Laser scanning multiphoton microscopy allows imaging deep in samples, as the nonlinearity causes the excitation to be strongly confined in the axial direction, that is, multiphoton microscopy comes with intrinsic sectioning of the signal. However, the resolution in laser scanning microscopy is given by how tightly one can focus the beam, which is the so-called Abbe limit, given by the wavelength $\lambda$, divided by twice the numerical aperture NA of the focusing element (objective). Thus, compared to single photon excited microscopy, the two- and three-photon excited microscopy the resolution worsens by a factor $\sqrt{2}$ or $\sqrt{3}$ respectively. To overcome the Abbe resolution limit, several approaches have been developed for single photon excited microscopy [1]–[5]. Of those methods, structured illumination microscopy [2] and image scanning microscopy (ISM) [4] can be applied with virtually any fluorescent marker, which makes them the most widely applicable super-resolution methods. ISM is implemented with laser scanning confocal microscopy, and recently it was demonstrated to be applicable to two-photon microscopy [6], achieving deep imaging with a resolution better than what is achieved with confocal microscopy.

We demonstrate sub-diffraction-limited simultaneous two- and three-photon imaging, using a high-power ultrafast fiber laser and single-photon avalanche detector array. Despite the 3-times longer excitation wavelength, our demonstration outperforms standard confocal microscopy of the same samples.

Multiphoton laser scanning microscopy is a powerful tool for deep imaging of thick biological samples. Image scanning microscopy (ISM) has demonstrated significant improvements in the signal-to-noise ratio in confocal laser scanning microscopy, while at the same time improving upon the effectively attainable resolution. Two-photon excitation (2PE), combined with ISM, has been shown to allow for deep tissue imaging with enhanced resolution compared to 2PE microscopy. Three-photon excitation (3PE) has enabled record imaging depth and contrast for multiphoton imaging, due to the superior suppression of out-of-focus signal generation. In this paper, we demonstrate super-resolution 3PE ISM. This is achieved using a single-photon avalanche detector array, and 1040-nm pulses for 3PE of blue fluorescence. This method enables subdiffraction limited resolution imaging of biological samples stained with blue fluorescent markers, such as mouse myocardial and spinal cord tissues stained with 4′,6-diamidino-2-phenylindole. Deconvolution improves the resolving power further and allows for imaging with better than λ/8 resolution with respect to the 3PE wavelength λ. With the ISM pixel reassignment procedure, we demonstrate a resolution enhancement of ∼1.6 laterally, compared to the resolution attained using a photomultiplier tube in a non-descanned detection arrangement, and a factor of ∼1.8 enhancement in axial resolution. The experimentally measured three-dimensional point spread function volume is shrunk ∼4.4-fold, which is close to the theoretically expected enhancement. Published by the American Physical Society 2024

Ammonium in the soil is converted into nitrate by nitrifying bacteria or archaea. While nitrate is readily available for plants, it is prone to leaching and contributes to eutrophication. In addition, when the soil conditions become anaerobic, nitrate can be reduced to nitrous oxide, a powerful greenhouse gas. Therefore, slowing nitrification in agricultural soil offers some benefits by reducing nitrogen loss and decreasing water and air pollution. Since nitrogen is a limiting nutrient for most ecological niches, many plants have evolved specialized compounds that reduce nitrification. One such compound, sorgoleone, which is secreted from the root hair of sorghum, has been relatively well studied due to its allelopathic function, with most enzymes involved in its biosynthesis elucidated. However, the secretion mechanisms remain unknown. Previous studies reported numerous lipidic vesicles in the sorghum root hair and speculated that they are involved in sorgoleone storage or secretion, but their roles remain unclear. Also, the subcellular organelles that are involved in sorgoleone synthesis have not been identified. In the present study, we found that the expression of sorgoleone biosynthesis enzymes is induced in a specific root zone, indicating that the secretion is developmentally regulated. The accumulation of internal vesicles preceded the peak of sorgoleone biosynthesis and secretion, indicating that the vesicles play a role in precursor storage rather than secretion. Moreover, our data suggest that enzymes that catalyze the first three steps, SbDES2, SbDES3, and SbARS1, interact with each other to form a multi-enzyme complex on the endoplasmic reticulum surface.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

The availability of an in vitro canine cell line would reduce the need for dogs for primary in vitro cell culture and reduce overall cost in pre-clinical studies. An immortalized canine muscle cell line, named Myok9, from primary myoblasts of a normal dog has been developed by the authors. Immortalization was performed by SV40 viral transfection of the large T antigen into the primary muscle cells. Proliferation assays, growth curves, quantitative PCR, western blotting, mass spectrometry, and light microscopy were performed to characterize the MyoK9 cell line at different stages of growth and differentiation. The expression of muscle-related genes was determined to assess myogenic origin. Myok9 cells expressed dystrophin and other muscle-specific proteins during differentiation, as detected with mass spectrometry and western blotting. Using the Myok9 cell line, new therapies before moving to pre-clinical studies to enhance the number and speed of analyses and reduce the cost of early experimentation can be tested now. This cell line will be made available to the research community to further evaluate potential therapeutics.

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits.

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Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z ring stabilization is not well-understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission EM. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly.

Phloem-derived amino acids are the major source of nitrogen supplied to developing seeds. Amino acid transfer from the maternal to the filial tissue requires at least one cellular export step from the maternal tissue prior to the import into the symplasmically isolated embryo. Some members of UMAMIT (usually multiple acids move in an out transporter) family (UMAMIT11, 14, 18, 28, and 29) have previously been implicated in this process. Here we show that additional members of the UMAMIT family, UMAMIT24 and UMAMIT25, also function in amino acid transfer in developing seeds. Using a recently published yeast-based assay allowing detection of amino acid secretion, we showed that UMAMIT24 and UMAMIT25 promote export of a broad range of amino acids in yeast. In plants, UMAMIT24 and UMAMIT25 are expressed in distinct tissues within developing seeds; UMAMIT24 is mainly expressed in the chalazal seed coat and localized on the tonoplast, whereas the plasma membrane-localized UMAMIT25 is expressed in endosperm cells. Seed amino acid contents of umamit24 and umamit25 knockout lines were both decreased during embryogenesis compared with the wild type, but recovered in the mature seeds without any deleterious effect on yield. The results suggest that UMAMIT24 and 25 play different roles in amino acid translocation from the maternal to filial tissue; UMAMIT24 could have a role in temporary storage of amino acids in the chalaza, while UMAMIT25 would mediate amino acid export from the endosperm, the last step before amino acids are taken up by the developing embryo.

The green colonial microalga Botryococcus braunii produces large amounts of liquid hydrocarbons that can be converted into transportation fuels. Colony cells are held together by a complex extracellular matrix (ECM) made up of a cross-linked long-chain hydrocarbon network around which liquid hydrocarbons are stored, a retaining wall for holding hydrocarbons within the cross-linked hydrocarbon network, and a polysaccharide fibrillar sheath radiating from the retaining wall and surrounding the entire colony. Analysis of “shells” shed from cell apical regions during cell division and containing the retaining wall and polysaccharide fibers shows association of a single protein where the fibers meet the retaining wall, suggesting involvement of this protein in polysaccharide fiber formation. Here we use peptide mass fingerprinting and bioinformatics to identify this protein called polysaccharide associated protein (PSAP). PSAP does not show similarity to any protein in databases, but contains several Proline-rich domains. Staining studies confirm PSAP as a glycoprotein, and mass spectrometry analysis identified ten N-linked glycosylation sites comprising seven different glycans containing mainly mannose and N-acetylglucosamine. Three of these glycans also contain fucose, with one of these glycans being unusual since it also contains arabinose. Additionally, four hydroxyproline residues have short O-linked glycans of mainly arabinose and galactose, with one also containing a 6-deoxyhexose. PSAP secretion and localization to shell material is confirmed using western blot analysis and microscopy. These studies indicate PSAP contains unique glycans and suggest its involvement in ECM polysaccharide fiber biosynthesis.

Metabolic dysfunction in Duchenne muscular dystrophy (DMD) is characterized by reduced glycolytic and oxidative enzymes, decreased and abnormal mitochondria, decreased ATP, and increased oxidative stress. We analyzed glucose metabolism as a potential disease biomarker in the genetically homologous golden retriever muscular dystrophy (GRMD) dog with molecular, biochemical, and in vivo imaging.

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.

This special issue focuses on articles that report on research in plants and fungi, offering topics not usually encountered in animal cells, such as the study of photosynthetic phenomena like chlorophyll fluorescence or chloroplast biology, or the analysis of tip-growing cells. Some of the contributions use already established experimental techniques but apply them to plants and fungal cells dealing with the specific challenges present in such samples. Experimental design and execution often goes hand-in-hand with instrument capabilities. Steinbach et al. present the CellFinder program that provides a text-based macro language aimed at controlling a commercial confocal microscope via external hardware devices offering much enhanced flexibility in imaging protocols. These authors demonstrate the capabilities of CellFinder by studying changes in chlorophyll fluorescence at controlled levels of actinic light in dark-adapted cyanobacteria. Schulthaus et al. present their studies of actin cytoskeleton dynamics in tip-growing cells of a filamentous fungus. They employ time-lapse fluorescence microscopy to learn how different actin structures relate to each other and describe a subapical actin web that has not been documented in any filamentous fungus to date. Fungi can serve as an excellent heterologous system to study protein dynamics. To this end, Terbush et al. present Schizosaccharomyces pombe as a valuable system for analyzing the chloroplast division complex. The in vivo analysis of chloroplast division proteins is particularly challenging due to light scattering in plant tissue, the high auto-fluorescence background as well as the low fluorescence signal from proteins expressed at native levels. In addition, interpretation is often rendered more difficult if the results depend on data obtained in the presence of intrinsic interacting proteins. Expression of chloroplast division proteins in S. pombe effectively and elegantly overcame these challenges and provided an optically conducive system for microscopy in which to study the assembly networks of Arabidopsis thaliana FtsZ1 and FtsZ2 proteins and the modulation of their assembly by regulatory proteins. Among other results, Terbush et al. present fluorescence recovery after photobleaching (FRAP) analysis of FtsZ and ARC6 protein turnover. Such experiments in plants proved to be very difficult for those researchers who tried. Protein dynamics is the focal point of another paper. Petrášek et al. analyze the dynamic of plasma membrane proteins using fluorescence correlation techniques such as Raster image correlation spectroscopy and number and brightness analysis providing very useful information on the mobility and aggregation of membrane proteins that is not accessible by FRAP experiments at comparable time and distance scales. Lastly, Banerjee et al. evaluate ratiometric imaging with fluorescence resonance energy transfer (FRET)-based phosphate biosensors in different cells and tissues of A. thaliana. They derive tissueand cell-specific spectral correction factors and define linear ranges for donor, acceptor, and FRET emission intensities. This study provides a useful guide for quantitative ratiometric study in live plants. Often, plant-specific obstacles or ways of overcoming them have contributed to solutions in other living systems and/or R&D areas. Therefore, it is hoped that the contributions addressing specific topics in plant and fungal research presented in this special issue may also be useful to a wide range of researchers across the disciplines.

A prior work describing a computer vision system for measuring maturity using longitudinal views of cotton fibers reported observing a large variation in maturity within a single fiber. This paper describes the use of confocal microscopy as an independent measuring method to validate those findings. Individual cotton fibers are imaged from end to end by a confocal microscope producing hundreds of image volumes of cotton fiber segments, each ∼150 µm in length. From these volumes, virtual fiber cross-sections are extracted, processed using the level set method, and measured according to AATCC standards. The results demonstrate with both visual and quantitative analysis that fiber maturity can exhibit large variations within a single fiber.

Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present the in planta analysis of Arabidopsis FtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.