- Department of Chemistry, Saint Mary’s University, Halifax, NS, Canada
The advancement of non-invasive quantitative optical diagnosis techniques such as polarization-sensitive second harmonic generation microscopy (PSHG) for diseases such as cancer presents opportunities for improving disease understanding and survival rates. Here, novel and developing techniques in PSHG microscopy applied for the differentiation of cancerous or diseased tissues are presented, including circular dichroism, modulation of laser linear polarization, detection of outgoing linear laser polarization, and double-Stokes Mueller. Typically, initial cancer diagnosis is performed by visual inspection of stained biopsy or surgical resection tissue sections under bright-field microscopy, however, early diagnosis is challenging due to variability in morphological interpretation of the tissues, and because cancer initiation regions can be small and easy to miss. Therefore, pathologists could benefit in identifying cancer on biopsy or surgical resection sections by using unbiased quantitative automated technologies with high spatial resolution and improved disease specificity that can check the entire slide pixel-by-pixel. Second harmonic generation microscopy offers the opportunity to measure ultrastructural alterations in collagenous scaffolds of organ tissues virtually background free on submicron-sized tissue regions. The approach is particularly interesting for cancer diagnosis applications, because during cancer initiation and progression, the collagen in the affected tissue extracellular matrix is often deregulated and becomes disorganized. This mini-review contains a thorough summary of PSHG techniques that have interrogated diseased tissues, and discusses their technical variations and successes in disease discrimination.
Introduction
Second harmonic generation (SHG) or frequency doubling is a nonlinear optical process which occurs efficiently in a microscope when two laser photons of wavelength λ interact with matter to produce light at λ/2 (Figure 1B). Only non-central symmetric materials at both molecular and macromolecular scales can produce SHG and therefore, microcrystalline structures are required for SHG. In animals, most SHG occurs from fibrous collagenous connective tissues or myosin in muscle tissues, both having a non-central microcrystalline structure. Since the SHG is emitted due to the presence and ultrastructure of the muscle or collagen interacting with the laser, no dyes or sample modification procedures are needed, and the signal can be interpreted as a direct indicator of structural sample changes.
FIGURE 1. Typical microscope schematics for forward- (A) and epi-detection (C) configurations for polarization-sensitive second harmonic generation (SHG) microscopy. The energy state diagram of SHG (B) along with the coordinate system, where Z-X is the image plane and the laser propagates along Y, for an arbitrarily oriented fiber (D) is shown. Abbreviations: SM-scanning mirrors, PSG-polarization state generator, EO-excitation microscope objective, CO-collection microscope objective, PSA-polarization state analyzer, F-filter, PMT-photomultiplier tube detector and DM-dichroic mirror. Example images obtained using PI-SHG (E), CD-SHG (F), DSMP-SHG (G) and PIPO-SHG (H). SHG intensity (E-i), fitted
SHG is energy conserving and consequently does not photobleach, differing significantly from non-parametric processes, such as fluorescence, where absorbed Stokes energy often leads to sample damage and photobleaching. For biological imaging, lasers with wavelength outside of the tissue absorption spectrum are typically chosen, therefore reduced heat is deposited into the sample allowing for long duration functional in vivo studies.
The SHG intensity and polarization can be written:
Historical Interpretation of the PSHG Parameters Pertaining to Collagen
Researchers, Freund et al. [3–6], pioneered the use of SHG to investigate the structure of collagenous biological tissue by utilizing different laser polarizations. They focused a Q-switched Nd:YAG laser onto rat tail tendon, collecting SHG in the forward direction, similar to Figure 1A, but without scan mirrors and at different scattering angles. They found an SHG peak at the 0° scattering angle, indicative of the macroscopic ordered polar structure of collagen, and found that χ(2) of tendon exhibited cylindrical symmetry, since rotation about the tendon axis did not appreciably change SHG parameters. They attributed the signal predominantly to C-N bonds in the amino acids, arguing these are the likely dominant polarizable, noncentrosymmetric and non-mobile candidates. They used a polarization state analyzer (PSA) consisting of a polarizer to directly measure
Noting the important SHG theoretical work of Dick [7], two groups, Plotnikov et al. [8] and Tiaho et al. [9], attributed
Applications of SHG Microscopy for Determining the Ultrastructure of Diseased Collagenous Tissues
Polarization-In SHG Microscopy
Pioneered in 1979, polarization-in SHG (PI-SHG) microscopy utilizes a polarization state generator (PSG) to rotate the laser linear polarization state. One method is to use a half-wave plate (HWP) in a motorized rotating mount, located before the excitation objective lens (Figures 1A,C). An SHG intensity image is recorded at each HWP angle, typically with 10–30 steps in the range 0°–90°. Since SHG is predominantly forward-directed, most epi-detected photons require backscattering, which is less efficient than forward-detection [11], and results in depolarization of a fraction of the SHG. Parameters
PI-SHG microscopy was used to distinguish normal and diseased tissue regions in several tissues (Table 1) with statistical significance, via the χ(2)zzz values of osteosarcoma, breast cancer and melanoma tissues [12], as well as the
TABLE 1. A summary of the PSHG microscopy techniques used for quantifying the differences in collagen structure in diseased tissue and the corresponding parameters measured with those techniques. The following abbreviation is used NR: Not reported.
To avoid delays due to nonlinear data fitting, a fast Fourier transform (FFT) algorithm has been developed to extract
Several groups have also investigated the excitation anisotropy
Polarization analysis of the outgoing SHG has also been incorporated using a PSA typically located after the collection objective in a forward-detection geometry (Figure 1A), or after a dichroic mirror that separates SHG in epi-detection (Figure 1C). While variable PSAs are reviewed in Double Stokes Mueller Polarimetric SHG (DSMP-SHG) Microscopy and Polarization-In, Polarization-Out SHG (PIPO-SHG) Microscopy sections, stationary PSAs are also used. In one implementation, SHG intensity through a polarizer parallel (
Interestingly, analysis of the PSHG data has also been achieved through Fourier projection of the PSHG image stacks onto two phasor plots referred to as microscopic multiparametric analysis by phasor projection of PSHG (µMAPPS). This technique has been used to compare the
PI-SHG microscopy was also used to image submucosa of esophageal squamous cell carcinoma (ESCC). The
Circular Dichroism Second Harmonic Generation Microscopy
In circular dichroism SHG (CD-SHG) microscopy, two SHG images are obtained using left-handed (IL) and right-handed (IR) circularly polarized laser light, with no PSA in the SHG detection path (Figures 1A,C). The CD-SHG intensity (ICD-SHG) is the normalized difference of the two quantities,
CD-SHG microscopy was applied to determine differences between normal and osteogenesis imperfecta skin tissues as well as idiopathic pulmonary fibrosis human lung tissue where variations in ICD-SHG were statistically significant [43, 47, 48]. Another study used CD-SHG microscopy to find differences between 4 ovarian tissue classifications. The mean ICD-SHG value for normal ovarian tissue was significantly higher than the other tissues [49].
Double Stokes Mueller Polarimetric SHG Microscopy
Double Stokes Mueller polarimetric SHG (DSMP-SHG) microscopy, introduced in 2015 [50, 51], is an alternative method to performing PSHG microscopy. DSMP-SHG microscopy aims to obtain all possible polarization information in the smallest amount of measurements. Stokes vectors are used to describe the laser polarization
DSMP-SHG has the advantage that 6 laboratory frame susceptibility components can be extracted from double Mueller matrix components:
Polarization-In, Polarization-Out SHG Microscopy
Polarization-in, polarization-out SHG (PIPO-SHG) uses a simplified PSA compared to DSMP-SHG as well as the same PSG as in the PI-SHG technique, and hence is typically implemented using a forward-detection geometry (Figure 1A). The PSA measures the linear polarization state of the SHG signal at different angles of the linear polarization of the laser. The simplest PSA consists of a linear polarizer in a motorized rotation mount. SHG images are typically captured with at least 8 PSG angles and 8 PSA angles, hence at least 64 images are recorded for the technique, resulting in ∼20 min acquisition times in comparison to PI-SHG which takes ∼1.5 min [53]. The additional dimensionality of the data is thought to produce higher accuracy fitting, although at the time of writing this manuscript no study has performed the comparison with the other techniques. Fitting of the SHG intensity versus PSA and PSG angles yields δ and
The PIPO-SHG measurements also allow the DOLP of the SHG signal to be obtained, where the SHG light is fully linearly polarized when
A PIPO-SHG microscopy study of pathology slides of lung samples from patients with non-small cell lung carcinoma revealed significant differences in the mean
PIPO-SHG microscopy was also applied to study a microarray slide containing 3 pathological human breast cancer types (Figure 1H) with the overexpression/absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2: triple +, double +, and triple -. The mean and median
Discussion
Automated digital histopathology using PSHG microscopy is a promising technology for diagnosis of disease in histopathological samples, however, implementation requires fast imaging and data analysis balanced with maintaining high measurement accuracy to obtain diagnosis based on the fewest amount of analyzed pixels. To reduce imaging time due to the different laser polarizations needed, liquid crystal or electro-optic modulators can be used [64–66], or beams with different polarizations can be interleaved. The clever use of fewer input polarizations could also be used, such as in Stokes-Mueller SHG [67] which uses four polarizations in a PI-SHG setup, or advanced polarimeters with multiple detectors can be used for simultaneous detection of the SHG polarization states [68]. In another technique, polarimetry analysis can also be performed using circularly polarized laser excitation, only requiring a single scan [53, 69]. Fourier techniques could be used for faster data analysis [16, 23, 24], while imaging rates can be increased using higher repetition rate pulsed lasers combined with faster scanners such as spinning mirrors, or by increasing the field of view using a wide-field imaging approach [70]. With these developments, it is reasonable that PSHG microscopy may be implemented in a modified hospital pathology slide scanner. Furthermore, since SHG requires no dyes, it can be implemented as an epi-detection setup in an endoscope for in vivo quantitative imaging as a biopsy tool [71–73].
It is evident that PSHG imaging can quantitatively differentiate certain diseased tissues based on their ultrastructure in pathological sample slides however, care must be given to PSHG image quality and the image analysis methods used. For instance, when assessing the quality of PSHG images, it has been found that using SHG intensity as a criterion is only suitable in specific instances [74]. Furthermore, while statistical discrimination based on many samples has been validated in different tissues, the efficacy of diagnosis in individual regions remains unclear and must be addressed. Improved differentiation could be obtained via additional implementation of complementary SHG intensity analysis techniques, such as texture analysis [56, 75–77], the Hough transform [78] and the structure tensor [79, 80]. These analysis techniques could be extended to PSHG
Author Contributions
The authors contributed to manuscript writing (RC, DT) and editing (RC, AJ, MH, DT).
Funding
The research was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants Program (RGPIN-2018-05444), Canada’s Research Support Fund, and Saint Mary’s University.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s Note
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Keywords: cancer, optical pathology, medical imaging, nonlinear optical polarimetry, nonlinear optical microscopy
Citation: Cisek R, Joseph A, Harvey M and Tokarz D (2021) Polarization-Sensitive Second Harmonic Generation Microscopy for Investigations of Diseased Collagenous Tissues. Front. Phys. 9:726996. doi: 10.3389/fphy.2021.726996
Received: 17 June 2021; Accepted: 10 August 2021;
Published: 30 August 2021.
Edited by:
Nirmal Mazumder, Manipal Academy of Higher Education, IndiaReviewed by:
Stefan G. Stanciu, Politehnica University of Bucharest, RomaniaMehdi Alizadeh, Vilnius University, Lithuania
Francisco Avila, Universidad de Zaragoza de, Spain
Copyright © 2021 Cisek, Joseph, Harvey and Tokarz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Danielle Tokarz, ZGFuaWVsbGUudG9rYXJ6QHNtdS5jYQ==