scholarly journals Label-free multiphoton imaging allows brain tumor recognition based on texture analysis—a study of 382 tumor patients

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Ortrud Uckermann ◽  
Roberta Galli ◽  
Georg Mark ◽  
Matthias Meinhardt ◽  
Edmund Koch ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Recurrent Glioblastoma ◽  
Image Resolution ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Linear Discriminant ◽  
High Grade Astrocytoma ◽  
Texture Parameters

Abstract Background Label-free multiphoton microscopy has been suggested for intraoperative recognition and delineation of brain tumors. For any future clinical application, appropriate approaches for image acquisition and analysis have to be developed. Moreover, an evaluation of the reliability of the approach, taking into account inter- and intrapatient variability, is needed. Methods Coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation were acquired on cryosections of brain tumors of 382 patients and 28 human nontumor brain samples. Texture parameters of those images were calculated and used as input for linear discriminant analysis. Results The combined analysis of texture parameters of the CARS and TPEF signal proved to be most suited for the discrimination of nontumor brain versus brain tumors (low- and high-grade astrocytoma, oligodendroglioma, glioblastoma, recurrent glioblastoma, brain metastases of lung, colon, renal, and breast cancer and of malignant melanoma) leading to a correct rate of 96% (sensitivity: 96%, specificity: 100%). To approximate the clinical setting, the results were validated on 42 fresh, unfixed tumor biopsies. 82% of the tumors and, most important, all of the nontumor samples were correctly recognized. An image resolution of 1 µm was sufficient to distinguish brain tumors and nontumor brain. Moreover, the vast majority of single fields of view of each patient’s sample were correctly classified with high probabilities, which is important for clinical translation. Conclusion Label-free multiphoton imaging might allow fast and accurate intraoperative delineation of primary and secondary brain tumors in combination with endoscopic systems.

MULTIPHOTON MICROSCOPY: A NEW APPROACH, IN PHYSIOLOGICAL STUDIES AND PATHOLOGICAL DIAGNOSIS FOR OPHTHALMOLOGY

2009 ◽  
Vol 02 (01) ◽  
pp. 45-60 ◽  
Author(s):  
CHIU-MEI HSUEH ◽  
WEN LO ◽  
SUNG-JAN LIN ◽  
TSUNG-JEN WANG ◽  
FUNG-RUNG HU ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Healing Process ◽  
Multiphoton Microscopy ◽  
In Vivo Studies ◽  
Wound Healing Process ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Morphological Alterations ◽  
Corneal Scar

Multiphoton microscopy (MPM), with the advantages of improved penetration depth, decreased photo-damage, and optical sectioning capability, has become an indispensable tool for biomedical imaging. The combination of multiphoton fluorescence (MF) and second-harmonic generation (SHG) microscopy is particularly effective in imaging tissue structures of the ocular surface. This work is intended to be a review of advances that MPM has made in ophthalmic imaging. The MPM not only can be used for the label-free imaging of ocular structures, it can also be applied for investigating the morphological alterations in corneal pathologies, such as keratoconus, infected keratitis, and corneal scar. Furthermore, the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty (CK) can also be studied with MPM. Finally, qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation. With additional development, multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.

scholarly journals Label-Free Multiphoton Microscopy: Much More Than Fancy Images

2021 ◽  
Vol 22 (5) ◽  
pp. 2657
Author(s):  
Giulia Borile ◽  
Deborah Sandrin ◽  
Andrea Filippi ◽  
Kurt I. Anderson ◽  
Filippo Romanato
Keyword(s):  
Harmonic Generation ◽  
Biomedical Research ◽  
Quantitative Methods ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Basic Research ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Third Harmonic ◽  
Research Fields

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

scholarly journals Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

2018 ◽  
Vol 315 (6) ◽  
pp. F1613-F1625 ◽  
Author(s):  
Milica Bugarski ◽  
Joana Raquel Martins ◽  
Dominik Haenni ◽  
Andrew M. Hall
Keyword(s):  
Mitochondrial Function ◽  
Redox State ◽  
Kidney Diseases ◽  
Multiphoton Microscopy ◽  
Kidney Tissue ◽  
Kidney Cortex ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Imaging Approach ◽  
Lactate And Pyruvate

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

scholarly journals Quantitative evaluation of the human vocal fold extracellular matrix using multiphoton microscopy and optical coherence tomography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fouzi Benboujja ◽  
Christopher Hartnick
Keyword(s):  
Extracellular Matrix ◽  
Optical Coherence Tomography ◽  
Optical Imaging ◽  
Vocal Fold ◽  
Second Harmonic ◽  
Layer Structure ◽  
Multiphoton Microscopy ◽  
Optical Coherence ◽  
Multiphoton Imaging ◽  
Laryngeal Diseases

AbstractIdentifying distinct normal extracellular matrix (ECM) features from pathology is of the upmost clinical importance for laryngeal diagnostics and therapy. Despite remarkable histological contributions, our understanding of the vocal fold (VF) physiology remains murky. The emerging field of non-invasive 3D optical imaging may be well-suited to unravel the complexity of the VF microanatomy. This study focused on characterizing the entire VF ECM in length and depth with optical imaging. A quantitative morphometric evaluation of the human vocal fold lamina propria using two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and optical coherence tomography (OCT) was investigated. Fibrillar morphological features, such as fiber diameter, orientation, anisotropy, waviness and second-order statistics features were evaluated and compared according to their spatial distribution. The evidence acquired in this study suggests that the VF ECM is not a strict discrete three-layer structure as traditionally described but instead a continuous assembly of different fibrillar arrangement anchored by predominant collagen transitions zones. We demonstrated that the ECM composition is distinct and markedly thinned in the anterior one-third of itself, which may play a role in the development of some laryngeal diseases. We further examined and extracted the relationship between OCT and multiphoton imaging, promoting correspondences that could lead to accurate 3D mapping of the VF architecture in real-time during phonosurgeries. As miniaturization of optical probes is consistently improving, a clinical translation of OCT imaging and multiphoton imaging, with valuable qualitative and quantitative features, may have significant implications for treating voice disorders.

scholarly journals Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dimitrios Kapsokalyvas ◽  
Rodrigo Rosas ◽  
Rob W. A. Janssen ◽  
Jo M. Vanoevelen ◽  
Miranda Nabben ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Light Sheet ◽  
Three Dimensions ◽  
Fluorescence Signal ◽  
Label Free ◽  
Microscopy Imaging ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy ◽  
3D Image Reconstruction

AbstractImaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy—imaging the sample from different angles followed by 3D image reconstruction—was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

scholarly journals Nerve regeneration in the cephalopod mollusc Octopus vulgaris: label-free multiphoton microscopy as a tool for investigation

2018 ◽  
Vol 15 (141) ◽  
pp. 20170889 ◽  
Author(s):  
Pamela Imperadore ◽  
Ortrud Uckermann ◽  
Roberta Galli ◽  
Gerald Steiner ◽  
Matthias Kirsch ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Scar Tissue ◽  
Octopus Vulgaris ◽  
Chemical Information ◽  
Label Free ◽  
Molecular Machinery ◽  
Staining Methods ◽  
Species Specific ◽  
Anti Stokes

Octopus and cephalopods are able to regenerate injured tissues. Recent advancements in the study of regeneration in cephalopods appear promising encompassing different approaches helping to decipher cellular and molecular machinery involved in the process. However, lack of specific markers to investigate degenerative/regenerative phenomena and inflammatory events occurring after damage is limiting these studies. Label-free multiphoton microscopy is applied for the first time to the transected pallial nerve of Octopus vulgaris . Various optical contrast methods including coherent anti-Stokes Raman scattering (CARS), endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) have been used. We detected cells and structures often not revealed with classical staining methods. CARS highlighted the involvement of haemocytes in building up scar tissue; CARS and TPEF facilitated the identification of degenerating fibres; SHG allowed visualization of fibrillary collagen, revealing the formation of a connective tissue bridge between the nerve stumps, likely involved in axon guidance. Using label-free multiphoton microscopy, we studied the regenerative events in octopus without using any other labelling techniques. These imaging methods provided extremely helpful morpho-chemical information to describe regeneration events. The techniques applied here are species-specific independent and should facilitate the comparison among various animal species.

scholarly journals Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

2021 ◽  
Author(s):  
Lars Rishøj ◽  
Iván Coto Hernández ◽  
Siddharth Ramachandran ◽  
Nate Jowett
Keyword(s):  
Peripheral Nerve ◽  
Harmonic Generation ◽  
Second Harmonic ◽  
Yellow Fluorescent Protein ◽  
Multiphoton Microscopy ◽  
Third Harmonic Generation ◽  
Second Harmonic Generation Signal ◽  
Label Free ◽  
Third Harmonic ◽  
Third Harmonic Generation Microscopy

AbstractConventional histomorphometry of peripheral nerve entails lengthy chemical processing, ultrathin sectioning in resin, and imaging by light or electron microscopy. Multiphoton microscopy techniques exist enabling label-free and in vivo imaging of histological samples. Third-harmonic-generation microscopy has recently been demonstrated effective for imaging the myelin sheath of peripheral nerve axons in animal models. Herein, we characterize use of second and third harmonic generation microscopy for label-free imaging of murine and human peripheral nerve via a novel multicolor multiphoton microscope based on a single excitation wavelength at 1300 nm. Second harmonic generation signal from collagen centered about 650 nm delineates neural connective tissue, while third harmonic general signal centered about 433 nm delineates myelin and other lipids. In transgenic mice expressing yellow fluorescent protein linked to the thy1 promoter, three-photon-excitation with emission peak at 527 nm delineates axoplasm. We compare label-free multiphoton imaging of murine and human peripheral nerve against conventional chemical stains and discuss clinical implications of this approach in guiding intraoperative decision making in nerve transfer procedures.

scholarly journals Diagnosing Hirschsprung disease by detecting intestinal ganglion cells using label-free hyperspectral microscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marcos A. Soares de Oliveira ◽  
Laura Galganski ◽  
Sarah Stokes ◽  
Che -Wei Chang ◽  
Christopher D. Pivetti ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Second Harmonic ◽  
Hirschsprung Disease ◽  
Principal Component ◽  
Distal Colon ◽  
Support Vector ◽  
Label Free ◽  
Accurate Identification ◽  
Linear Discriminant ◽  
Multiple Biopsies

AbstractHirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

scholarly journals NI-78 * LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

2014 ◽  
Vol 16 (suppl 5) ◽  
pp. v155-v156
Author(s):  
O. Uckermann ◽  
R. Galli ◽  
K. Geiger ◽  
E. Koch ◽  
G. Schackert ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Multiphoton Microscopy ◽  
Label Free

scholarly journals Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching

2020 ◽  
Vol 10 (20) ◽  
pp. 7173
Author(s):  
Yifan Qin ◽  
Deying Chen ◽  
Yuanqin Xia
Keyword(s):  
Real Time ◽  
Imaging System ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Cost Effective ◽  
Axial Direction ◽  
Three Dimensions ◽  
Mouse Lung ◽  
Label Free

Multiphoton microscopy is a well-established technique for biomedical applications, but real-time multidepth multimodal multiphoton microscopy using non-imaging detection has barely been discussed. We demonstrate a novel label-free imaging system capable of generating multimodal multiphoton signals at different focal planes simultaneously. Two spatially overlapped and temporally interlaced beams are obtained by applying cost-effective electro-optic modulator (EOM)-based fast-switching light paths. The switching beams have different divergence properties, enabling imaging at different depths into samples. The EOM is synchronized to the pixel clock from the microscope, achieving pixel-to-pixel focus-switching. The capability of the imaging system is demonstrated by performing real-time multidepth two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging of freshly excised mouse lung lobes. TPF and SHG images are acquired at two wavelength ranges. One is between 415 and 455 nm, and the other is between 495 and 635 nm. The microenvironment of pulmonary alveoli is depicted by the distributions of both elastin fibers visualized by TPF and collagen fibers illustrated by SHG. Macrophages residing inside apparent alveolar lumens are also identified by TPF, which shows that the imaging system is capable of localizing biological objects in three dimensions and has the potential of monitoring in vivo cellular dynamics in the axial direction.

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