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 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 Multiphoton Microscopy for Ophthalmic Imaging

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Emily A. Gibson ◽  
Omid Masihzadeh ◽  
Tim C. Lei ◽  
David A. Ammar ◽  
Malik Y. Kahook
Keyword(s):  
Harmonic Generation ◽  
Second Harmonic ◽  
Imaging Techniques ◽  
Multiphoton Microscopy ◽  
Tissue Imaging ◽  
Third Harmonic ◽  
Two Photon ◽  
Disease Mechanisms ◽  
Ophthalmic Imaging ◽  
Anti Stokes

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

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 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.

scholarly journals Label-Free Fried Starchy Matrix: Investigation by Harmonic Generation Microscopy

Sensors ◽  
2019 ◽  
Vol 19 (9) ◽  
pp. 2024
Author(s):  
Agathe Chouët ◽  
Sylvie Chevallier ◽  
Romain Fleurisson ◽  
Catherine Loisel ◽  
Laurence Dubreil
Keyword(s):  
Harmonic Generation ◽  
Second Harmonic ◽  
Nile Red ◽  
Food Products ◽  
Starch Granules ◽  
Label Free ◽  
Third Harmonic ◽  
Starchy Food ◽  
Innovative Methodology ◽  
Safranin O

An innovative methodology based on non-destructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix and topography analysis of food products. Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Fluorescence of starch and oil respectively stained with Safranin O and Nile red was observed from non-linear microscopy. By using sequential scanning and specific emission filters, it was possible to merge fluorescence and harmonic generation signals. Second harmonic generation (SHG) generated by starch granules was superposed with safranin fluorescence, whereas third harmonic generation (THG), not restricted to the superposition with Nile red fluorescent signal, was used to investigate the topography of the fried product. By these experiments, starch granule mapping and topography of the starchy fried product were obtained without any destructive preparation of the sample. This label-free approach using harmonic generation microscopy is a very promising methodology for microstructure investigation of a large panel of starchy food products.

High-power ultrafast thin-disk multipass amplifiers for efficient laser-based manufacturing

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Marwan Abdou Ahmed ◽  
Christoph Roecker ◽  
André Loescher ◽  
Florian Bienert ◽  
Daniel Holder ◽  
...  
Keyword(s):  
Harmonic Generation ◽  
State Of The Art ◽  
Second Harmonic ◽  
Average Power ◽  
Polycrystalline Diamond ◽  
Third Harmonic Generation ◽  
Thin Disk ◽  
Ultrafast Laser ◽  
Third Harmonic ◽  
Laser Systems

Abstract Thin-disk multipass amplifiers represent one of the most powerful approaches to scale the average and peak powers of ultrafast laser systems. The present paper presents the amplification of picosecond and femtosecond pulses to average powers exceeding 2 and 1 kW, respectively. Second-harmonic generation in lithium-triborate crystals with powers higher than 1.4 kW and 400 W at a wavelength of 515 nm with picosecond and femtosecond pulse durations, respectively, are also reported. Furthermore, third-harmonic generation was demonstrated with output powers exceeding 250 W at a wavelength of 343 nm. Finally, processing of silicon, metals, and polycrystalline diamond with fs pulses at an average power of 1 kW is presented to demonstrate removal rates that are improved by orders of magnitude as compared to state-of-the-art techniques.

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