Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

Source at https://doi.org/10.1515/nanoph-2017-0112. Licensed CC BY-NC-ND 4.0.The dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy

Nanoscopy of bacterial cells immobilized by holographic optical tweezers

Diekmann R, Wolfson D, Spahn C, Heilemann M, Schüttpelz M, Huser T. Nanoscopy of bacterial cells immobilized by holographic optical tweezers. Nature Communications. 2016;7(1): 13711

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon

Label-free superior contrast with c-band ultra-violet extinction microscopy

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample’s refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup

Structured Illumination Microscopy of Biological Structures

Abstract of presentation held at Norwegian Electro-Optics Meeting, Henningsvær, Norway, 2-4 May 2018.Resolution in optical microscopy has long been limited to the Abbe diffraction limit, i.e. about 250 nm laterally for visible wavelengths on a very good microscope. In the last two decades several techniques have been devised to circumvent this limit: an achievement which was recognized with the 2014 Nobel Prize in Chemistry. Structured Illumination Microscopy (SIM) was the first of these techniques to become commercially available, and continues to be the only super-resolution technique which is practically compatible with living cells, while also requiring the least modification to conventional sample-labeling protocols. SIM utilizes Moiré patterns and frequency shifting to improve resolution 2X in each dimension, as well as significantly improve the contrast for the mid-range spatial frequencies. These advances have unlocked a new realm of biological inquiry: the combination of the high biochemical specificity of fluorescent probes with resolution previously only possible with electron microscopy now enables the direct study of sub-organelle colocalization and the dynamics of living cells. Here, we will present both the basics of the SIM technique as well as a sampling of its biological applications from our lab at UiT, including sub-mitochondrial localization and dynamics, sieve-like nanostructures in liver cells, and large-scale visualization of super-resolved cardiac tissue sections, as well as discuss the practical limitations and implications of this work

Uptake and Degradation of Bacteriophages by Liver Sinusoidal Endothelial Cells

Bacteriophages (briefly, “phages”) are viruses which target bacteria, and are non-infectious to eukaryotic cells. It is estimated that more than 30 billion phages cross into the human body from the gut each day1, and eventually need to be cleared from the blood circulation. The liver plays a central role in pathogen clearance, and liver sinusoidal endothelial cells (LSECs), which form the lining of the numerous capillaries in the liver, are therefore on the front lines for this removal process. However, despite their strategic location and efficiency in removing small (<200 nm) particles2, LSECs have historically been poorly studied in terms of removal of phages. We hypothesized that LSECs play a critical role in the removal of phages from the bloodstream through endocytic uptake and lysosomal degradation, and used GFP-labeled T4 bacteriophages as a model system to study this clearance process. Uptake and trafficking of phages in primary cultured LSECs was monitored by deconvolution microscopy on both short (1 hour) and long (24 hours) term timescales, and structured illumination microscopy was used to confirm the identity of the LSECs using their unique, sub-diffraction scale morphological features: tiny holes called fenestrations. After being taken up by the cells, the phages were rapidly transported to late endosomes/lysosomes, as confirmed by colocalization studies with an LSEC-specific lysosomal vital marker. Challenging the LSEC cultures with radiolabeled phages for up to 24 hours showed that the phages were degraded about 4h after being taken up by the cells, with degradation products being increasingly released to the spent medium up to about 18h after uptake. In conclusion, our novel finding that LSECs internalize and degrade bacteriophages lends support to the hypothesis that LSECs play an important role in the clearance of blood borne phages

Stimulation of exosome release by extracellular DNA is conserved across multiple cell types

This is the submitted manuscript version of the following article Iliev, D., Strandskog, G., Nepal, A., Aspar, A., Olsen, R., Jørgensen, J., ... Mironova, R. (2018). Stimulation of exosome release by extracellular DNA is conserved across multiple cell types. The FEBS Journal, 285(16), 3114-3133. https://doi.org/10.1111/febs.14601. Published version available at https://doi.org/10.1111/febs.14601.Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA – their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions

Vertically integrated laser radar imagers -- performance and analysis

Massachusetts Institute of Technology's Lincoln Laboratory has developed laser radar systems using timing circuitry combined with Geiger-mode avalanche photodiodes (GM-APD) to produce reliable three-dimensional images. The Laboratory has recently been working with silicon-on-insulator technology to create a vertically integrated sensor array (VISA) that stacks an array of GM-APDs on top of two layers of timing circuitry. Through extensive laboratory testing and simulation of this device, the world's first image from a vertically integrated array was produced

Designing Educational Media in Suan Phung Nature Education Park

This project produced tools to increase the educational resources offered to visitors in Suan Phung Nature Education Park in Ratchaburi, Thailand. Interviews, surveys, and extensive archival research provided us with the necessary information on the park, its programs, conservation, and qualities of our target audience. We succeeded in complementing the park's existing educational program by producing a video about the park and conservation, as well as by recommending additional activities, programs, and exhibits for future implementation

Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

The dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy