Maxime Rivard

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.

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

We image the relative orientation of organized groups of noncentrosymmetric molecules (like collagen or myosin) at the micron scale in biological tissues by combining interferometry and Second Harmonic Generation (SHG) microscopy.

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 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.

Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy

The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.

External amplification of OCT swept-sources for challenging applications: from 10 mW to more than 120 mW

For bioimaging applications, commercial swept-sources currently provide enough power (tens of milliwatts) insuring good imaging condition without damaging the tissues. For industrial applications, more power is needed since the amount of light collected can be very low due to challenging measurement conditions or due to poor sample reflectivity. To address this challenge, we explore three different setups to externally amplify the output of a commercial swept-source: a booster semiconductor optical amplifier (BOA), an erbium-doped fiber amplifier (EDFA) and a combination of both. These external amplification setups allow the exploration of emerging OCT applications without the need to develop new hardware.

High Contrast Interferometric Second Harmonic Generation Microscopy Probes the Polarity of Fibrils in Complex Collagenous Tissues

We implemented for the first time Interferometric SHG microscopy, using femtosecond pulses, to image the relative fibrils polarity in cartilage and show that SHG intensity depends on the local ratio of fibrils with opposite polarities.

Interferometric Second Harmonic Generation microscopy for tissue imaging

The results obtained with interferometric second harmonic generation (I-SHG) microscopy show that this technique is complementary to standard SHG microscopy since it provides additional information about the structural organization of tissues containing large groups of noncentrosymmetric proteins.

Imaging tendon and muscle with Interferometric Second Harmonic Generation microscopy (I-SHG)

SHG microscopy, used to image noncentrosymmetric structural organization, cannot measure the relative SHG phase between the structures. Using I-SHG microscopy, we show that the relative phase between structures in tendon and muscle can be imaged.