Periodicum Biologorum

Celebrating 40 years of the Croatian Microscopy Society

Danijela Poljuha, Goran Kovačević — Tue, 14 Nov 2023 00:00:00 +0100

We are delighted to present a special issue of Periodicum biologorum,
dedicated to the 30th anniversary of the Croatian Microscopy Society
as an independent Society and the 40th anniversary of its foundation.

Croatian Microscopy Society 1983-2022

Danijela Poljuha — Tue, 14 Nov 2023 00:00:00 +0100

This paper provides a brief overview of the history of the Croatian Microscopy Society on the occasion of its 30th anniversary as an independent society and the 40th anniversary of its foundation. The overview chronologically details the period of the Society's work as the Section for Electron Microscopy of the Croatian Natural Science Society (1983-1992), followed by its independent activity first as the Croatian Society for Electron Microscopy (1992-2006) and finally as the Croatian Microscopy Society (2006-present). The review concludes with recent trends and policies in the Society's development. The review is an extended excerpt from the opening speech held by the Chair of the 4th Croatian Microscopy Congress and a member of the Society’s board, on the occasion of the Society's anniversary, May 18th 2022.

Proposal of the Croatian Microscopy Society for awarding the "Spiridion Brusina" Medal to Professor Miran Čeh, PhD, Jožef Stefan Institute, Ljubljana, Slovenia

Andreja Gajović — Tue, 14 Nov 2023 00:00:00 +0100

Cooperation with Professor Miran Čeh started back in 2007, when a member of the Croatian Microscopy Society (CMS), Andreja Gajović, joined the group of Prof. Miran Čeh for post-doctoral training to Jožef Stefan Institute. Since then, Prof. Čeh collaborated extensively with Croatian scientists through a number of bilateral projects, and in this way participated and still participates in the education of Croatian scientists on high resolution transmission electron microscopy (HRTEM). Moreover, Milivoj Plodinec, a member of CMS, spent a sixmonth TEM training (2011/2012) at the Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia (Croatian Science Foundation's scholarship for doctoral students, project title "Titanate nanostructures - synthesis and high-resolution transmission electron microscopy (TinaTEM)"). Training was performed under the mentorship of Prof. Čeh in the field of synthesis of nanomaterials and their characterization, using, primarily, basic, and advanced techniques of electron microscopy. Plodinec gathered significant knowledge in scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED) and energy dispersive X-ray spectroscopy (EDS) and respected techniques. He transferred the acquired knowledge to the Ruđer Bošković Institute, where he was working on the functionalization of Ti0₂ nanostructures for various applications.

SEM and TEM investigations of materials at the Centre for Electron Microscopy and Microanalysis (CEMM)

Miran Čeh, Jitka Hreščak, Sandra Drev — Tue, 14 Nov 2023 00:00:00 +0100

Modern state-of-the-art electron microscopy (EM) techniques are among the most versatile and useful analytical methods for nondestructive morphological, structural, and chemical characterization of materials. Both, scanning electron microscopy techniques (SEM) and transmission electron microscopy techniques (TEM), in essence complementary analytical methods, are capable of providing insight into the surface morphology, structure and chemical composition of materials from micrometer range to sub-atomic range. The importance of electron microscopy techniques in materials science have been readily recognized by the researchers of the Jozef Stefan Institute very early after its foundation in 1949 which resulted in the purchase of the first transmission electron microscopy Carl Zeiss EM-8 already in 1954. During the following years many researchers and institute directors have put a lot of effort into building and developing EM research infrastructure at the institute with accompanying expertise.

Forensic potential of pollen grains: A case study from continental Croatia (town of Donja Stubica)

Ivana Horjan Zanki, Dario Hruševar, Božena Mitić — Tue, 14 Nov 2023 00:00:00 +0100

Background and Purpose: Pollen grains can come as “ invisible” traces during forensic palynological expertise. As forensic palynology is not carried out in Croatia at all, the main aims of our work were to establish an initial palynological collection for a limited area in Croatia and determine its forensic potential.

Materials and Methods: For our case study, we chose a typical town from continental Croatia, Donja Stubica, in which there had been no previous floristic studies. The field survey was conducted during 2005, 2007, and 2021. Floristic and palynological analysis was carried out with an emphasis on the forensic potential of pollen grains.

Results: A total of 141 representative plant taxa with pollen samples were collected. The taxonomic analysis showed that the area for the case study was chosen well. Palynological analysis showed that the highest number of taxa had pollen grains that were spheroidal in shape (60%), medium sized (70%), and colporate (41%). The pollen of the majority of taxa had excellent or very important forensic potential, due to their dispersal by insects (60%), a combination of two dispersal methods (23%), and autogamy (1%). In addition, due to the limited distribution of certain rare plants (e.g., Iris sibirica subsp. sibirica in Croatia), their pollen has an even greater forensic potential than the similar pollen of related, more widespread taxa.

Conclusion: The generated palynological collection, due to the great forensic potential, could be used in the future as a comparative, and at least partly as a reference forensic collection, applicable in specific cases of legal pollen expertise in Croatia.

Morphology of extrafloral nectaries of Ailanthus altissima (Mill.) Swingle (Simaroubaceae)

Tue, 14 Nov 2023 00:00:00 +0100

Background and Purpose: Ailanthus altissima (Mill.) Swingle (Tree of Heaven) is a highly invasive, widespread, and widely investigated plant species native to the broader area of China. Extrafloral nectaries are major components of the A. altissima secretory system, but the knowledge of their morphology and role in the tree's physiology is limited. This research aims to explore the morphology of extrafloral nectaries, compare it to previous findings, and discuss their possible role and function.

Materials and Methods: Extrafloral nectaries on leaves have been monitored through different phases of leaf development, from June to August 2015 and 2022. The nectaries' morphology was investigated using Zeiss Axioscope 5 and BOECO BSZ-405 light microscopes, and FEG QUANTA 250 FEI scanning electron microscope, operating at 7 kV and pressure of 60–100 Pa, without samples pretreatment.

Results: Our investigation revealed the absence of earlier reported pores or ducts on the top of the glands. However, it supports one of the first, systematical investigations of A. altissima extrafloral nectaries conducted in Croatia a century ago, which was until recently forgotten by history. We evaluated our findings against prior theories and assessed the nectaries' potential role and purpose in disposing surplus sugars.

Conclusions: The nectar in A. altissima is not secreted via an opening on the leaves but rather through epidermal tissue tearing.

Ultrastructural and immunofluorescence features of the epidermal cells and its secretory granules in the amphioxus Branchiostoma lanceolatum L.

Nives Kević, Ivana Restović, Ivana Bočina — Tue, 14 Nov 2023 00:00:00 +0100

Background and purpose: In the vertebrate family, the epidermis of terrestrial animals is keratinized while in aquatic forms and amphibians can produce mucus. Amphioxus, a cephalochordate, is an important animal model in the study of chordate phylogeny. Major cytoskeletal and extracellular matrix proteins in epidermal cells of amphioxus (Branchiostoma lanceolatum L.) were investigated using transmission electron microscopy and immunofluorescence.

Materials and methods: Amphioxus specimens were fresh caught in the Adriatic Sea. Tissue samples were fixed and prepared for transmission electron microscopy and immunofluorescence techniques.

Results: The epidermis consisted of one-layered columnar epithelium. Epithelial cells contained voluminous nucleus, a well-developed supranuclear Golgi apparatus, abundant vesicles and apical secretory vesicles surrounded by cytokeratin filaments. A single row of smaller vesicles also appeared close to the basal cell membrane. Strong positive immunolocalization of hyaluronic acid and collagen type I was observed in the apical domain of the plasma membrane of amphioxus, respectively. The positive signal of hyaluronan and collagen proteins referred to the secretory granules in the apical domain of the epidermal cell. Strong positive labelling of aggrecan was seen around the cell nucleus, deep below the apical domain, suggesting that aggrecan is not located in the secretory granules. A strong signal of the cytokeratin 10 protein was seen throughout the cytoplasm of the epidermal cells.

Conclusion: Due to its morphology and histochemical composition, epidermal cells of amphioxus may contribute to course surface protection and the mucous layer in amphioxus may serve as a physical barrier between the body and its environment.

Expression of dendrin, neurofilament and glial fibrillary acidic protein in the brain of the dogfish Scyliorhinus canicula L.

Tue, 14 Nov 2023 00:00:00 +0100

Background and purpose: Dendrin is a brain and renal protein that is supposed to be involved in cytoskeletal modifications at the synapse and a part of the slit diaphragm and podocytes. Here, we aimed to investigate dendrin expression in dogfish brain since this newly discovered protein was never reported in fish. We compared the expression of dendrin to those of glial (GFAP) and neuronal (NF) proteins, which have already been described in the dogfish brain.

Materials and methods: Histological and immunofluorescent techniques were performed on tissue samples. The obtained data were statistically analyzed.

Results: Our results have shown that dendrin is expressed in all observed parts of the dogfish brain. In the forebrain, both observed parts (telencephalon and olfactory lobes) expressed dendrin. Regarding the percentage area of dendrin expression, it is expressed more in olfactory lobes than in the telencephalon. Compared with GFAP and NF expression, the expression of dendrin significantly differs in both parts of the forebrain. The highest dendrin expression was noticed in the midbrain. In dogfish midbrain, the difference in expression of dendrin in comparison to those of GFAP and NF was even more significant. The percentage area of dendrin expression in the hindbrain (cerebellum and medulla oblongata) was smaller than those in the forebrain and midbrain, contrary the percentage area of intermediate filaments GFAP and NF were significantly higher.

Conclusion: These results are the first report on dendrin expression in the dogfish brain opening the path for future studies on its role and function.

Retro is the new modern: Contemporary application of gold impregnation staining on brain cryosections for digital image analysis

Tue, 14 Nov 2023 00:00:00 +0100

Background and purpose: Since the times of Golgi and Cajal, impregnation with gold or silver has been used to visualize microscopic details of the nervous tissue. Although immunohistochemistry has largely replaced impregnation techniques, they are still used, and there is a growing interest in combining them with modern image analysis methods for quantitative studies in neuroscience. The aim of this research was to modify the gold chloride impregnation method published by Schmued to improve consistency of staining, to be adequate for digital image analysis.

Materials and methods: Brains of 8 six-month-old female Wistar rats were fixed in 4% PFA, cryoprotected in sucrose and flash‑frozen in liquid nitrogen. Neighboring sets of coronal sections were chosen for gold impregnation, Nissl staining and MAP2 immunohistochemistry. Whole-slide images and images of specific regions were taken for analysis.

Results: Myelinated fibers were stained dark reddish to brown on goldstained sections, and other tissue was yellowish, which gave an excellent contrast for digital image analysis. Gold staining was consistent in all regions, and no major artifacts were noticed. When compared to Nissl and MAP2, only myelinated structures were stained with gold impregnation.

Conclusions: Modified gold impregnation method is an alternative that’s on par with traditional myelin staining methods. The new, modified gold impregnation method gives a consistent and reproducible staining suitable for digital image analysis. It can be useful in morphometric evaluation of nervous tissue and investigation of neuropathological changes in nervous tissue, especially for quantitative studies.

Interactions of variously coated gold and silver nanoparticles with a bis(triarylborane) photodyanmic therapy (PDT)-dye; their cellular uptake, cytotoxicity and photo-activity

Tue, 14 Nov 2023 00:00:00 +0100

Background and purpose: Diethynylarene-linked bis(triarylborane) tetracations can be used as probes for fluorimetric and Raman sensing of biomacromolecules, as well as promising theragnostic agents. Among them, bis(triarylborane) fluorophore (TAB3), when bonded to Ag nanoparticles (NP), stood out with specific properties such as Raman signal enhancemen of the TAB3 dye in a cuvette. However, TAB3 dye - nanoparticle composites have not been studied in biological systems. For this reason, questions arose as to whether different types of metal nanoparticles (Au or Ag-based) with different coatings (negatively charged citrate or neutral PVP) could be efficiently stained with the TAB3 dye in a cuvette. The aim of this research was to examine Au and Ag nanoparticles of similar size (20-25 nm) with different stabilizers for their cellular uptake, cytotoxicity in the dark and under visible light radiation, to characterize the interactions of nanoparticles with the TAB3 fluorophore, and to study NP-TAB3 composites in cells, evaluate their intracellular staining, as well as possible photoinduced release and biological activity.

Materials and methods: The binding constants of Au- and Ag- based nanoparticles with TAB3 were determined by fluorimetric titrations. The cytotoxic effect of NPs was determined by the survival of A549 cells (MTT assay). Cellular uptake of both NP and NP-TAB3 composites were performed by live cell imaging experiments.

Results: The Au- or Ag-based NPs with different coatings bind to the TAB3 with high affinity. These NPs, as well as TAB3-NP complexes, efficiently enter living human cells, accumulating in cytoplasm with no apparent selectivity for a particular organelle. Even prolonged 3-day treatment with the NPs studied did not show any toxic effect on the cells. Bioimaging studies in cells revealed that the TAB3-NP complex does not intracellularly dissociate; the previously reported photo-bioactivity of TAB3 is completely inhibited by binding to NPs.

Conclusion: Au- and Ag NPs were non-covalently stained by TAB3, irrespective of the different coatings, with similar binding affinities. Emission from TAB3 is strongly quenched by the NPs, but not completely. Experiments on living human cells revealed that neither free NPs, nor their composites with TAB3, were toxic. Bioimaging studies by confocal microscopy revealed that all NPs efficiently enter living cells within 90 min. Colocalization experiment with simultaneous collection of data in the reflection and fluorescence modes demonstrated that the TAB3 dye remained bound to NPs inside cells. Strong irradiation of TAB3-NP inside cells with a 457 nm laser did not yield any damage to the cells, at variance with our previously shown very strong photo-bioactivity of the TAB3 dye alone. Thus, binding of a chromophore to a nanoparticle can inhibit the chromophore’s ability to undergo photo-induced singlet oxygen production, consequently blocking its photo-bioactivity.

Application of thermal analysis methods in biology and medicine

Jelena Macan — Tue, 14 Nov 2023 00:00:00 +0100

Thermal analysis methods are widely used in the characterization of substances and materials in chemistry and engineering. But they also find their application in life sciences: biology and medicine. This paper exhaustively and critically reviews the application of the most commonly used thermal analysis methods for the characterization of organs and tissues of plants, animals, and humans. The methods are suitable for differentiating between several types of water in a cell, optimizing treatment, storage, or cultivation conditions, following plant or animal development, medical diagnostics and modelling, and more. Expertise developed in the characterization of synthetic materials and molecules can be transferred to that of biological tissues and biomolecules and opens a perspective for interdisciplinary research. Still, researchers should take into consideration the inherent complexity of biological samples, as well as inevitable changes when isolating the tissue from the living organism.

Use of atomic force microscopy for characterization of model membranes and cells

Anja Sadžak, Lucija Mandić, Suzana Šegota, Vida Strasser — Tue, 14 Nov 2023 00:00:00 +0100

Background: To provide a fundamental understanding of the potential and use of atomic force microscopy (AFM) in medicine and the life sciences, this work presents a thorough description of imaging and non-imaging atomic force microscopy modes for characterizing model membranes and cells at the nanoscale.

Methods: The imaging and non-imaging AFM modes are described with examples in terms of the characterization of topographic, morphological, and nanomechanical sample properties.

Results: AFM imaging of supported lipid bilayers (SLBs) revealed the effects of temperature and medium composition on SLB topography in the gel and fluid phases, and on the bilayer thickness. Non-imaging AFM showed the strengthening of the SLB in both phases by the ion binding process.
Imaging of neuronal and neuroblastoma cells with and without treatment revealed morphological changes in shape, volume, roughness, and Feret dimension. Non-imaging AFM showed the change in cell elasticity induced by the treatment with H₂O₂ with and without quercetin and by the treatment with copper and myricetin. The measurements of cells elasticity revealed a reorganization of the cytoskeleton and filament structures.

Conclusions: Diverse applications of imaging and non-imaging AFM can provide important information about the underlying processes in biologically relevant systems. AFM, as a complementary technique to other biomedical methods, allows screening and monitoring of physiological changes at the nanoscale.

Tissue optical clearing methods for microscopy: A review of their application in neuroscience

Dominik Hamer, Daniela Petrinec, Monika Berecki, Anton Glasnović, Srećko Gajović — Wed, 25 Oct 2023 00:00:00 +0200

Recent advances in microscopy have enabled cellular-resolution imaging of thick tissue samples or even whole organs. The natural opacity of organs and tissues acts as a barrier to light penetration and must be removed to visualise structures of interest on a three-dimensional scale. Tissue optical clearing methods achieve sample transparency while also preserving fluorescently labelled epitopes. This innovative approach to sample preparation effectively enhances traditional histological sections and, with the aid of light sheet microscopy, enables optical sectioning and three-dimensional reconstruction of entire organs, even whole brains. Light sheet microscopy of optically cleared brain samples is a valuable method in neuroscience that is used in neuro-oncology, traumatic brain injury, ischemic brain injury, and neurodegenerative disease research.
In this review, we describe tissue optical clearing methods used to achieve optical transparency in brain samples. This quickly developing field has a significant potential for producing cutting-edge uses in neuroscience research.

Beginners guide to sample preparation techniques for transmission electron microscopy

Ena Dumančić, Lea Vojta, Hrvoje Fulgosi — Tue, 14 Nov 2023 00:00:00 +0100

Background purpose: The revolution in microscopy came in 1930 with the invention of electron microscope. Since then, we can study specimens on ultrastructural and even atomic level. Besides transmission electron microscopy (TEM), for which specimen preparation techniques will be described in this article, there are also other types of electron microscopes that are not discussed in this review.

Materials and methods: Here, we have described basic procedures for TEM sample preparation, which include tissue sample preparation, chemical fixation of tissue with fixatives, cryo-fixation performed by quick freezing, dehydration with ethanol, infiltration with transitional solvents, resin embedding and polymerization, processing of embedded specimens, sectioning of samples with ultramicrotome, positive and negative contrasting of samples, immunolabeling, and imaging.

Conclusion: Such collection of methods can be useful for novices in transmission electron microscopy.

A young researcher’s guide to three-dimensional fluorescence microscopy of living cells

Vedrana Filić, Igor Weber — Tue, 14 Nov 2023 00:00:00 +0100

Three-dimensional imaging of fast intracellular processes by fluorescence microscopy should provide decent spatial and high temporal resolution while minimizing fluorophore bleaching and cytotoxicity. We give a condensed introductory overview of three contemporary methods mostly used for imaging of living cells in 3D and compare their performance in terms of temporal and spatial resolution, imaging flexibility and specimen photodamage: point-scanning confocal microscopy, spinning-disc confocal microscopy, and lattice light-sheet microscopy. While point-scanning instruments are unsurpassed in terms of confocal performance, flexibility and configurability of their optical path, spinning-disc and lattice light-sheet optical designs excel in acquisition speed and low levels of light-inflicted specimen deterioration.

Attendance of extracurricular activities in the field of natural sciences and the attractiveness of the content offered for extracurricular activities in biology in elementary schools

Damir Sirovina, Daniela Petrinec, Petra Tramontana, Goran Kovačević — Tue, 14 Nov 2023 00:00:00 +0100

Background and purpose: For many years, students’ interest in the field of natural sciences has declined, which consequently results in decreasing numbers of professional staff in the mentioned field. The aim of this study was to examine the attendance of extracurricular activities in the field of natural sciences by elementary school students (age 13 and 14) in Zagreb and whether the teachers' expectations are in line with the students' preferences for the content offered as part of extracurricular activities in biology.

Material and methods: Using an anonymous online survey (Google Forms), data were collected on participation in extracurricular activities in the field of natural sciences and the attractiveness of the content of extracurricular activities. The respondents were 90 students in the 7th grade and 46 teachers of biology in Zagreb, 2020.

Results: Participation in extracurricular activities in the field of natural sciences among the surveyed students was very low. Students were attracted to content that includes simple practical work in the field of biology, including microscopy, but the perception of their interest by teachers is not in accordance with the real interests of the students.

Conclusion: The results of this research show that the share of the science field in extracurricular activities is low, and that the surveyed teachers are not aware of the students' wishes regarding the content offered in extracurricular activities. The conducted research can be a good basis for organizing and planning extracurricular activities in elementary school.