Abstract
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.
Highlights
Light microscopy is the method of choice for visualizing cells in their biological or physiological context
The classic light microscopy techniques such as Kohler illumination and phase contrast can be applied without prior labeling, allowing the observation of unperturbed cells or tissues. 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 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
The expression of GFP or GFP fusion proteins was linked to induction of apoptosis [1], dilated cardiomyopathy in transgenic mice [2], impairment of actin-myosin interactions [3,4], inhibition of polyubiquitination [5], and cytokine induction [6]
Summary
Light microscopy is the method of choice for visualizing cells in their biological or physiological context. Collagen fibers and sarcomeres Striated muscle and collagen fibers were inducers of SHG and THG (Figure 1a,b) and forward signals were generally stronger than backward signals, as described before [11,25,26,27,28].
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