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Perspectives in Laser Scanning Microscopy
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Perspectives in Laser Scanning Microscopy

A special issue of Applied Sciences (ISSN 2076-3417). This special issue belongs to the section "Optics and Lasers".

Deadline for manuscript submissions: closed (30 September 2021) | Viewed by 19949

Special Issue Editor

Dr. Stefano Selci

E-Mail Website
Guest Editor
Institute for Photonics and Nanotechnologies, ARTOV C.N.R., Via del Fosso del Cavaliere 100, 00133 Roma, Italy
Interests: scanning electron microscopy; optics; optoelectronics; experimental physics; solid state physics; condensed matter physics; optical physics; photonics; thin films and nanotechnology; nanotechnology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Laser Scanning Microscopy (LSM) is a broad term that includes many specific techniques. For instance, Confocal Microscopy is a way to perform LSM, usually applied to biology and medicine investigations, but with uses also in materials engineering and many other applications. Multiphoton excitation fluorescence imaging, second and third harmonic generation imaging techniques, Raman based microscopies and many other variations of LSM systems are routinely sought for many purposes. One reason of such a widespread adoption of LSM is for sure the availability of new laser sources (e.g., supercontinuum lasers) that have widened their capacities both in the spectral realm as well as in the time domain, so that femtosecond sources can be obtained and used with the same easiness of standard CW sources available ten years ago. So far, so good. Actually, it seems that some further steps are needed to overcome some current limitations. One example for all.

It is well established that Reflectance Confocal Microscopy (RCM) can be a valuable asset for diagnosing malignant skin lesions in dermatology. For instance, the American Food and Drug Administration (FDA) has already validated the RCM inspection for skin cancer; however, there is no real substitution of standard histopathologic examination of derma samples. That it is partially due to some depth limitation in the RCM vision in the derma thickness, and partially due to the long and difficult training of highly specialized RCM operators, but, in the end, diagnoses are obtained through traditional histopathologists’ analyses. Why? Is this due to some technical insurmountable limitation, or to the lack of some intrinsic capability of the microscope’s understanding of the observed sample? Will some kind of Artificial Intelligence be needed, eventually?

In this Special Issue, researchers involved in LSM are kindly requested to expose their ideas regarding experimental or technical issues, new theoretical analysis for crunching data and images untangling, as well as needed, yet missing, LSM abilities, in order to outline the future of LSM.

Dr. Stefano Selci
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Applied Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Confocal laser scanning microscopy
  • Skin
  • Fluorescent visualization
  • Fluorescent probes
  • Fluorescence correlation spectroscopy
  • Fluorescence lifetime imaging microscopy
  • Nanoscale imaging
  • Scattering scanning near-field optical microscopy
  • Raman microscopy
  • Reflectance Microscopy
  • Laser sources
  • Statistical data analysis
  • Optical microscopy

Published Papers (6 papers)

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Research

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18 pages, 3626 KiB  
Article
Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline
by Jeemol James, Jonas Enger and Marica B. Ericson
Appl. Sci. 2021, 11(5), 2122; https://doi.org/10.3390/app11052122 - 27 Feb 2021
Viewed by 2175
Abstract
Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) [...] Read more.
Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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Review

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24 pages, 23486 KiB  
Review
Two-Photon Imaging to Unravel the Pathomechanisms Associated with Epileptic Seizures: A Review
by Luqman Khan, Rick van Lanen, Govert Hoogland, Olaf Schijns, Kim Rijkers, Dimitrios Kapsokalyvas, Marc van Zandvoort and Roel Haeren
Appl. Sci. 2021, 11(5), 2404; https://doi.org/10.3390/app11052404 - 8 Mar 2021
Cited by 4 | Viewed by 2663
Abstract
Despite extensive research, the exact pathomechanisms associated with epileptic seizure formation and propagation have not been elucidated completely. Two-photon imaging (2PI) is a fluorescence-based microscopy technique that, over the years, has been used to evaluate pathomechanisms associated with epileptic seizures and epilepsy. Here, [...] Read more.
Despite extensive research, the exact pathomechanisms associated with epileptic seizure formation and propagation have not been elucidated completely. Two-photon imaging (2PI) is a fluorescence-based microscopy technique that, over the years, has been used to evaluate pathomechanisms associated with epileptic seizures and epilepsy. Here, we review previous applications of 2PI in epilepsy. A systematic search was performed in multiple literature databases. We identified 38 publications that applied 2PI in epilepsy research. These studies described models of epileptic seizure propagation; anatomical changes and functional alterations of microglia, astrocytes, and neurites; and neurometabolic effects that accompany seizures. Moreover, various neurovascular alterations that accompany seizure onset and ictal events, such as blood vessel responses, have been visualized using 2PI. Lastly, imaging and quantitative analysis of oxidative stress and the aggregation of lipofuscin in the neurovasculature have been accomplished with 2PI. Cumulatively, these papers and their reported findings demonstrate that 2PI is an especially well-suited imaging technique in the domain of epilepsy research, and these studies have significantly improved our understanding of the disorder. The application of 2PI provides ample possibilities for future research, most interestingly on human brains, while also stretching beyond the field of epilepsy. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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16 pages, 1480 KiB  
Review
Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy
by Stefania Guida, Federica Arginelli, Francesca Farnetani, Silvana Ciardo, Laura Bertoni, Marco Manfredini, Nicola Zerbinati, Caterina Longo and Giovanni Pellacani
Appl. Sci. 2021, 11(5), 1979; https://doi.org/10.3390/app11051979 - 24 Feb 2021
Cited by 14 | Viewed by 3549
Abstract
Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis [...] Read more.
Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 μm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and non-melanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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18 pages, 3227 KiB  
Review
Review on Complete Mueller Matrix Optical Scanning Microscopy Imaging
by Aymeric Le Gratiet, Ali Mohebi, Fabio Callegari, Paolo Bianchini and Alberto Diaspro
Appl. Sci. 2021, 11(4), 1632; https://doi.org/10.3390/app11041632 - 11 Feb 2021
Cited by 23 | Viewed by 4119
Abstract
Optical scanning microscopy techniques based on the polarization control of the light have the capability of providing non invasive label-free contrast. By comparing the polarization states of the excitation light with its transformation after interaction with the sample, the full optical properties can [...] Read more.
Optical scanning microscopy techniques based on the polarization control of the light have the capability of providing non invasive label-free contrast. By comparing the polarization states of the excitation light with its transformation after interaction with the sample, the full optical properties can be summarized in a single 4×4 Mueller matrix. The main challenge of such a technique is to encode and decode the polarized light in an optimal way pixel-by-pixel and take into account the polarimetric artifacts from the optical devices composing the instrument in a rigorous calibration step. In this review, we describe the different approaches for implementing such a technique into an optical scanning microscope, that requires a high speed rate polarization control. Thus, we explore the recent advances in term of technology from the industrial to the medical applications. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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18 pages, 18002 KiB  
Review
Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
by Xue Wang, Xinchao Lu and Chengjun Huang
Appl. Sci. 2021, 11(3), 1002; https://doi.org/10.3390/app11031002 - 22 Jan 2021
Cited by 3 | Viewed by 3074
Abstract
By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser [...] Read more.
By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser scanning coherent Raman scattering microscopy, second harmonic generation microscopy and scanning localized surface plasmon microscopy are discussed with their fundamentals, features and recent progress. The applications of label-free biological imaging of these laser scanning microscopies are also introduced. Finally, the performance of the microscopies is compared and the limitation and perspectives are summarized. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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16 pages, 4415 KiB  
Review
Laser Scanning versus Wide-Field—Choosing the Appropriate Microscope in Life Sciences
by Herbert Schneckenburger and Verena Richter
Appl. Sci. 2021, 11(2), 733; https://doi.org/10.3390/app11020733 - 13 Jan 2021
Cited by 4 | Viewed by 3574
Abstract
Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size [...] Read more.
Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens. Full article
(This article belongs to the Special Issue Perspectives in Laser Scanning Microscopy)
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