Second Harmonic Generation Microscopy in Biological Tissues

Second harmonic generation (SHG) microscopy is a label-free, nonlinear optical imaging technique that exploits the frequency-doubling of incident photons in non-centrosymmetric structures. It provides intrinsic contrast for ordered biomolecules such as fibrillar collagen, myosin and microtubules without the need for exogenous dyes. By delivering pulsed near-infrared light and detecting the backward- or forward-scattered second harmonic signal, SHG microscopy achieves submicrometre resolution and millimetre-scale penetration in thick biological specimens. The addition of polarization-resolved measurements further enables quantitative mapping of molecular orientation and supramolecular organisation. In combination with complementary modalities—such as two-photon fluorescence, optical coherence microscopy or fluorescence lifetime imaging—SHG microscopy yields a comprehensive view of tissue architecture and dynamics. Its applications span ophthalmology, cancer diagnostics, fibrosis assessment, cardiac remodelling and fundamental studies of extracellular matrix biomechanics. The technique’s non-invasive nature and reduced phototoxicity make it attractive for in vivo and longitudinal studies, while advances in instrumentation and computational modelling continue to enhance sensitivity, specificity and interpretability of SHG signals.

Research from Nature Portfolio

Recent work has introduced a novel SHG-active organic dye that is both non-fluorescent and membrane-potential-sensitive, enabling purely SHG-based two-photon imaging at cellular membranes with minimal photodamage. This dye permits simultaneous and independent acquisition of SHG signals alongside conventional fluorescence from reporter molecules, facilitating true multimodal microscopy in live neuronal preparations. The development represents a key advance in decoupling SHG contrast from overlapping fluorescence and broadens the scope for real-time functional imaging of electrical activity and membrane dynamics in intact tissues.

Research from all publishers

A polarization-resolved SHG study of intact human corneas has provided the first continuous, quantitative characterisation of stromal collagen lamellae over the full corneal thickness. By combining epi-detection with polarimetric analysis, researchers mapped a gradual anterior–posterior shift in lamellar orientation and revealed distinct disorder profiles in the anterior stroma. This work underscores the power of P-SHG for probing dense, unsectioned tissues with native curvature.

A general multiscale numerical framework has been established to link atomic-scale peptide bonds to micrometre-scale SHG measurements in organised protein assemblies. The model accurately predicts the second-order hyperpolarizability tensor and SHG efficiency for collagen, myosin and microtubule networks, and it has been validated in live zebrafish larvae. This approach bridges molecular structure and macroscopic optical response, offering a predictive tool for interpreting SHG contrast in complex biological systems.

An integrated multimodal microscope combining polarization-sensitive SHG, polarization-sensitive optical coherence microscopy and two-photon fluorescence lifetime imaging has been demonstrated for fresh, unsectioned tissues. By co-registering these contrasts from a single excitation source, the system delivers quantitative, multiparametric characterisation of collagen subtypes and their organisation in situ. This platform paves the way for comprehensive studies of extracellular matrix heterogeneity in health and disease.

Second Harmonic Generation Microscopy in Biological Tissues publication trend

The graph below shows the total number of articles in second harmonic generation microscopy in biological tissues across all publications each year (not limited to Nature Index journals).

Technical terms

Second Harmonic Generation (SHG): A nonlinear optical process in which two photons of the same frequency combine to generate a single photon at twice the frequency, providing intrinsic contrast for non-centrosymmetric structures.

Polarization-resolved SHG (P-SHG): An SHG modality that analyses the polarization state of the emitted signal to map molecular orientation and structural anisotropy.

Nonlinear Susceptibility (χ(2)): The second-order tensor describing the efficiency of a material’s second harmonic response under an applied optical field.

Hyperpolarizability Tensor (β): A molecular-level tensor representing the elementary source of SHG in proteins and other harmonophores, linking atomic structure to macroscopic signal.

Multimodal Imaging: The simultaneous acquisition of multiple complementary imaging contrasts (e.g., SHG, two-photon fluorescence, optical coherence) in a co-registered manner.

References

1. Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy. Light: Science & Applications (2023).
2. Modeling and Predicting Second-Harmonic Generation from Protein Molecular Structure. Physical Review X (2024).
3. Label-free multimodal polarization-sensitive optical microscope for multiparametric quantitative characterization of collagen. Optica (2024).
4. Multimodal two-photon imaging using a second harmonic generation-specific dye. Nature Communications (2016).
5. Second harmonic generation microscopy: a powerful tool for bio-imaging. Biophysical Reviews (2023).