Second harmonic generation microscopy: a powerful tool for bio-imaging.

Second harmonic generation (SHG) microscopy is an important optical imaging technique in a variety of applications. This article describes the history and physical principles of SHG microscopy and its more advanced variants, as well as their strengths and weaknesses in biomedical applications. It also provides an overview of SHG and advanced SHG imaging in neuroscience and microtubule imaging and how these methods can aid in understanding microtubule formation, structuration, and involvement in neuronal function. Finally, we offer a perspective on the future of these methods and how technological advancements can help make SHG microscopy a more widely adopted imaging technique.

Calibration procedure for enhanced mirror artifact removal in full-range optical coherence tomography using passive quadrature demultiplexing.

Significance: Passive quadrature demultiplexing allows full-range optical coherence tomography (FR-OCT). However, imperfections in the wavelength- and frequency-response of the demodulation circuits can cause residual mirror artifacts, which hinder high-quality imaging on both sides of zero delay. Aim: We aim at achieving high mirror artifact extinction by calibrated postprocessing of the FR-OCT signal. Approach: We propose a mathematical framework for the origin of the residual mirror peaks as well as a protocol allowing the precise measurement and correction of the associated errors directly from mirror measurements. Results: We demonstrate high extinction of the mirror artifact over the entire imaging range, as well as an assessment of the method's robustness to time and experimental conditions. We also provide a detailed description of the practical implementation of the method to ensure optimal reproducibility. Conclusion: The proposed method is simple to implement and produces high mirror artifact extinction. This may encourage the adoption of FR-OCT in clinical and industrial systems or loosen the performance requirements on the optical demodulation circuit, as the imperfections can be handled in postprocessing.

Fast interferometric second harmonic generation microscopy.

We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample. Finally, we used fast interferometric second harmonic generation microscopy to acquire phase images in tendon. Our results show that the method exposed here, using a laser scanning system, allows to recover the polarity of collagen fibrils, similarly to standard I-SHG (using a sample scanning system), but with an imaging time about 40 times shorter.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy.

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.

Smart textile plasmonic fiber dew sensors.

We propose a novel Surface Plasmon Resonance (SPR)-based sensor that detects dew formation in optical fiber-based smart textiles. The proposed SPR sensor facilitates the observation of two phenomena: condensation of moisture and evaporation of water molecules in air. This sensor detects dew formation in less than 0.25 s, and determines dew point temperature with an accuracy of 4%. It can be used to monitor water layer depth changes during dew formation and evaporation in the range of a plasmon depth probe, i.e., 250 nm, with a resolution of 7 nm. Further, it facilitates estimation of the relative humidity of a medium over a dynamic range of 30% to 70% by measuring the evaporation time via the plasmon depth probe.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 µm, while in the transverse direction it is ∼1-15 µm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano-cylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced.

Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

Double-clad fiber coupler for endoscopy.

We present a double-clad fiber coupler (DCFC) for use in endoscopy to reduce speckle contrast, increase signal collection and depth of field. The DCFC is made by fusing and tapering two all silica double-clad fiber (DCF) and allows achromatic transmission of >95% of core illumination (1265nm - 1325nm) as well as collection of >42% of inner cladding diffuse light. Its potential for endoscopy is demonstrated in a spectrally encoded imaging setup which shows speckle reduction by a factor 5, increased signal collection by a factor 9 and enhanced depth of field by 1.8 times. Separation by the DCFC of single- and multi-mode signals allows combining low-speckle reflectance images (25.5 fps) with interferometrically measured depth profiles (post-processed) for of small three-dimensional (3D) features through an all-fiber low loss instrument.

The structural origin of second harmonic generation in fascia.

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.