Second Harmonic Generation Light Sheet Microscopy (SHG-LSM): A new tool for label-free 3D bioimaging

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher J. Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Second Harmonic ◽  
Light Sheet ◽  
Label Free ◽  
Light Sheet Microscopy

scholarly journals Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon I. R. Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Biological Samples ◽  
3D Imaging ◽  
Second Harmonic ◽  
Three Dimensional ◽  
Light Sheet ◽  
Optical Methods ◽  
Label Free ◽  
Light Sheet Microscopy

AbstractLight sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

scholarly journals Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy

2021 ◽  
Author(s):  
Kamdin Mirsanaye ◽  
Leonardo Uribe Castaño ◽  
Yasmeen Kamaliddin ◽  
Ahmad Golaraei ◽  
Renaldas Augulis ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Tumor Tissue ◽  
Second Harmonic ◽  
Tissue Microarrays ◽  
Cancer Diagnostics ◽  
Label Free ◽  
Second Harmonic Generation Microscopy ◽  
Trained Classifier ◽  
Omission Rate

The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor tissue from normal with 94.2% accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.

scholarly journals Label-free imaging of membrane potentials by intramembrane field modulation, assessed by Second Harmonic Generation Microscopy

2021 ◽  
Author(s):  
Yovan de Coene ◽  
Stijn Jooken ◽  
Olivier Deschaume ◽  
Valérie Van Steenbergen ◽  
Pieter Vanden Berghe ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Second Harmonic ◽  
Hek293t Cell ◽  
Cellular Function ◽  
Membrane Voltage ◽  
Live Cells ◽  
Voltage Sensitivity ◽  
Label Free

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, we show that endogenous second harmonic generation (SHG) from live cells is highly sensitive to changes in membrane potential. Simultaneous electrophysiological control of a living (HEK293T) cell, through whole-cell voltage clamp reveals a linear relation between the SHG intensity and membrane voltage. Our results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. We further provide a conceptual framework that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the membrane potential without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

scholarly journals Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

2016 ◽  
Vol 09 (01) ◽  
pp. 1640003 ◽  
Author(s):  
Chunqiang Li ◽  
Riikka K. Pastila ◽  
Charles P. Lin
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Immune Cells ◽  
Ex Vivo ◽  
Second Harmonic ◽  
Label Free ◽  
Optical Signals ◽  
Two Photon

Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs), mast cells, vascular smooth muscle cells (SMCs). Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG) were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs) which participate in the degradation of collagen.

scholarly journals Depth resolved label-free multimodal optical imaging platform to study morpho-molecular composition of tissue

2019 ◽  
Vol 18 (5) ◽  
pp. 997-1008 ◽  
Author(s):  
Marco Andreana ◽  
Ryan Sentosa ◽  
Mikael T. Erkkilä ◽  
Wolfgang Drexler ◽  
Angelika Unterhuber
Keyword(s):  
Optical Coherence Tomography ◽  
Raman Scattering ◽  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Structural Information ◽  
Second Harmonic ◽  
Label Free ◽  
Two Photon ◽  
Non Invasive ◽  
Anti Stokes

The presented multi-modal platform combines optical coherence tomography, two-photon excited fluorescence, second harmonic generation and anti-Stokes Raman scattering to provide molecular and structural information of tissue in a fast and non-invasive manner.

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