher wavelengths.
The advantage of SHG microscopy compared to conventional fluorescence microscopy (and 2-photon microscopy) is that it is a label-free technique. This means that no fluorescent dyes have to be used to label the structure of interest. This fact implies that the molecular structure of the imaged specimen remains unchanged and also its metabolism is not altered. (Of course this is also true when autofluorescence of endogenous fluorophores like NADH or flavins is used for imaging.) Furthermore neither phototoxicity nor photobleaching should occur during imaging. These properties make SHG microscopy and ideal tool especially for live-cell imaging.
However, second harmonic generation requires a noncentrosommetric molecular structure and only highly ordered structures show sufficiently high cross-sections for SHG. In biological tissues mainly collagen (an extracellular protein), microtubules and myosin (intracellular proteins) produce sufficiently strong SHG signals for imaging. An application for SHG microscopy is for example non-invasive high resolution imaging of myosin in living muscle cells or the analysis of structural changes of the extracellular matrix.
Fluorescence is an incoherent process and the fluorophore is emitting in all directions. SHG on the other hand is a coherent process, meaning that the signals of adjacent molecules will interact with each other and phase matching has to be accounted for. Direction and polarization of the SHG signal depend therefore on the local distribution of the molecules in the focus. In the majority of cases the phase matching condition is fulfilled for the light co-propagating with the incident light and a strong SHG signal can be detected in forward direction. The phase matching condition for backward scattered light however, can only be fulfilled for thin specimens (i.e. thickness small compared to the wavelength). The analysis of forward-to-backward scattered intensities can thus give information about the thickness of the imaged structure. Furthermore, the analysis of the polarization dependency of the signal can yield information about the orientation distribution of the structures in the sample as well as their degree of organization.
SHG imaging system. Technically, a SHG microscope is comparable to a multi-photon setup: usually a laser-scanning microscope is equipped with a mode-locked ps- or fs-pulsed laser in the range between 700nm and 1000nm. In the laser-scanning microscope the incident IR-laser beam is focused via the objective lens onto the sample. The scanning mirrors in the microscope change the orientation of the beam so that the focal point scans the specimen in the lateral direction. This way the resulting 2D-image is acquired pixel-by-pixel. By changing the axial position of the microscope stage or of the objective, images from different focal planes can be obtained for 3D imaging. As mentioned earlier, the SHG-signal can either be detected in forward or in backward direction. The backscattered SHG-signal is descanned by the scanning mirrors and reflected by a suitable dicroic mirror on a photo multiplier tube (PMT) for detection. The SHG signal that propagates in the forward direction can be detected in a transmitted light configuration by a second objective, focusing on the same spot as the illuminating objective, and a non-descanned PMT. In both imaging pathways the laser light is blocked by short-pass filters in front of the PMTs. In order to make sure that no autofluorescence signal is detected, a narrow bandpass filter at half the laser wavelength can additionally be inserted into the light path.
Up to now large and expensive Titanium-Sapphire lasers have mainly been used for SHG imaging. TOPTICA’s FFproNIR (780nm, pulse width <100 fs, power >100 mW) is a fiber based turnkey laser system for simple and hands‑off operation. The FFproNIR has a small (A4/letter format) footprint and is an alignment-free all-fiber system which can easily be incorporated in SHG setups.