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Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (15 December 2021) | Viewed by 25925

Special Issue Editor

Dr. Kiryl D. Piatkevich

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Guest Editor
McGovern Institute for Brain Research, Cambridge, MA, USA
Interests: fluorescent biosensors; optogenetics; expension microscop; neuroengineering
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

We would like to kindly invite you to contribute to the Special Issue "Advanced Molecular Probes for Structural and Functional Imaging of Biological Systems" of International Journal of Molecular Sciences (Impact Factor 4.556). The Special Issue will elucidate the engineering, validation, and application of cutting-edge molecular probes for functional and structural imaging, from cells to intact animals. Novel imaging modalities enabling greater utility of such probes, as well as new biological findings gleaned from experiments performed using these probes, are also within the scope of the Special Issue.

Research articles may present the design and development of novel fluorescent proteins and biosensors as well as biochemical, spectroscopic, structural, and biophysical studies that help elucidate the molecular mechanisms of fluorescence and the biosensing of such probes. Application articles should demonstrate the use of novel molecular probes in complex samples or present new imaging modalities that expand the application of currently available probes. Papers may also focus on addressing important biological questions using advanced molecular probes. In addition, the Special Issue will provide a forum for sharing and discussing perspectives on the development of the field in the next several years. Perspectives should present the authors’ opinions on important new directions in molecular probe design and application. Reviews can cover recent advances in the field of fluorescent proteins and biosensors, or novel or emerging class of fluorescent proteins or biosensors.

Dr. Kiryl D. Piatkevich
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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • Fluorescent proteins
  • Fluorescent biosensors
  • In vivo microscopy
  • Super-resolution microscopy
  • Protein engineering

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Published Papers (7 papers)

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Research

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23 pages, 3240 KiB  
Article
Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It
by Mikhail Drobizhev, Rosana S. Molina and Jacob Franklin
Int. J. Mol. Sci. 2022, 23(2), 770; https://doi.org/10.3390/ijms23020770 - 11 Jan 2022
Cited by 6 | Viewed by 3043
Abstract
Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 [...] Read more.
Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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29 pages, 5787 KiB  
Article
LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift
by Oksana M. Subach, Anna V. Vlaskina, Yuliya K. Agapova, Pavel V. Dorovatovskii, Alena Y. Nikolaeva, Olga I. Ivashkina, Vladimir O. Popov, Kiryl D. Piatkevich, Maria G. Khrenova, Tatiana A. Smirnova, Konstantin M. Boyko and Fedor V. Subach
Int. J. Mol. Sci. 2021, 22(23), 12887; https://doi.org/10.3390/ijms222312887 - 28 Nov 2021
Cited by 11 | Viewed by 5878
Abstract
Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety [...] Read more.
Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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13 pages, 2387 KiB  
Article
Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus
by Luisa Galla, Nicola Vajente, Diana Pendin, Paola Pizzo, Tullio Pozzan and Elisa Greotti
Int. J. Mol. Sci. 2021, 22(18), 9945; https://doi.org/10.3390/ijms22189945 - 14 Sep 2021
Cited by 3 | Viewed by 2528
Abstract
Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in [...] Read more.
Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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14 pages, 7849 KiB  
Article
Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping
by Thai Pham, Christopher D. Nazaroff, Joshua Labaer and Jia Guo
Int. J. Mol. Sci. 2021, 22(16), 8644; https://doi.org/10.3390/ijms22168644 - 11 Aug 2021
Cited by 3 | Viewed by 3346
Abstract
Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or [...] Read more.
Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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10 pages, 10345 KiB  
Article
Pyridazino-1,3a,6a-Triazapentalenes as Versatile Fluorescent Probes: Impact of Their Post-Functionalization and Application for Cellular Imaging
by Doina Sirbu, Nicolas Chopin, Ivana Martinić, Moussa Ndiaye, Svetlana V. Eliseeva, Marie-Aude Hiebel, Stéphane Petoud and Franck Suzenet
Int. J. Mol. Sci. 2021, 22(12), 6645; https://doi.org/10.3390/ijms22126645 - 21 Jun 2021
Cited by 7 | Viewed by 2597
Abstract
Pyridazino-1,3a,6a-triazapentalenes (PyTAP) are compact fused 6/5/5 tricyclic scaffolds which exhibit promising fluorescent properties. Chemically stable, they can be post-functionalized using standard Pd-catalyzed cross-coupling chemistry. Several original PyTAP bearing additional unsaturated substituents in positions 2 and 8 were synthetized and their spectroscopic properties analyzed. [...] Read more.
Pyridazino-1,3a,6a-triazapentalenes (PyTAP) are compact fused 6/5/5 tricyclic scaffolds which exhibit promising fluorescent properties. Chemically stable, they can be post-functionalized using standard Pd-catalyzed cross-coupling chemistry. Several original PyTAP bearing additional unsaturated substituents in positions 2 and 8 were synthetized and their spectroscopic properties analyzed. They have been successfully tested as fluorescent probes for cellular imaging. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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17 pages, 31843 KiB  
Article
FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus
by Oksana M. Subach, Natalia V. Barykina, Elizaveta S. Chefanova, Anna V. Vlaskina, Vladimir P. Sotskov, Olga I. Ivashkina, Konstantin V. Anokhin and Fedor V. Subach
Int. J. Mol. Sci. 2021, 22(1), 111; https://doi.org/10.3390/ijms22010111 - 24 Dec 2020
Cited by 8 | Viewed by 3554
Abstract
Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, [...] Read more.
Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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Review

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27 pages, 2825 KiB  
Review
Analysis of Factors Affecting 5-ALA Fluorescence Intensity in Visualizing Glial Tumor Cells—Literature Review
by Marek Mazurek, Dariusz Szczepanek, Anna Orzyłowska and Radosław Rola
Int. J. Mol. Sci. 2022, 23(2), 926; https://doi.org/10.3390/ijms23020926 - 15 Jan 2022
Cited by 19 | Viewed by 3304
Abstract
Glial tumors are one of the most common lesions of the central nervous system. Despite the implementation of appropriate treatment, the prognosis is not successful. As shown in the literature, maximal tumor resection is a key element in improving therapeutic outcome. One of [...] Read more.
Glial tumors are one of the most common lesions of the central nervous system. Despite the implementation of appropriate treatment, the prognosis is not successful. As shown in the literature, maximal tumor resection is a key element in improving therapeutic outcome. One of the methods to achieve it is the use of fluorescent intraoperative navigation with 5-aminolevulinic acid. Unfortunately, often the level of fluorescence emitted is not satisfactory, resulting in difficulties in the course of surgery. This article summarizes currently available knowledge regarding differences in the level of emitted fluorescence. It may depend on both the histological type and the genetic profile of the tumor, which is reflected in the activity and expression of enzymes involved in the intracellular metabolism of fluorescent dyes, such as PBGD, FECH, UROS, and ALAS. The transport of 5-aminolevulinic acid and its metabolites across the blood–brain barrier and cell membranes mediated by transporters, such as ABCB6 and ABCG2, is also important. Accompanying therapies, such as antiepileptic drugs or steroids, also have an impact on light emission by tumor cells. Accurate determination of the factors influencing the fluorescence of 5-aminolevulinic acid-treated cells may contribute to the improvement of fluorescence navigation in patients with highly malignant gliomas. Full article
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems 2.0)
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