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Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology

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

Deadline for manuscript submissions: closed (31 March 2022) | Viewed by 35552

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Special Issue Editor

Dr. Chan-Gi Pack

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Guest Editor

1. Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
2. Convergence Medicine Research Center, Asan Medical Center, Seoul 05505, Republic of Korea
Interests: nanoparticle/drug-cell interaction; cellular drug delivery; protein dynamics and interaction; cellular biophysics; fluorescence correlation spectroscopy; optical diffraction tomography; immuno-gold EM; CLEM

Special Issue Information

Dear Colleagues,

Various imaging techniques have long provided researchers with specific and quantitative information about molecules and cells through a wide variety of imaging modalities, from conventional microscopic and nonmicroscopic techniques to advanced and super-resolution level techniques. Today, the development and improvement of such imaging techniques allow for the establishment of next-generation biological and biomedical techniques. Moreover, the synthesis of established imaging techniques combined with analytical techniques provides new insight into various fields of life science. This Special Issue aims to serve as a guideline of various imaging and analytical methods of cells and provide a wide range of their applications.

Research on applications is expected to target specific biological and biophysical questions, which include quantification and functional analyses of the molecular and cellular states in fixed and live cells. Moreover, the content of the Special issue covers reports uncovering the molecular mechanisms underpinning biological processes encompassing various disciplines of biology and medicine.

We would like to welcome authors to submit research and review articles for the Special Issue “Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology”.

Dr. Chan-Gi Pack
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.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. 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

super-resolution microscopy
multiphoton microscopy
fluorescence correlation methods
fluorescence lifetime imaging
label-free microscopic imaging
electron microscopy
single-molecule imaging
fluorescent probe
cellular organelles
molecular function
molecular interaction
diffusion dynamics
disease- and drug-related subcellular phenomena

Published Papers (13 papers)

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Research

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14 pages, 2941 KiB

Open AccessArticle

Iron Accumulation and Changes in Cellular Organelles in WDR45 Mutant Fibroblasts

by Hye Eun Lee, Min Kyo Jung, Seul Gi Noh, Hye Bin Choi, Se hyun Chae, Jae Hyeok Lee and Ji Young Mun

Int. J. Mol. Sci. 2021, 22(21), 11650; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111650 - 28 Oct 2021

Cited by 8 | Viewed by 2787

Abstract

Iron overload in the brain, defined as excess stores of iron, is known to be associated with neurological disorders. In neurodegeneration accompanied by brain iron accumulation, we reported a specific point mutation, c.974-1G>A in WD Repeat Domain 45 (WDR45), showing iron accumulation in the brain, and autophagy defects in the fibroblasts. In this study, we investigated whether fibroblasts with mutated WDR45 accumulated iron, and other effects on cellular organelles. We first identified the main location of iron accumulation in the mutant fibroblasts and then investigated the effects of this accumulation on cellular organelles, including lipid droplets, mitochondria and lysosomes. Ultrastructure analysis using transmission electron microscopy (TEM) and confocal microscopy showed structural changes in the organelles. Increased numbers of lipid droplets, fragmented mitochondria and increased numbers of lysosomal vesicles with functional disorder due to WDR45 deficiency were observed. Based on correlative light and electron microscopy (CLEM) findings, most of the iron accumulation was noted in the lysosomal vesicles. These changes were associated with defects in autophagy and defective protein and organelle turnover. Gene expression profiling analysis also showed remarkable changes in lipid metabolism, mitochondrial function, and autophagy-related genes. These data suggested that functional and structural changes resulted in impaired lipid metabolism, mitochondrial disorder, and unbalanced autophagy fluxes, caused by iron overload. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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11 pages, 7146 KiB

Open AccessArticle

Insight into the Characteristics of Novel Desmin-Immunopositive Perivascular Cells of the Anterior Pituitary Gland Using Transmission and Focused Ion Beam Scanning Electron Microscopy

by Depicha Jindatip, Rebecca Wan-Yan Poh and Ken Fujiwara

Int. J. Mol. Sci. 2021, 22(16), 8630; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168630 - 11 Aug 2021

Cited by 3 | Viewed by 2166

Abstract

Recently, another new cell type was found in the perivascular space called a novel desmin-immunopositive perivascular (DIP) cell. However, the differences between this novel cell type and other nonhormone-producing cells have not been clarified. Therefore, we introduced several microscopic techniques to gain insight into the morphological characteristics of this novel DIP cell. We succeeded in identifying novel DIP cells under light microscopy using desmin immunocryosection, combining resin embedding blocks and immunoelectron microscopy. In conventional transmission electron microscopy, folliculostellate cells, capsular fibroblasts, macrophages, and pericytes presented a flat cisternae of rough endoplasmic reticulum, whereas those of novel DIP cells had a dilated pattern. The number of novel DIP cells was greatest in the intact rats, though nearly disappeared under prolactinoma conditions. Additionally, focused ion beam scanning electron microscopy showed that these novel DIP cells had multidirectional processes and some processes reached the capillary, but these processes did not tightly wrap the vessel, as is the case with pericytes. Interestingly, we found that the rough endoplasmic reticulum was globular and dispersed throughout the cytoplasmic processes after three-dimensional reconstruction. This study clearly confirms that novel DIP cells are a new cell type in the rat anterior pituitary gland, with unique characteristics. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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17 pages, 38046 KiB

Open AccessArticle

Comparative Analysis of Single-Molecule Dynamics of TRPV1 and TRPV4 Channels in Living Cells

by Yutaro Kuwashima, Masataka Yanagawa, Mitsuhiro Abe, Michio Hiroshima, Masahiro Ueda, Makoto Arita and Yasushi Sako

Int. J. Mol. Sci. 2021, 22(16), 8473; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168473 - 06 Aug 2021

Cited by 4 | Viewed by 2688

Abstract

TRPV1 and TRPV4, members of the transient receptor potential vanilloid family, are multimodal ion channels activated by various stimuli, including temperature and chemicals. It has been demonstrated that TRPV channels function as tetramers; however, the dynamics of the diffusion, oligomerization, and endocytosis of these channels in living cells are unclear. Here we undertook single-molecule time-lapse imaging of TRPV1 and TRPV4 in HEK 293 cells. Differences were observed between TRPV1 and TRPV4 before and after agonist stimulation. In the resting state, TRPV4 was more likely to form higher-order oligomers within immobile membrane domains than TRPV1. TRPV1 became immobile after capsaicin stimulation, followed by its gradual endocytosis. In contrast, TRPV4 was rapidly internalized upon stimulation with GSK1016790A. The selective loss of immobile higher-order oligomers from the cell surface through endocytosis increased the proportion of the fast-diffusing state for both subtypes. With the increase in the fast state, the association rate constants of TRPV1 and TRPV4 increased, regenerating the higher-order oligomers. Our results provide a possible mechanism for the different rates of endocytosis of TRPV1 and TRPV4 based on the spatial organization of the higher-order structures of the two TRPV channels. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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15 pages, 4835 KiB

Open AccessArticle

Image-Based Method to Quantify Decellularization of Tissue Sections

by Maria Narciso, Jorge Otero, Daniel Navajas, Ramon Farré, Isaac Almendros and Núria Gavara

Int. J. Mol. Sci. 2021, 22(16), 8399; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168399 - 05 Aug 2021

Cited by 7 | Viewed by 2921

Abstract

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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15 pages, 3119 KiB

Open AccessArticle

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif

by Chan-Gi Pack, Keehoon Jung, Bjorn Paulson and Jun Ki Kim

Int. J. Mol. Sci. 2021, 22(15), 8293; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22158293 - 02 Aug 2021

Viewed by 2381

Abstract

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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12 pages, 2950 KiB

Open AccessArticle

Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy

by Markéta Dalecká, Ján Sabó, Lenka Backová, Daniel Rösel, Jan Brábek, Aleš Benda and Ondřej Tolde

Int. J. Mol. Sci. 2021, 22(15), 7805; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22157805 - 21 Jul 2021

Cited by 5 | Viewed by 2235

Abstract

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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13 pages, 4015 KiB

Open AccessArticle

Adhesion of Triple-Negative Breast Cancer Cells under Fluorescent and Soft X-ray Contact Microscopy

by Paulina Natalia Osuchowska, Przemysław Wachulak, Wiktoria Kasprzycka, Agata Nowak-Stępniowska, Maciej Wakuła, Andrzej Bartnik, Henryk Fiedorowicz and Elżbieta Anna Trafny

Int. J. Mol. Sci. 2021, 22(14), 7279; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22147279 - 06 Jul 2021

Cited by 2 | Viewed by 2547

Abstract

Understanding cancer cell adhesion could help to diminish tumor progression and metastasis. Adhesion mechanisms are currently the main therapeutic target of TNBC-resistant cells. This work shows the distribution and size of adhesive complexes determined with a common fluorescence microscopy technique and soft X-ray contact microscopy (SXCM). The results presented here demonstrate the potential of applying SXCM for imaging cell protrusions with high resolution when the cells are still alive in a physiological buffer. The possibility to observe the internal components of cells at a pristine and hydrated state with nanometer resolution distinguishes SXCM from the other more commonly used techniques for cell imaging. Thus, SXCM can be a promising technique for investigating the adhesion and organization of the actin cytoskeleton in cancer cells. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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17 pages, 46992 KiB

Open AccessArticle

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis

by Anna Ligasová and Karel Koberna

Int. J. Mol. Sci. 2021, 22(13), 7098; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22137098 - 01 Jul 2021

Cited by 1 | Viewed by 2933

Abstract

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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13 pages, 2530 KiB

Open AccessArticle

Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence Correlation Methods

by Bjorn Paulson, Yeonhee Shin, Akimitsu Okamoto, Yeon-Mok Oh, Jun Ki Kim and Chan-Gi Pack

Int. J. Mol. Sci. 2021, 22(12), 6433; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22126433 - 16 Jun 2021

Viewed by 2167

Abstract

Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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10 pages, 4056 KiB

Open AccessArticle

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy

by Hong-Lim Kim, Tae-Ryong Riew, Jieun Park, Youngchun Lee and In-Beom Kim

Int. J. Mol. Sci. 2021, 22(8), 4273; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22084273 - 20 Apr 2021

Cited by 5 | Viewed by 2903 | Correction

Abstract

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu’s method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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18 pages, 4884 KiB

Open AccessArticle

Vibrational Spectroscopic Investigation of Blood Plasma and Serum by Drop Coating Deposition for Clinical Application

by Jing Huang, Nairveen Ali, Elsie Quansah, Shuxia Guo, Michel Noutsias, Tobias Meyer-Zedler, Thomas Bocklitz, Jürgen Popp, Ute Neugebauer and Anuradha Ramoji

Int. J. Mol. Sci. 2021, 22(4), 2191; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22042191 - 22 Feb 2021

Cited by 5 | Viewed by 2623

Abstract

In recent decades, vibrational spectroscopic methods such as Raman and FT-IR spectroscopy are widely applied to investigate plasma and serum samples. These methods are combined with drop coating deposition techniques to pre-concentrate the biomolecules in the dried droplet to improve the detected vibrational signal. However, most often encountered challenge is the inhomogeneous redistribution of biomolecules due to the coffee-ring effect. In this study, the variation in biomolecule distribution within the dried-sample droplet has been investigated using Raman and FT-IR spectroscopy and fluorescence lifetime imaging method. The plasma-sample from healthy donors were investigated to show the spectral differences between the inner and outer-ring region of the dried-sample droplet. Further, the preferred location of deposition of the most abundant protein albumin in the blood during the drying process of the plasma has been illustrated by using deuterated albumin. Subsequently, two patients with different cardiac-related diseases were investigated exemplarily to illustrate the variation in the pattern of plasma and serum biomolecule distribution during the drying process and its impact on patient-stratification. The study shows that a uniform sampling position of the droplet, both at the inner and the outer ring, is necessary for thorough clinical characterization of the patient’s plasma and serum sample using vibrational spectroscopy. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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Review

Jump to: Research, Other

20 pages, 3358 KiB

Open AccessReview

Label-Free Multiphoton Microscopy: Much More Than Fancy Images

by Giulia Borile, Deborah Sandrin, Andrea Filippi, Kurt I. Anderson and Filippo Romanato

Int. J. Mol. Sci. 2021, 22(5), 2657; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052657 - 06 Mar 2021

Cited by 42 | Viewed by 4814

Abstract

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. Full article

(This article belongs to the Special Issue Applications of Advanced Imaging and Analytical Microscopic Techniques on Molecular and Cell Biology)

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