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Technical Advances in Light Microscopy
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Technical Advances in Light Microscopy

A special issue of Methods and Protocols (ISSN 2409-9279).

Deadline for manuscript submissions: closed (15 May 2019) | Viewed by 36587

Special Issue Editor

Dr. Pablo Loza-Alvarez

E-Mail Website
Guest Editor
ICFO—Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Castelldefels, 08860 Barcelona, Spain
Interests: light sheet microscopy; super-resolution microscopy; multiphoton microscopy

Special Issue Information

Dear Colleagues,

The different imaging modalities, including confocal, multiphoton, light sheet, and super-resolution, etc., can be collectively called advanced light microscopy technologies. Each technique has specific advantages and drawbacks that appeal to different researchers, depending on their specific applications and needs. In fact, despite the latest efforts, biomedical researchers are still looking for novel ways for visualising organisms, tissues, cells and subcellular components. In each case, higher resolutions, larger fields of view and penetration depths, faster imaging speeds, higher signal to noise ratios and better sensitivities, etc. keep to be sought. On top of that, all of these are to be compatible with in vivo imaging. For example, multiscale imaging (from a few nm to mm) in neurobiology would allow observation of how far synapses are interconnected in the brain. Fast dynamic observations of processes in the whole sample could be used for a precise tracking of dividing cells during long development periods. Light-based manipulation of the samples, including optogenetics and photoactivation could enable the unravelling of protein transport during transduction of light into signals on retinas, and so on. To address these issues, a multi-disciplinary cutting-edge research effort is necessary. This should combine different scientific disciplines such as optics and photonics, engineering, medicine, chemistry and mathematics. In this Special Issue on “Technical Advances in Light Microscopy”, we welcome original research and review articles dealing with novel technical implementations in light microscopy used to tackle important bio-medical questions. Approaches include the implementation of multimodal microscopy imaging, the use of novel scientific instrumentation, the use of novel fluorescent markers, the development of modelling and algorithms for image quantification and analysis and so on.

Dr. Pablo Loza-Alvarez
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. Methods and Protocols 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 1800 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

  • Light sheet microscopy (SPIM, DSLM, DiSPIM, etc.)
  • Nonlinear microscopy (TPEF, SHG, THG, etc.)
  • Super resolution microscopy (STED, STORM, PALM, structured illumination etc.)
  • Lattice light sheet microscopy
  • Optogenetics
  • Photoexcitation and photoablation
  • Optical tweezers
  • Adaptive Optics
  • Wavefront shaping
  • Beam engineering
  • Nonlinear and specialised dyes
  • Deconvolution algorithms
  • Inverse problem solving
  • Hyperspectral imaging
  • Machine learning

Published Papers (7 papers)

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Research

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13 pages, 2969 KiB  
Article
Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate
by Viktoras Lisicovas, Bala Murali Krishna Mariserla, Chakradhar Sahoo, Reuben T. Harding, Michael K. L. Man, E Laine Wong, Julien Madéo and Keshav M. Dani
Methods Protoc. 2019, 2(3), 65; https://doi.org/10.3390/mps2030065 - 28 Jul 2019
Viewed by 3870
Abstract
Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse [...] Read more.
Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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13 pages, 2109 KiB  
Article
Highly Sensitive Shack–Hartmann Wavefront Sensor: Application to Non-Transparent Tissue Mimic Imaging with Adaptive Light-Sheet Fluorescence Microscopy
by Javier Morgado Brajones, Gregory Clouvel, Guillaume Dovillaire, Xavier Levecq and Corinne Lorenzo
Methods Protoc. 2019, 2(3), 59; https://doi.org/10.3390/mps2030059 - 11 Jul 2019
Cited by 5 | Viewed by 3767
Abstract
High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which [...] Read more.
High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which useful imaging can be done. Adaptive optics (AOs) is a method capable of measuring and correcting aberrations in different kinds of fluorescence microscopes, thereby improving the performance of the optical system. We have incorporated a wavefront sensor adaptive optics scheme to SPIM (WAOSPIM) to correct aberrations induced by optically-thick samples, such as multi-cellular tumor spheroids (MCTS). Two-photon fluorescence provides us with a tool to produce a weak non-linear guide star (NGS) in any region of the field of view. The faintness of NGS; however, led us to develop a high-sensitivity Shack–Hartmann wavefront sensor (SHWS). This paper describes this newly developed SHWS and shows the correction capabilities of WAOSPIM using NGS in thick, inhomogeneous samples like MCTS. We report improvements of up to 79% for spatial frequencies corresponding to cellular and subcellular size features. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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14 pages, 5075 KiB  
Article
Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples
by Marco Marchetti, Enrico Baria, Riccardo Cicchi and Francesco Saverio Pavone
Methods Protoc. 2019, 2(2), 51; https://doi.org/10.3390/mps2020051 - 20 Jun 2019
Cited by 16 | Viewed by 4900
Abstract
Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality [...] Read more.
Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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Review

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12 pages, 4418 KiB  
Review
The DIVER Microscope for Imaging in Scattering Media
by Alexander Dvornikov, Leonel Malacrida and Enrico Gratton
Methods Protoc. 2019, 2(2), 53; https://doi.org/10.3390/mps2020053 - 21 Jun 2019
Cited by 21 | Viewed by 5507
Abstract
We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive [...] Read more.
We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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Other

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12 pages, 2206 KiB  
Letter
Enhanced Light Sheet Elastic Scattering Microscopy by Using a Supercontinuum Laser
by Diego Di Battista, David Merino, Giannis Zacharakis, Pablo Loza-Alvarez and Omar E. Olarte
Methods Protoc. 2019, 2(3), 57; https://doi.org/10.3390/mps2030057 - 5 Jul 2019
Cited by 10 | Viewed by 5170
Abstract
Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of [...] Read more.
Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of contrast available in every light sheet microscope. Interestingly, elastic scattering can potentially disclose valuable information from the structure and composition of the sample at different spatial scales. However, when coherent scattered light is detected with a camera sensor, a speckled intensity is generated on top of the native imaged features, compromising their visibility. In this work, we propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images: (i) polarization filtering; (ii) reducing the temporal coherence of the excitation laser light; and, (iii) reducing the spatial coherence of the light sheet. Finally, we show how these strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing, fluorescent labelling. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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15 pages, 7403 KiB  
Protocol
Protocol for the Design and Assembly of a Light Sheet Light Field Microscope
by Jorge Madrid-Wolff and Manu Forero-Shelton
Methods Protoc. 2019, 2(3), 56; https://doi.org/10.3390/mps2030056 - 4 Jul 2019
Cited by 6 | Viewed by 9434
Abstract
Light field microscopy is a recent development that makes it possible to obtain images of volumes with a single camera exposure, enabling studies of fast processes such as neural activity in zebrafish brains at high temporal resolution, at the expense of spatial resolution. [...] Read more.
Light field microscopy is a recent development that makes it possible to obtain images of volumes with a single camera exposure, enabling studies of fast processes such as neural activity in zebrafish brains at high temporal resolution, at the expense of spatial resolution. Light sheet microscopy is also a recent method that reduces illumination intensity while increasing the signal-to-noise ratio with respect to confocal microscopes. While faster and gentler to samples than confocals for a similar resolution, light sheet microscopy is still slower than light field microscopy since it must collect volume slices sequentially. Nonetheless, the combination of the two methods, i.e., light field microscopes that have light sheet illumination, can help to improve the signal-to-noise ratio of light field microscopes and potentially improve their resolution. Building these microscopes requires much expertise, and the resources for doing so are limited. Here, we present a protocol to build a light field microscope with light sheet illumination. This protocol is also useful to build a light sheet microscope. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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14 pages, 5801 KiB  
Protocol
Comparison of Different Polarization Sensitive Second Harmonic Generation Imaging Techniques
by Mehdi Alizadeh, Masood Ghotbi, Pablo Loza-Alvarez and David Merino
Methods Protoc. 2019, 2(2), 49; https://doi.org/10.3390/mps2020049 - 7 Jun 2019
Cited by 7 | Viewed by 3229
Abstract
Polarization sensitive second harmonic generation (pSHG) microscopy is an imaging technique able to provide, in a non-invasive manner, information related to the molecular structure of second harmonic generation (SHG) active structures, many of which are commonly found in biological tissue. The process of [...] Read more.
Polarization sensitive second harmonic generation (pSHG) microscopy is an imaging technique able to provide, in a non-invasive manner, information related to the molecular structure of second harmonic generation (SHG) active structures, many of which are commonly found in biological tissue. The process of acquiring this information by means of pSHG microscopy requires a scan of the sample using different polarizations of the excitation beam. This process can take considerable time in comparison with the dynamics of in vivo processes. Fortunately, single scan polarization sensitive second harmonic generation (SS-pSHG) microscopy has also been reported, and is able to generate the same information at a faster speed compared to pSHG. In this paper, the orientation of second harmonic active supramolecular assemblies in starch granules is obtained on by means of pSHG and SS-pSHG. These results are compared in the forward and backward directions, showing a good agreement in both techniques. This paper shows for the first time, to the best of the authors’ knowledge, data acquired using both techniques over the exact same sample and image plane, so that they can be compared pixel-to-pixel. Full article
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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