Konstantin Popov

Imaging and modeling collagen architecture from the nano to micro scale

The collagen meshwork plays a central role in the functioning of a range of tissues including cartilage, tendon, arteries, skin, bone and ligament. Because of its importance in function, it is of considerable interest for studying development, disease and regeneration processes. Here, we have used second harmonic generation (SHG) to image human tissues on the hundreds of micron scale, and developed a numerical model to quantitatively interpret the images in terms of the underlying collagen structure on the tens to hundreds of nanometer scale. Focusing on osteoarthritic changes in cartilage, we have demonstrated that this combination of polarized SHG imaging and numerical modeling can estimate fibril diameter, filling fraction, orientation and bundling. This extends SHG microscopy from a qualitative to quantitative imaging technique, providing a label-free and non-destructive platform for characterizing the extracellular matrix that can expand our understanding of the structural mechanisms in disease.

Image formation in CARS and SRS: effect of an inhomogeneous nonresonant background medium

We investigate the role of a spatially inhomogenous nonresonant background medium on several Raman-based imaging modalities. In particular, we consider a small resonant bead submerged in a spatially heterogeneous nonresonant χ(3) background. Using detailed 3D electrodynamic simulations, we compare coherent anti-Stokes Raman scattering (CARS), frequency-modulated CARS, amplitude-modulated stimulated Raman scattering (SRS), and frequency-modulated SRS. We find that only FM-SRS is background-free.

Analysis of forward and backward Second Harmonic Generation images to probe the nanoscale structure of collagen within bone and cartilage

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees. We analyze the intensity dependence on polarization and discuss the differences between Forward and Backward images in both tissues. Focusing on articular cartilage, we observe an increase in Forward/Backward ratio from the cartilage surface to the bone. Coupling these results to numerical simulations reveals the evolution of collagen fibril diameter and spatial organization as a function of depth within cartilage.

Image formation in CARS microscopy: effect of the Gouy phase shift

Image formation in Coherent Anti-Stokes Raman Scattering (CARS) microscopy of sub-wavelength objects is investigated via a combined experimental, numerical and theoretical study. We consider a resonant spherical object in the presence of a nonresonant background, using tightly focused laser pulses. When the object is translated along the laser propagation axis, we find the CARS signal to be asymmetric about the laser focal plane. When the object is located before the focus, there is a distinct shadow within the image, whereas the brightest signal is obtained when the object is behind the focus. This behaviour is caused by interference between resonant and nonresonant signals, and the Gouy phase shift is responsible for the observed asymmetry within the image.

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.

Femtosecond laser induced surface swelling in poly-methyl methacrylate

We show that surface swelling is the first step in the interaction of a single femtosecond laser pulse with PMMA. This is followed by perforation of the swollen structure and material ejection. The size of the swelling and the perforated hole increases with pulse energy. After certain energy the swelling disappears and the interaction is dominated by the ablated hole. This behaviour is independent of laser polarization. The threshold energy at which the hole size coincides with size of swelling is 1.5 times that of the threshold for surface swelling. 2D molecular dynamics simulations show surface swelling at low pulse energies along with void formation below the surface within the interaction region. Simulations show that at higher energies, the voids coalesce and grow, and the interaction is dominated by material ejection.

Spatial-spectral coupling in coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is a third-order nonlinear optical technique which permits label-free, molecule-specific hyperspectral imaging. The interference between coherent resonant and non-resonant terms leads to well known distortions in the vibrational spectrum, requiring the use of retrieval algorithms. It also leads to spatial imaging distortions, largely due to the Gouy phase, when objects are smaller than the Rayleigh range. Here we consider that the focal position and spectral contributions to the nonlinear image formation are intrinsically coupled and cannot be corrected by conventional retrieval methods.

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.

Spatial-spectral coupling in hyperspectral CARS microscopy image formation

Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy has provided an imaging tool for extraction of 3-dimensional volumetric information, as well as chemically-sensitive spectral information. These techniques have been used in a variety of different domains including biophysics, geology, and material science. The measured CARS spectrum results from interference between the Raman response of the sample and a non-resonant background. We have collected four dimensional data sets (three spatial dimensions, plus spectra) and extracted Raman response from the CARS spectrum using a Kramers-Kronig transformation. However, the three dimensional images formed by a CARS microscope are distorted by interference, some of which arises because of the Gouy phase shift. This type of interference comes from the axial position of the Raman resonant object in the laser focus. We studied how the Gouy phase manifests itself in the spectral domain by investigating microscopic diamonds and nitrobenzene droplets in a CARS microscope. Through experimental results and numerical calculation using finite-diference time-domain (FDTD) methods, we were able to demonstrate the relationship between the spatial configuration of the sample and the CARS spectral response in three dimensional space.

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.