Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy.

Second and Third Harmonic Generation (SHG and THG) microscopy is
based on optical effects which are induced by specific inherent
physical properties of a specimen. As a multi-photon laser scanning
approach which is not based on fluorescence it combines the
advantages of a label-free technique with restriction of signal
generation to the focal plane, thus allowing high resolution 3D
reconstruction of image volumes without out-of-focus background
several hundred micrometers deep into the tissue. While in
mammalian soft tissues SHG is mostly restricted to collagen fibers
and striated muscle myosin, THG is induced at a large variety of
structures, since it is generated at interfaces such as refraction
index changes within the focal volume of the excitation laser.
Besides, colorants such as hemoglobin can cause resonance
enhancement, leading to intense THG signals. We applied SHG and THG
microscopy to murine (Mus musculus) muscles, an established model
system for physiological research, to investigate their potential
for label-free tissue imaging. In addition to collagen fibers and
muscle fiber substructure, THG allowed us to visualize blood vessel
walls and erythrocytes as well as white blood cells adhering to
vessel walls, residing in or moving through the extravascular
tissue. Moreover peripheral nerve fibers could be clearly
identified. Structure down to the nuclear chromatin distribution
was visualized in 3D and with more detail than obtainable by bright
field microscopy. To our knowledge, most of these objects have not
been visualized previously by THG or any label-free 3D approach.
THG allows label-free microscopy with inherent optical sectioning
and therefore may offer similar improvements compared to bright
field microscopy as does confocal laser scanning microscopy
compared to conventional fluorescence microscopy.