Second harmonic generation is a coherent process. Because SHG is a coherent process, in this cartoon we illustrate how at least four aspects of collagen’s homeostasis may influence collagen’s raw TPM SHG signal, an antibody Stain for quantifying collagen levels, and the ratio of forward-scattered to backward-scattered SHG (F/B) for collagen. SHG is sensitive to the regular ordering of scatterers, so conversion of disordered collagen triple helices into ordered fibrils, or de novo synthesis/breakdown of ordered fibrils can alter the detected SHG. Compaction of fibrils can also alter the detected SHG by changing the spacing of fibrils and hence altering interference properties. Lastly, the diameter of ordered fibrils dictates the F/B ratio for collagen SHG.⁴⁴ Readers should appreciate that this overview, simplified for pictorial presentation and clarity, does not reflect all possible signal changes. For example, organization or compaction changes could alter antibody stain intensity, depending on how antibody binding epitopes get rearranged.

Non-linear TPEF and SHG compared. Shown are the Perrin–Jablonski fluorescence diagram for TPEF (left), and the energy-level diagram for SHG (right). In TPEF, two photons of incident frequency x_i and wavelength k_i (in red) are simultaneously absorbed by a fluorophore, to produce a single photon (in green) whose wavelength is greater than one-half the irradiating wavelength k_i (i.e., with some net energy loss). In SHG, two photons of frequency x_i and wavelength k_i (in red) interacting simultaneously with a non-centrosymmetric target, combine to form a single photon (in blue) whose wavelength is exactly half that of the incident wavelength k_i (i.e., no net energy loss). Hence, 2P irradiation of an SHG generating target at 810 nm will emit SHG photons at 405 nm, whereas TPEF emission might occur at ~500 nm for the same target excited at 810 nm. Diagram is reproduced and modified with kind permission from Pantazis et al.,⁸³ with acknowledgment to National Academy of Sciences, USA.

Tumor collagen produces strong SHG. Collagen in an ex vivo E0771 mammary tumor produces a strong SHG signal in response to near-IR two-photon irradiation, showing the fibrous structure characteristic of highly ordered collagen fibers. All work was approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).

Quantifying tumor collagen SHG. (a) Collagen in an ex vivo E0771 mammary tumor can be immunohistochemically (IHC) labeled and quantified with an anti-collagen antibody (pseudocolored red). (b) Collagen produces a strong SHG signal (pseudocolored red) (same field as in (a)). (c) Dividing the SHG (b) image signal by the IHC (a) image signal (SHG/IHC) provides a new “ratiometric” image (c) which indicates levels of SHG relative to total collagen on a pixel-by-pixel basis. Hence circled regions indicate where this ratio is clearly different (i.e., high or low pixel values), suggesting differences in relative collagen organization in these regions. Increased SHG signal relative to immunolabeling may represent a conversion of disordered triple helices to ordered fibrils, or compaction of fibrils.⁵¹ An increase in both signals may represent de novo synthesis of ordered triple helices. Green signal is a nuclear counterstain. All work was approved by and performed in accordance with the guidelines of the IACUC. Diagram is modified from Brown et al.,⁸ with permission, and acknowledgment to Nature Publishing Group.