Light microscopy is the method of choice for visualizing cells in their biological or physiological context. The classic light microscopy techniques such as Köhler illumination and phase contrast can be applied without prior labeling, allowing the observation of unperturbed cells or tissues. However, these techniques are not suitable for thick tissues and they do not allow high resolution three-dimensional reconstruction of image volumes, since the microscopic image also contains data from above and below the focal plane. This limitation can be circumvented by restricting image generation to photons originating from the focal plane as achieved by confocal laser scanning microscopy or by two-photon excited fluorescence microscopy. Both approaches require a fluorescent structure, and thus except for a few autofluorescent structures staining with structure specific fluorescent dyes such as DNA stains or fluorescent antibodies is necessary. Images from adjacent focal planes then can be combined to generate a three-dimensional image volume. For staining of living cells or tissues, however, it often remains unclear how far the staining perturbs physiologic functions of cells or tissues. For example, the expression of GFP or GFP fusion proteins was linked to induction of apoptosis 
, dilated cardiomyopathy in transgenic mice 
, impairment of actin-myosin interactions 
, inhibition of polyubiquitination 
, and cytokine induction 
Second and Third Harmonic Generation (SHG and THG) microscopy 
combine the advantages of optical sectioning and label-free visualization: Signal generation is restricted to the focal spot of the incoming laser while the need for potentially damaging fluorescent labeling is avoided. In the SHG process two simultaneously incoming photons are transformed into one emitted photon with exactly half the wavelength. SHG signals are generated at dense, non-centrosymmetric structures. In mammalian soft tissues, this equals mostly myosin in striated muscles and thick collagen type I, II and III fibers. In some special cases, dense microtubule bundles also can be visualized 
. SHG microscopy is comparatively widespread, since detection is possible using standard two-photon microscopes with an excitation wavelength twice that of an active detection channel.
In THG, the energy of three simultaneously incoming photons is combined to emit one photon with one third the wavelength. THG is more complex to realize because detection of the signal in the visible range above 400 nm requires an excitation wavelength of above 1200 nm, a wavelength range not provided by standard two-photon microscopes. Detection in the visible range is not an absolute requirement dictated by physical theory, but rather by the optical elements of standard microscopes which have limited transmission for UV light. In addition, Rayleigh scattering with accompanying light loss in tissues increases with the 4th
power with shorter wavelengths 
. THG microscopy is more versatile than SHG microscopy since THG is induced at interfaces such as refraction index changes within the focal volume of the excitation laser 
. Refraction indices change for example between cell nuclei and cytoplasm or cytoplasm and interstitial fluid. They are the basis for classical microscopy techniques such as phase contrast and DIC which are, however, less suitable for deep tissue 3D imaging since the contrast generating process is not limited to the focal plane. An additional source for THG which is of practical relevance in biological microscopy is resonance enhancement by light absorbing structures. For example hemoglobin induces a strong THG signal when illuminated with 1275 nm light, due to its absorption around 425 nm 
. In summary, THG offers for unlabeled preparations what confocal and two-photon induced fluorescence did for fluorescent specimens, i.e. providing a technique with optical sectioning capability, with exclusion of out-of-focus signal and with 3D visualization.
While SHG and THG are well-established phenomena in the optical sciences, the biomedical community is less aware of their potential. To evaluate the capabilities of these label-free techniques, we applied THG and SHG microscopy to an established tissue model. The rodent cremaster muscle can be prepared from the scrotum as a thin, light permeable sheet and thus became widely-used for in vivo physiological research with conventional bright field microscopy (e.g. 
). In this well characterized tissue we investigated which structures were identified by SHG and THG and which level of detail could be achieved. The maximal achievable depth was determined in thigh muscles.