Polarization resolved second harmonic microscopy

Mazumder, Nirmal; Deka, Gitanjal; Wu, Wei; Gogoi, Ankur; Zhuo, Guan‐Yu; Kao, Fu Jen

doi:10.1016/j.ymeth.2017.06.012

Cited by 26 publications

(18 citation statements)

References 68 publications

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“…Having shown that SR-SHG is possible using the PNJ-approach and the FOV can be expanded, we sought to investigate ways to achieve ultra-high resolution and develop a technique that can be used for distinction at the nanoscale level. Hence we examined the property that in SHG, signals are dependent on the relative orientation of harmonophores vs the polarisation of the excitation [34] offering a measure of the disorder of a sample. We used a mouse tail tendon that contains a high density of aligned collagen fibres to demonstrate this sensitivity to polarisation ( Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Johnson

Karvounis

Singh

et al. 2020

Preprint

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Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. Here we achieve super-resolution SHG (SR-SHG) for the first time. We use the photonic nanojet (PNJ) phenomena to achieve a resolution of ~λ/6 with respect to the fundamental wavelength, a ~2.7-fold improvement over diffraction-limited SHG under the same imaging conditions. Crucially we find that the polarisation properties of excitation are maintained in a PNJ allowing the resolution to be further enhanced by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we are able to demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery. List of abbreviationsSecond Harmonic Generation (SHG), Super-resolution (SR), Barium Titanate Glass (BTG), Photonic Nanojet (PNJ), Point Spread Function (PSF) Calculation of Polarisation anisotropyPolarisation anisotropy images were created using a home written Fiji macro in which the key steps are as follows: A difference image was generated by (Ipar -Iperp), this was divided by a total intensity image (Ipar + 2Iperp). The total intensity image was used to select the region of the image containing signal and for display all pixels outside of this region were set to 0. Only the regions containing signal were used for further anisotropy analysis. Collagen fibre analysisCollagen fibre analysis was performed using CT-FIRE V1.3. SHG intensity images were converted to 8bit format before being batch processed using default parameters. Four parameters were measured to describe fibre morphology, width, length, straightness and angle.

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Section: Resultsmentioning

confidence: 99%

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Johnson

Karvounis

Singh

et al. 2020

Preprint

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“…Second-harmonic generation (SHG), a second-order non-linear scattering process, generates only from non-centrosymmetric structures [1,88] and the SHG intensity depends on the relative orientation between the incoming polarization state of light and the second-order hyperpolarizability of the sample [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107]. In SHG, two photons with the same frequency are combined and generate a single photon of precisely double the energy of incident photons (i.e., half the wavelength and twice the frequency).…”

Section: Second Harmonic Generation (Shg) Microscopymentioning

confidence: 99%

“…For Stokes vector measurement, the SHG signals are analyzed using a polarization state analyzer (PSA), specifically, a four-channel Stokes-polarimeter. The basic principles of Stokes polarimetry using four channels are discussed in detail [107,111,112]. The forward propagating SHG signal is limited to the thickness of the samples, however, backscattered SHG measurement demonstrates the potential of the technique for in-vivo imaging by the use of fiber-optics based endoscopic probe [103,[112][113][114].…”

Section: Second Harmonic Generation (Shg) Microscopymentioning

confidence: 99%

“…The method investigates the molecular organization through the characterization of the polarization properties of SH signal from KDP crystal, collagen type-I, starch granules, skeletal muscle fiber, etc. [107]. Hence, polarization-resolved SHG imaging offers additional parameters including circular dichroism, susceptibility, Stokes vectors and can be used as measuring parameters in the detection of subtle changes in tissue structure associated with collagenrelated diseases including fibrosis, cancer, wound healing, osteoarthritis, etc.…”

Section: Second Harmonic Generation (Shg) Microscopymentioning

confidence: 99%

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Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

et al. 2019

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Non-linear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free non-linear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The non-linear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved non-linear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

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“…have employed a polarization‐resolved second harmonic generation imaging system for the imaging of biological structures such as collagens . Polarized Raman spectroscopy is another popular technique for anisotropic material characterizations …”

Section: Introductionmentioning

confidence: 99%

Single‐Shot Optical Anisotropy Imaging with Quantitative Polarization Interference Microscopy

Zhou

Takiguchi

et al. 2018

Laser & Photonics Reviews

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Optical anisotropy measurement is essential for material characterization and biological imaging. In order to achieve single-shot mapping of the birefringence parameters of anisotropic samples, a novel polarized light imaging concept is proposed, namely quantitative polarization interference microscopy (QPIM). QPIM can be realized through designing a compact polarization-resolved interference microscopy system that captures interferograms bearing sample’s linear birefringence information. To extract the retardance and the orientation angle maps from a single-shot measurement, a mathematical model for QPIM is further developed. The QPIM system is validated by measuring a calibrated quarter-wave plate, whose fast-axis orientation angle and retardance are determined with great accuracies. The single-shot nature of QPIM further allows to measure the transient dynamics of birefringence changes in material containing anisotropic structures. This application is demonstrated by capturing transient retardance changes in a custom-designed parallel-aligned nematic liquid crystal-based device.

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Polarization resolved second harmonic microscopy

Cited by 26 publications

References 68 publications

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

Single‐Shot Optical Anisotropy Imaging with Quantitative Polarization Interference Microscopy

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