Probing the Molecules that Control Protein Levels in Your Cells

Fluorescence Correlation Spectroscopy (FCS) provides insights into mRNA binding proteins

Proteins are critical for countless roles in our bodies including structure, function, and regulation of our tissues and organs. To create proteins from the genes in our DNA, an intermediate molecule, mRNA, is needed. mRNA is made from DNA in the nucleus and then transported out of the nucleus where it is translated into protein.

Dr. Finn Cilius Nielsen and his team at the Center for Genomic Medicine, Denmark, work to understand the molecules which control mRNAs. Dr. Nielsen’s lab recently published an article using superresolution microscopy, fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS, respectively) to better understand these molecules.

We spoke with Àngels Mateu-Regué, a graduate student in Dr. Nielsen’s lab and lead author on the publication, about their findings and use of this technique.

Can you briefly describe your area of research and the findings in your article?

mRNAs are coated with a wide-range of proteins, forming complexes known as messenger ribonucleoprotein particles or “mRNPs”. These proteins regulate mRNA transport, translation, stability, etc. It had been hypothesized that mRNAs could be transported in groups, thus coordinating transport and translation of different mRNAs in space and time.

To answer these questions, we used a combination of superresolution microscopy and fluorescence correlation spectroscopy (FCS). Using these techniques, we could see that mRNAs exist as single molecules in cells and that the association between different RNA-binding proteins happen via mRNA-dependent interactions. Therefore, coordinate protein synthesis is unlikely to be a result of assembly of different mRNAs in the same mRNP granule.

How did fluorescence correlation spectroscopy (FCS) contribute to your findings?

We characterized the dynamic behavior of the mRNA-protein complexes using two GFP-tagged RNA binding proteins present in these particles: IMP1 and YBX1. Through the analysis of their autocorrelation curves, we learned that mRNP granule dynamics is not described by a simple diffusion model. We could conclude that mRNP granules diffuse in at least two different motions in live cells. Data visualization and quantification of diffusion was very easy to perform. GFP alone, in contrast, gave a very fast and simple 3D diffusion compared to the two other factors.

Additionally, we were also able to count the number of proteins bound to a complex by comparing the brightness of monomeric GFP with the brightness of the complex.

How did you use Fluorescence Cross-Correlation Spectroscopy (FCCS)?

We wanted to study the nature of the interaction between the above mentioned proteins: IMP1 and YBX1. Specifically, we wanted to see whether the interaction was direct or RNA-mediated (both proteins bound to the same mRNA). This time, we used a mCherry-tagged YBX1 and we co-transfected cells with GFP-IMP1 or a GFP-IMP1 mutant that is not able to bind to RNA. It was very clear to see that when we used wild type GFP-IMP1 and mCherry-YBX1, fluorescence signals coming from GFP or mCherry were synchronous, and that resulted in a positive cross-correlation curve. On the other hand, when we used the GFP-IMP1 mutant, we could clearly see that the cross-correlation curve was completely flat, indicating that the interaction between the two proteins is RNA-dependent.

Fluorescence Cross-Correlation Spectroscopy (FCCS) cytoplasmic measurement of HeLa cells co-transfected with GFP-IMP1 (green autocorrelation curve) and mCherry-YBX1 (red autocorrelation curve) showing in vivo interaction between the two RNA-binding proteins (positive cross-correlation curve). When a RNA-binding impaired IMP1 mutant is used (IMP1_KH1-4mut), cross-correlation with YBX1 is negative (right graph).

What are the main advantages of FCS and FCCS compared to other commonly used techniques?

The possibilities of FCS are enormous in terms of measuring how proteins diffuse in live cells. Having such a direct, fast and easy measurement about protein dynamics and stoichiometry in a single cell in vivo is already a major advantage of the technique. Using the same equipment, FCS and FCCS allow a precise and well defined biochemical characterization of macromolecular complexes combined with imaging in live cells at the same time.

One could think about endless possibilities – not only measuring protein diffusion in normal conditions but also how the diffusion (or biological activity) of a particular protein changes, for instance upon the addition of drugs, etc.

Regarding FCCS, the advantage of protein-protein interaction studies inside the cells (their native environment) is by itself of major importance. The analysis with this technique is performed directly in intact live cells, in which the real protein-protein interactions can be captured. This is in contrast to extensively used protein-protein interaction methods, such as immunoprecipitation.

Àngels Mateu-Regué analyzing fluorescence correlation spectroscopy (FCS) data.

FCS / FCCS can be seen as an intimidating fluorescence microscopy modality. What would you recommend to a researcher new to these techniques?

After trying FCS and FCCS, our understanding and our view about the technique has radically changed. We could not do anything other than say to other researchers: go for it!

As a start, we would recommend to read a few broad spectrum FCS and FCCS reviews or explanatory material. It is very important to understand and incorporate the theory behind the technique in order to understand the outcome and be able to troubleshoot in your experiments later. In the same line, we would also suggest that you get some training if possible, in order to familiarize with the technique and try it out with very simple things. For example, these simple things could be an antibody coupled with an Alexa Fluor fluorophore diluted in PBS, a cell lysate (or even a cell) expressing GFP alone. It might be easier for researchers who already work with fluorophores and fluorescent proteins.

Learn More

  • Read the full article from Dr. Nielsen’s group: “Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.” Link

FCS Reviews:

  • Wikipedia: Fluorescence Correlation Spectroscopy Link
  • Nature Protocols: Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms Link

ZEISS Technology with FCS capabilities:

  • ZEISS LSM 980 laser scanning confocal microscope Link

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Studying Viral Entry into Cells

Researchers combine confocal and atomic force microscopy to better understand this critical step of viral infections

Viruses cannot reproduce on their own. They require the internal machinery of host cells for replication and propagation. One of the challenges that successful viruses have overcome is evolving complex, multi-step mechanisms to bind to host cell surfaces and then transport themselves inside the cell.

Prof. Dr. David Alsteens, Research Associate of the FNRS

Dr. David Alsteens and his group at the Louvain Institute of Biomolecular Science and Technology, Belgium, along with colleagues at the University of Pittsburgh, USA, have recently published work in Nature Communications focused on better understanding how one type of virus binds to the cell surface. Their work is particularly interesting as they were able to study this for the first time in living cells by using a combination of confocal and atomic force microscopy (AFM).

Dr. Alsteens was kind enough to answer some questions about his use of confocal microscopy, AFM and his research.

Please broadly describe the research goals of your lab. How did this publication fit into your overall research?

The ‘NanoBiophysics lab’ aims at better understanding the complex biological processes that take place on the cell surface under relevant physiological conditions. To this end we combine AFM and point scanning confocal microscopy to localize molecules, receptors and cells while mapping their nanomechanical properties or biophysical interactions between the AFM probe and the biological sample. We have been using force-distance based-AFM for several years already to study a variety of biological samples at both molecular and cellular levels to better understand how single-molecule interactions can drive biological processes.

Some key applications of my lab include, among others, imaging G-protein coupled receptors while quantifying their ligand-binding free-energy landscape, studying the first entry steps of viruses to animal cells (Reovirus, Rotavirus, Herpesvirus, Ebola virus-like particles) with a focus on the understanding of the dynamics of the interactions established at the surface of living cells.

Our recent publication perfectly fits into the main research’s axes of my lab. Since my postdoc stay at ETH Zürich (Basel, Switzerland), we further developed the combination of the latest generation of Bio-AFM with a high-resolution optical microscope. The ambition is to develop a new methodology in biophysics and virology to study virus entry at high-resolution, directly on living cells, and to provide quantitative information on the molecular details underlying the early steps of cell infection.

In our recent work, we combine the latest generations of confocal laser scanning microscope (CLSM) and AFM to follow the early steps of single virus entry. We also succeeded to describe these first steps in a quantitative manner and provided the kinetic and energetic parameters of the virus-receptors interactions.

The Alsteens Lab in front of their confocal – AFM equipment

Could you describe in layman’s terms how your combination of confocal and AFM technologies contributed to the findings in this publication?

Thanks to an atomic force microscope coupled to a confocal microscope, we investigated how reoviruses interact with mammalian cell surfaces in cell culture conditions. A very sharp tip is functionalized with a single virus and approached to the cell surface until soft contact, enabling it to interact with the cell surface component including glycans and receptors (Figure 1).

Figure 1: Confocal microscopy image of an AFM tip bearing a single virus (green spot) on a layer of CHO cells.

The combination with the confocal microscope permits us to follow in real time the position of the AFM tip on the cell and the localization of some fluorescently labelled receptors. Upon retraction of the AFM tip, the bounded virus starts to detach from the receptors and the deflection of the tip during the retraction movement is monitored. The deflection is directly proportional to the force acting between the virus and the receptors, thus enabling us to quantitate the binding strength. The measured binding forces are then analyzed in detail and gives access to parameters that describe the interaction on a kinetic and energetic manner. We can also determine the number of bonds that have been formed between the virus and the cell (Figure 2).

Figure 2: Probing reovirus binding to CHO cells expressing JAM-A. (a) Cartoon of the experiment highlighting that CHO cells (having no JAM-A) are fluorescently labeled. (b) Confocal image showing an AFM tip on the top of two adjacent cells expressing or not JAM-A receptors. (c) FD-based AFM height image and corresponding adhesion channels. The adhesion map reveals that most adhesion (bright pixels) are localized on the cells expressing JAM-A receptors.

Altogether, this study allowed us to make a major breakthrough in understanding the binding mechanism of the reovirus to cells. We showed for the first time that reovirus early binding to cell surface is regulated by glycans, known as attachment factors (sialic acid for reoviruses). Until now, those interactions were often neglected and seen as unspecific interactions, the role of which was to concentrate the virus at the cell surface. However, these links are much more than ‘simple tethers or unspecific step’ as previously thought. Our study highlights a physiologically relevant interplay between the attachment factors (α-linked sialic acid glycans [α-SA]) and the specific entry receptor (junctional adhesion molecule A [JAM-A]). Our in vitro and cellular experiments revealed a cooperative effect. The α-SA binding to the viral glycoprotein, which is engaged with low affinity, serves as the initial attachment event and further triggers a conformational change into the viral glycoprotein. The glycoprotein adopts a more extended conformation that facilitate its specific interactions with the high-affinity JAM-A receptor. Moreover, we discovered that short sialylated glycans induce an enhancement of reovirus receptor binding, due to a conformational change in the glycoprotein. This leads to an increased binding avidity and ultimately infectivity, which can be applied for future vaccine and oncolytic treatments.

You mention in the publication that these insights could lead to the use of reoviruses as oncolytic agents – could you elaborate on that?

The main means of cancer treatment such as chemotherapy, radiotherapy, and even targeted kinase inhibitors and mAbs are limited by lack of efficacy, cellular resistance, and toxicity. Oncolytic viral therapy offers a novel therapeutic strategy that has the potential to dramatically improve clinical outcomes by having a wide spectrum of anticancer activity with minimal human toxicity.

Reovirus is a leading candidate for therapeutic development and currently in phase III trials. Reovirus selectively targets transformed cells with activated Ras signaling pathways. Ras genes are some of the most frequently mutated oncogenes in human cancer and it is estimated that at least 30% of all human tumors exhibit aberrant Ras signaling. By targeting Ras-activated cells, reovirus can directly lyse cancer cells, disrupt tumor immunosuppressive mechanisms, reestablish multicellular immune surveillance, and generate robust antitumor responses. Reovirus phase I clinical trials have shown indications of efficacy, and several phase II/III trials are ongoing at present. Reovirus’ extensive preclinical efficacy, replication competency, and low toxicity profile in humans have placed it as an attractive anticancer therapeutic for ongoing clinical testing. Despite advances in our understanding of the host and viral determinants that underlie reovirus replication and killing of transformed cells, many gaps in knowledge remain. With all these developments but also open questions in mind, our study provides unique opportunities to manipulate reovirus binding efficiency and infectivity for vaccine and oncolytic applications.

Learn More

Read the full article “Glycan-mediated enhancement of reovirus receptor binding” in Nature Communications

Learn about the ZEISS technology used in this publication:

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Cellular Therapy Opens up New Perspectives

Professor Alp Can from Ankara University speaks about his research

Professor Alp Can is the director of the Department of Histology and Embryology at Ankara University School of Medicine. Moreover, he is also responsible for the microscopic multi-imaging facility, which hosts many ZEISS microscopes at various levels.

Prof. Can’s main research topic is to investigate the cellular properties of human umbilical cord mesenchymal stromal cells with regard to using them in cellular therapies. In this interview we asked him three questions concerning his research area.

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Prof. Alp Can from Ankara University investigates the cellular properties of human umbilical cord mesenchymal stromal cells with a ZEISS microscope.

What are the big issues in your research area?

Prof. Alp Can“Cellular therapy is a relatively new issue, but seems very promising as different types of cells are under extensive focus in many animal experiments and clinical trials. As they are tested in various organisms and tissue compartment their novel properties are recognized and give credence to new therapy regimens.”

What do you think are your most significant research accomplishments?

Prof. Alp Can“Cellular development processes starting from the very early stages of zygote formation up to the differentiation of cell clusters into specific tissues are very thrilling and exciting topics as they still hide many unanswered questions. Recently, induced pluripotent stem cell technology opened insights to the novel gene control and regulatory mechanisms, which may solve some common health problems that has never been tested before. In that sense, high-end microscopy is still one of the most import research and diagnostic tool, as preclinical researchers and physicians can see the in vivo live and diseased tissues/cells in front of their very eyes.”

Human umbilical cord mesenchymal stromal cells after they were induced for adipogenic transdifferentiation in vitro for 35 days. Green signal refers to F-actin filaments, red signal lipid droplets and blue signal for DNA. Image courtesy of Alp Can, Ankara University, Turkey.
Human umbilical cord mesenchymal stromal cells in culture. Cells were labeled with anti-vimentin (yellow) and Hoechst 33342 (magenta). Image courtesy of Alp Can, Ankara University, Turkey.

If you had unlimited resources, what would you do with them?

Prof. Alp Can“My team is working on the cellular and subcellular features of human fetal cells which give promises to be used in the regenerative medicine. I think the era we live in is the “era of regeneration and rejuvenation” as human beings are getting older while they want to pursue maintaining their health status till the end of their last day. So, my ultimate effort in the lab would be to find out evidence-based and reproducible remedies to human aging, which in fact starts at birth!”

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Automated Microscope for Gentle and Fast Confocal 4D Imaging

Enhancing ZEISS Celldiscoverer 7 with ZEISS LSM 900 for optical sectioning

The proven ZEISS Celldiscoverer 7 is a fully integrated high-end imaging system with various incubation and detection options. It combines the easy-to-use automation of a boxed microscope with the image quality and flexibility of a classic inverted research microscope.  To get better data from three-dimensional samples, it is now possible to add ZEISS LSM 900 with Airyscan 2 for confocal imaging.

Learn more about this powerful combination:

Connecting widefield and confocal – the best of both worlds

Life sciences research often calls for optical sectioning to image samples with best possible contrast and resolution. By adding ZEISS LSM 900 with Airyscan 2 to ZEISS Celldiscoverer 7, users get the ease-of-use and automation from a fully integrated microscope platform and the superb confocal image quality and flexibility of the ZEISS LSM 9 family with Airyscan 2. The new Multiplex mode allows the user to perform superresolution 3D imaging with up to 1.5x higher resolution. Additionally, researchers can easily separate multiple labels with spectral imaging.

Understand the technology behind:

A flexible, integrated microscope

ZEISS Celldiscoverer 7 simplifies the laboratory setup and makes work more comfortable. All components are optimized for hassle-free automated imaging. New users and multi-user facilities especially enjoy the in-built automation and usability features when setting up complex experiments. Users can expect better data in shorter times, with less training and maintenance. As the user’s requirements grow, they can expand ZEISS Celldiscoverer 7 with confocal technology, external cameras, deconvolution, and additional environmental control – whatever they need for the challenge of live cell observation.

Observe primary lung fibroblasts stained with mitotracker red (mitochondria) and a DNA marker (nuclei). Sample courtesy of S. Gawrzak and M. Jechlinger, EMBL, Heidelberg, Germany:

More information on ZEISS Celldiscoverer 7

ZEISS Celldiscoverer 7 expands the possibilities of automated microscopy: Flexible: ZEISS Celldiscoverer 7 comes with various incubation and detection options, so you can tailor the system to your applications Better data: the optional ZEISS LSM 900 with Airyscan 2 allows fast and gentle superresolution imaging; Autocorr objectives, Autofocus and Autoimmersion make your work easier: you always get images with crisp contrast and high resolution in large fields of view Reproducible: automatic calibration routines make sure each experiment delivers reproducible data; with barcode recognition, you can identify your sample, sample carrier and even the type of experiment

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New Multiplex Mode for ZEISS Airyscan 2 Enables Fast and Gentle Confocal Microscopy

ZEISS LSM 9 family for life sciences research introduced

Life sciences research can be demanding, and if you are involved in neuroscience, cancer research or other cell- or organism-based disciplines, you’ll often need microscopy data for your work. Emerging technologies such as CRISPR / Cas open up innovative ways of thinking and allow you to ask altogether new scientific questions, deeply affecting your imaging experiments. To monitor life as undisturbed as possible requires low labeling density for your biological models—for example, 3D cell culture, spheroids, organoids or even whole organisms—and this calls for 3D imaging that combines optical sectioning with low phototoxicity and high speed.

The new Multiplex mode

The new Multiplex mode for ZEISS Airyscan 2 delivers more information in less time. Smart illumination and detection schemes allow parallel pixel acquisition for fast and gentle confocal microscopy. Researchers can now image their most challenging three-dimensional samples with high framerates in superresolution. The speed and sensitivity gain can be used to gently capture either fixed samples in larger fields of view with higher throughput and without bleaching, or dynamic processes in living specimens with minimal disturbance.

The Multiplex mode is available for the whole ZEISS LSM 9 familyZEISS LSM 980 is the flexible research platform with complementary multiphoton and superresolution capabilities. ZEISS LSM 900 is a very compact system that delivers image quality without complexity.

ZEISS LSM 980 with Airyscan 2 is the ideal platform for confocal 4D imaging that combines optical sectioning with low phototoxicity and high speed.

ZEISS LSM 980

The new ZEISS LSM 980 with Airyscan 2 is the ideal research platform for confocal 4D imaging. The entire beam path is optimized for simultaneous spectral detection of multiple weak labels with the highest light efficiency. Researchers benefit from the full flexibility of a point scanning confocal and the speed and gentleness of the sensitive ZEISS Airyscan 2 detector. The new Multiplex mode combines an elongated excitation laser spot and parallel pixel readout of this area detector. This allows acquiring up to eight image lines in a single sweep. Users can gently image larger fields of view with superresolution in shorter acquisition times than ever before.

Watch the product trailer:

ZEISS LSM 900

ZEISS LSM 900 with Airyscan 2 is a very compact confocal microscope for high-end imaging. This system has a genuinely small footprint, concentrating on the essence of a confocal and leaving out needless complexity. It fits easily into labs or imaging facilities and is optimized for ease of use. ZEISS LSM 900 can be combined with ZEISS Celldiscoverer 7 for automated confocal imaging with high efficiency.

Observe a cell division of LLC-PK1 cells, alpha-tubulin (mEmerald, green) and H2B (mCherry, red).  With the new Multiplex mode for ZEISS Airyscan a Z-stack of 52 slices was captured every 40 seconds for a total of 40 minutes:

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Why Do Dopamine Neurons Die Particularly Fast in a Specific Brain Area?

Research team from York uses confocal microscopy from ZEISS to investigate neurodegeneration processes in Parkinson’s disease

People with Parkinson’s lose control of their muscles, resulting in changes of speech and gait. It is caused by the death of a specific population of dopamine-producing neurons in the substantia nigra – a specific brain area that plays an important role in movement.

In a recent paper, a research team from the University of York studied Drosophila dopamine neurons using ZEISS LSM 780 in the Bioscience Technology Facility to investigate basic processes of neurodegenerative diseases. Inherited mutations in the LRRK2 (Leucine-rich-repeat kinase2) protein are the most common known cause of Parkinson’s. The authors focused on this LRRK2-G2019S mutation. Rabs – proteins involved in transmitting signals within cells – are a plausible LRRK2 substrate leading to neurodegeneration, as they act as molecular switches interacting with a range of proteins regulating supply and delivery of cargo to membranes.

Rab10 is expressed (magenta) in the dopaminergic neurons (green) controlling vision (MC neurons, white, Aii) and proboscis movement (TH-VUM, Aiii). With a higher magnification objective, the TH-VUM is seen to have green cytoplasm (anti-tyrosine hydroxylase antibody) and magenta nucleus (Rab10-RFP). However, other dopamine neurons are not Rab10 positive (green arrows). This distribution is confirmed using a second expression system (B)

Using ZEISS LSM 780 generated crisp, detailed images of the distribution of proteins, like Rab10, both in the fly brain, and within specific brain neurons.

Dr. Chris Elliott, lead author of the publication

The research team proposed that variations in Rab expression contribute to differences in neurodegeneration seen in Parkinson’s. These findings could potentially lead to new treatments, targeted to specific brain regions.

Access the paper here for free

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Spotlight on Cell Ultrastructure

New correlative approach combines superresolution confocal and scanning electron imaging

Researchers from the Department of Cell Biology, theme Nanomedicine, and the ‘Microscopy Imaging Center’ at the Radboudumc in Nijmegen, Netherlands recently developed and optimized a pipeline for correlative imaging using superresolution (SR) microscopy and scanning electron microscopy (SEM).

ZEISS Airyscan and SEM correlative imaging pipeline. (1) ITO-coated coverslips are marked on three edges for stage calibration on the Zeiss LSM880 and Sigma 300. (2) Cells are seeded on the marked coverslips for at least 3 h. (3) Live cells are sonicated to prepare VPMs and immediately fixed. (4) Sample is immunolabeled for the proteins of interest, fixed for the second time and fiducials are added. (5) The sample is imaged with Airyscan microscopy. (6) The sample is dehydrated, critical point dried and then sputtered with 5 nm chromium. (7) The sample is imaged with SEM microscopy. (8) Fiducials are used to align the LM and SEM image using Matlab.

This state-of-the-art imaging approach allows the correlative visualization of up to three cellular components by SR fluorescence microscopy and the cellular ultrastructure by SEM.

In other words, one can fluorescently label up to three different proteins and accurately determine their localization with respect to specific cellular ultrastructures.

Ben Joosten, cell biologist and part of the research team at the Radboudumc
VPM preparation and CPD procedure preserve podosome organization. (A) DCs were seeded on glass coverslips and after VPM preparation, cells were fixed and stained for actin (cyan), vinculin (green) and zyxin (magenta). After CPD, DCs were imaged by SEM (gray). Shown are representative images of all three channels in three dimensions and the corresponding SEM image. Insets depict two representative podosomes within the cluster. (B) Shown are the SEM-LM overlays for all three channels for the same cells as in (A) Scale bar = 5 μm.

Narrowing the resolution gap

Correlative light and electron microscopy (CLEM) was so far performed using conventional light microscopy (LM) and electron microscopy (EM). Although this offered unique and complementary information from the same cell or tissue sample, the interpretation of those correlative images was challenged by the fact that the lateral resolution of conventional LM (~250 nm) is much worse than the lateral resolution of EM (~2 nm). This is referred to as the “resolution gap”.

The Radboundumc research team: Top row from left: Marieke Willemse, Jack Fransen; Bottom row from left: Ben Joosten, Koen van den Dries and Alessandra Cambi

Our correlative imaging pipeline, called SR-CLEM, narrows this gap as it combines the ultrastructure provided by the ZEISS Sigma SEM with super-resolved fluorescent images acquired with ZEISS LSM 800 with Airyscan (lateral resolution of ~140 nm).

Ben Joosten, cell biologist and part of the research team at the Radboudumc

He and his colleague Koen van den Dries used SR-CLEM to study the nanoscale architecture of podosomes, small cytoskeletal structures used by leukocytes to transmigrate basement membranes or by osteoclasts to remodel bone tissue. The results of the study are published in Frontiers in Immunology, the most-cited open-access journal in immunology.

The SR-CLEM approach is particularly interesting for elucidating the organization of complex multimolecular cellular structures as well as for characterizing microorganisms, nanomaterials or nanoparticles and their interaction with cells.

Jack Fransen, Associate Professor at the Radboudumc

Read the paper, published in Frontiers in Immunology, here

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The Lamprey Regenerates Its Spinal Cord Not Just Once – but Twice

Marine Biological Laboratory (MBL) scientists determine central nervous system regeneration with ZEISS microscopes

Spontaneous recovery from spinal cord injury is almost unheard of in humans and other mammals, but many vertebrates fare better. The eel-like lamprey, for instance, can fully regenerate its spinal cord even after it’s been severed – within three months the lamprey is swimming, burrowing, and flipping around again, as if nothing had happened.

In a new study, Marine Biological Laboratory (MBL) scientists report that lampreys recover and regenerate just as impressively after a second complete spinal cord injury at the same location. The study opens up a new path for identifying pro-regenerative molecules and potential therapeutic targets for human spinal cord injury.

Research insights

We’ve determined that central nervous system (CNS) regeneration in lampreys is resilient and robust after multiple injuries. The regeneration is nearly identical to the first time, both anatomically and functionally.

Jennifer Morgan, senior author and Director of the MBL’s Eugene Bell Center for Regenerative Biology and Tissue Engineering

Morgan’s lab has been focusing on the descending neurons, which originate in the brain and send motor signals down to the spinal cord. Some of these descending neurons regenerate after CNS injury in lamprey, while others die.

Using ZEISS Axio Imager M2 and ZEISS Axio Zoom.V16 stereo microscopes, as well as a ZEISS LSM confocal microscope, we were able to visualize the regenerating neurons in great detail. This allowed us to describe and quantify the regenerating neurons, thus enabling the major results of the study.

Jennifer Morgan
Longitudinal section of a lamprey spinal cord at 11 weeks post-injury, showing many regenerated axons (green) and a repaired central canal (blue tubelike structure). The original lesion site is in the center of the image. Credit: S. Allen and J. Morgan

We are beginning to isolate individual descending neurons and look at their transcriptional profiles (gene activity) to see if we can determine what makes some of them better at regenerating than others. The ‘good’ regenerators, for example, may express molecules that are known to promote growth during development. That’s one hypothesis.

Jennifer Morgan

Sea Lamprey and Regeneration – Jennifer Morgan from Marine Biological Laboratory on Vimeo.

Better strategies for treatments

Observing how the descending neurons respond to a second CNS injury can help the team tease out the factors required for repeated, resilient regeneration, which could have implications for designing better strategies for treatments aimed at promoting CNS re-growth after injury or disease.

Read the present study, published in PLOS ONE, here

Regeneration has been a core area of research at the Marine Biological Laboratory since its founding, particularly in the pioneering work of Nobel laureate Thomas Hunt Morgan, an embryologist and geneticist whose 1901 text “Regeneration” is a classic in the field. Over a century later Morgan’s lab in particular has led many breakthroughs, including a 2018 study that found genes that aid spinal cord healing in lamprey are also present in mammals. In 2010, the Eugene Bell Center for Regenerative Biology and Tissue Engineering was established at MBL in honor of Eugene Bell (1919-2007), a pioneer of tissue engineering and a valued member of the MBL scientific community. Scientists in the Bell Center, in collaboration with colleagues at the University of Chicago and the Argonne National Laboratory, are providing new insights into the basic mechanisms of tissue growth, repair and regeneration in all metazoans that will permit novel approaches to the understanding, treatment and prevention of human disease.

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African Students Fascinated by the Possibilities of Confocal Microscopy

Microscopy and image analysis course at University of Ghana

Recently, the two-week course “Introduction to fluorescent microscopy and image analysis techniques” was held at the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) at University of Ghana at Legon. It helped 15 Master’s and PhD students from different universities in Ghana and Senegal gain a basic understanding of light microscopy techniques and computerized image analysis. The course consisted of both theoretical as well as practical sessions and was designed to enable students to use these applications in their biological and biomedical research.

The participants appreciated the opportunities that have become available to them with the first confocal microscope to be located in West Africa.

Basic optical principles, different light microscopy techniques, appropriate applications, benefits and limitations as well as advanced microscopy methods such as superresolution were the subject of the course. Participants gained practical experience on different sample preparation approaches. Hands-on sessions on ZEISS LSM 800 with Airyscan were also included. The participants gained awareness of the different image analysis approaches and software that can be used and what they can achieve.

“We received a high number of applications for the course and those students who were selected showed a great level of enthusiasm. Both verbal and written feedback after the course emphasized the excitement of the students for the techniques they had learnt about”, said Petra Stockinger, Scientific Officer at University of Gothenburg and one of the organizers of the course.

The participants of the microscopy and image analysis course at University of Ghana showed a great level of enthusiasm.

Thank you very much for the opportunity to be part of the training. I really enjoyed every bit of it and I learnt a lot of valuable skills.

Do more collab(oration)s with African universities. It would be great if you could offer this kind of opportunity to other African students.

Course participants

“For many students, it was the first time they used fluorescent microscopy and they were actually able to directly observe cellular features of trypanosoma parasites as well as of different stages of malaria infection with high resolution”, Petra said, summarizing the success of the course. “Finally, the students spent some time considering how the approaches they have learnt about could be applied to their current and future research.”

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A Journey Into a Human Kidney

ZEISS microscopes help uncover centuries old hidden secrets of human kidney stones

Kidney stones are hard deposits made of minerals and salts that can form inside your kidneys. They have been ascribed no medical value at all, and doctors usually discard them right away. A research team led by Bruce Fouke, a geology and microbiology professor at the University of Illinois, have now shown their complex structure and composition. These findings may lead to better diagnostics and treatment.

A layered history of the kidney’s physiology

Many doctors assume that kidney stones are homogeneous, insoluble, even boring. They either break them using ultrasound leading them to painful passage or more invasively surgically remove them once a pathological stone is brought in to a hospital setting. Recent finding schallenge this 150 year notion that kidney stones could not be dissolved at all, which set the mindset of medical professionals and physicians.

The Carl R. Woese Institute of Biology scientists (from left): Jessica Saw, Bruce Fouke, Mayandi Sivaguru Photo by L. Brian Stauffer

Mayandi Sivaguru (Associate Director of Core Facilities, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, a microscopist), Jessica Saw (an M.D. Ph.D. student from Mayo Clinic) and Bruce Fouke (Professor of Geology and Microbiology and Director of Carver Biotech Center) recently published a paper in the journal Scientific Reports: DOI: 10.1038/s41598-018-31890-9

The team found – unlike the conventional wisdom – that the Calcium oxalate stone which comprise over 70% of all kidney stones could not be dissolved. It is actually undergoing multiple steps of dissolution and recrystallization during the course of its growth.

Instead of looking at these stones as static lumps of crystals, imagine they have a record of daily, if not hourly and minute-by-minute record of bodily fluid, food and metabolism like a record of environment and climate in tree rings and other biomineralization settings in the nature.

Bruce Fouke, Professor of Geology and Microbiology and Director of Carver Biotech Center

Colorful snapshots

Dr. Fouke and his fellow researchers examined more than 50 kidney stone fragments from six Mayo Clinic patients.

A tiled (20×20, 400 images x 3 channels= ~1200 images) 405, 488 and 561 nm ex and their corresponding emission detected using the ZEISS LSM 880 confocal system showing a single stone could be actually a combination of 3 stone complex. Image provided by Mayandi Sivaguru, Jessica Saw and Bruce Fouke.

The team used a variety of optical modalities available at this ZEISS labs@location partner facility. In addition to existing optical techniques from diffraction limit to superresolution, the team has used combination of optical techniques which are never tried before to retrieve high-frequency layering information as in this example, where a phase contrast technique is coupled with either crossed nicols polarization or circular polarization, which enabled to both visualize and quantify high frequency nano-layering.

We left no crystals un-turned.

Mayandi Sivaguru, Associate Director of Core Facilities, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign

This is the first time the authors employed autofluorescence using both the confocal and superresolution modalities of ZEISS LSM 880 with Airyscan. Conventionally, people looked at kidney stones using brightfield, POL and SEM and TEM microscopy.

The images reveal triangles and other geometrics. The disruptive patterns in the stones showed that the vast majority of the material had dissolved and reformed over time.

Future treatment?

Finally, after looking at these human kidney stones under a multitude of optical and electron microscopes the team also provide clinical intervention strategies to start thinking about making new treatments by providing a roadmap for future implications.

The scientists argue that these understandings will help uncover hidden mechanisms of human diseases caused by both dissolution of crystals of calcium phosphate (of bones in the case of arthritis) and crystallization (in the case of arthrosclerosis), thereby treatments could be tailored to preventing them, eventually.

Clinical intervention strategies to dissolve the kidney stones inside kidney rather than using current painful passage or invasive surgeries in the pipeline. Image provided by Mayandi Sivaguru, Jessica Saw and Bruce Fouke.

Doctors often base patient care plans on the chemistry and molecular components of a patient’s urine, yet further research could allow doctors to take advantage of the changing composition of kidney stones themselves. Specific ingredients could then dissolve the stones completely– without painful passage or invasive procedures.

More information on ZEISS Airyscan

Read the New York Times article

A look into history

In 1868, Leslie Beale, a British scientist, first documented calcium oxalates are “difficult to dissolve”. That is where it all started over four years ago, when Prof. Fouke and this team started looking at human kidney stone samples from Mayo Clinic, Rochester patients. Approximately at the same time, Carl Zeiss , Ernst Abbe and Otto Schott make history by producing the first microscopes with science-based optics.

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A New Platform for Fast and Gentle 3D Superresolution Microscopy

ZEISS Elyra 7 with Lattice SIM expands the possibilities of structured illumination microscopy (SIM)

ZEISS Elyra 7 with Lattice SIM is a new flexible platform for fast and gentle 3D superresolution. Lattice SIM expands the possibilities of structured illumination microscopy (SIM): illuminating the sample with a lattice pattern rather than grid lines gives higher contrast and allows a more robust image reconstruction. Scientists can use 2x higher sampling efficiency to lower the illumination dosage to observe fast cellular processes in superresolution. High image quality is maintained even at high frame rates.

ZEISS Elyra 7 with Lattice SIM is the new flexible platform for fast and gentle 3D superresolution.

Fast and gentle superresolution imaging with Lattice SIM

Lattice SIM enables fast imaging of 3D volumes with up to 120 nm laterally. Thanks to greater light efficiency, the new Lattice SIM technology provides scientists with gentle superresolution imaging of living specimens with 255 frames per second. Using less light to illuminate the specimen means scientists can image longer with less bleaching of the sample. The novel Lattice SIM technology allows new mechanistic details to be uncovered and the finest subcellular structures in large fields of view to be quantified.

Understand how Lattice SIM works:

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Discovery in Telomere Biology Advances Understanding of Cancer, Ageing and Heart Disease

ZEISS Airyscan technology helps to reveal telomere structure

A team of Sydney scientists has made a ground-breaking discovery in telomere biology, with implications for conditions ranging from cancer to ageing and heart disease. The research project was led by Dr. Tony Cesare, Head of the Genome Integrity Unit at Children’s Medical Research Institute (CMRI) at Westmead, in collaboration with scientists from CMRI as well as UNSW Sydney’s Katharina Gaus. The unique area detector technology of ZEISS LSM 880 with Airyscan made it possible to image telomere structures.

What are telomeres?

Telomeres are DNA segments at the ends of every human chromosome. As we age, telomere length naturally decreases. Over the course of a lifetime, telomere shortening instructs ageing cells to stop dividing.

This normally functions as a critical barrier to stop cancer. However, some people are born with abnormally short telomeres and suffer from bone marrow failure, pulmonary fibrosis and high rates of cancer. Telomere length is also an important marker of disease risk for conditions such as cancer, heart disease and diabetes.

Telomere shortening causes chromosomes ends to resemble broken DNA. However, it has remained a mystery why telomeres change from healthy to unhealthy with age. This research has identified the underlying cause.

Confocal imaging with ZEISS Airyscan reveals: Telomere structure matters

We knew that telomeres regulate cellular ageing, but our new data explain the trigger that makes telomeres unhealthy. Telomeres normally form a loop structure, where the chromosome end is hidden. We found that when the telomere-loop unfolds, the chromosome end is exposed and the cell perceives this as broken DNA. It is not telomere length that matters, but telomere structure. The telomere-loop becomes harder to form as telomeres get short.

Dr. Tony Cesare, Head of the Genome Integrity Unit at Children’s Medical Research Institute (CMRI) at Westmead

Additionally, the team identified that telomeres can also change structure in response to some chemotherapeutic agents, which helps kill cancer cells.

The results of this study have also proven how important technological advances are in the field of research. Dr Cesare first developed his theories about telomere-loops in 2002 when studying for his PhD. However, the technology was not available at the time to easily visualize telomere-loops using microscopy.

However, the advent of superresolution microscopy, which was awarded the 2014 Nobel Prize in Chemistry, made it possible to see telomere-loops with a microscope. To complete this research, the team used superresolution microscopes at four Sydney research institutions, and purchased the first ZEISS LSM 880 with Airyscan in Australia.

ZEISS Airyscan allowed us to see ten times more detail than we had in the past. We could pass the physical limits of light and see the telomere-loop structure.

Dr. Tony Cesare
Mitotic chromosomes from the HT1080 6TG human fibrosarcoma cell line stained with propidium iodide to identify DNA content and telomere fluorescent in situ hybridization (green) to identify the repetitive telomere DNA sequence.

To complete the project, the team combined this breakthrough technology with powerful genetic models that mimic cellular aging.

We were only the second group in the world to see telomere-loops with superresolution microscopes and the first to determine their function. It took us four and a half years to complete the project. It has been an enormous effort that I didn’t think was feasible five years ago. We’ve shown that it’s not just telomere length, but telomere structure and telomere health that we need to understand. The next step is to ask, can we correlate human health with telomere health? Our work suggests there is more to the story than just measuring telomere length.

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Read the paper describing these studies, ‘Telomere Loop Dynamics in Chromosome End Protection’, published online by Molecular Cell here

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Cell Reports Focus: Brain Imaging

ZEISS & Cell Press present focus issue that celebrates the power of modern imaging to reveal new insights into the architecture and operation of nervous systems

The topics in this Cell Press focus issue on brain imaging range from the molecular framework of axons to the large-scale organization of whole brains, and the techniques include structured-illumination super resolution microscopy and serial two-photontomography.

Application notes on ZEISS LSM 880 Airyscan and ZEISS Cryo-Airyscan imaging are also included.

Download the free supplement here

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New Publication Shows the Potential of ZEISS Airyscan

Comparing Airyscan technology to conventional confocal imaging in live cell imaging

The recently published peer-reviewed paper “Exploring the Potential of Airyscan Microscopy for Live Cell Imaging” by Kseniya Korobchevskaya, B. Christoffer Lagerholm, Huw Colin-York and Marco Fritzsche from the University of Oxford, UK highlights the significant improvement of resolution and signal-to-noise at the same time of ZEISS Airyscan compared to conventional confocal imaging techniques. The article was published as part of the Special Issue Superresolution Optical Microscopy.

Biomedical research demands non-invasive and ultra-sensitive imaging techniques. Especially, our laboratory for Biophysical immunology at the MRC Human Immunology Unit and Kennedy Institute for Rheumatology at the University of Oxford relies on state-of-the-art imaging technology with extended spatial and temporal resolution as offered by the novel ZEISS Airyscan technology. In our recent paper, we demonstrate how Airyscan imaging successfully bridges the gap between conventional confocal and super-resolution microscopy.

Marco Fritzsche, University of Oxford

Abstract

Biological research increasingly demands the use of non-invasive and ultra-sensitive imaging techniques. The Airyscan technology was recently developed to bridge the gap between conventional confocal and super-resolution microscopy. This technique combines confocal imaging with a 0.2 Airy Unit pinhole, deconvolution and the pixel-reassignment principle in order to enhance both the spatial resolution and signal-to-noise-ratio without increasing the excitation power and acquisition time. Here, we present a detailed study evaluating the performance of Airyscan as compared to confocal microscopy by imaging a variety of reference samples and biological specimens with different acquisition and processing parameters. We found that the processed Airyscan images at default deconvolution settings have a spatial resolution similar to that of conventional confocal imaging with a pinhole setting of 0.2 Airy Units, but with a significantly improved signal-to-noise-ratio. Further gains in the spatial resolution could be achieved by the use of enhanced deconvolution filter settings, but at a steady loss in the signal-to-noise ratio, which at more extreme settings resulted in significant data loss and image distortion.

Time-lapse images of activating Rat Basophilic Leukaemia (RBL) cell. (a) Comparison of 1.25 AU confocal and Airyscan processed AF6.7 images. Scale bar is 10 µm. (b) Direct comparison of Region of Interest area (ROI; red rectangle in (a)) between confocal 1.25 AU and Airy processed images with AF4, AF6 and AF7, respectively. Scale bar is 5 µm. (c) Time-lapse of activating RBL cell at 0, 60 and 120 s, respectively. Green LifeAct-citrine (excitation at 488 nm), red SNAP-tag (excitation at 561 nm). (d) Intensity profiles from 1.25 AU (grey filled), AF7 (blue dots) and AF8 (red) images along the line indicated by white arrows in (b). Arrows indicate peaks from two separate actin fibres, which are only distinguishable at high AF strength and are not resolved at 1.25 AU.

The authors of this paper focused on the superresolution aspect. To learn more about the enhanced sensitivity and speed of ZEISS Airyscan read this free paper.

Watch this video and understand how ZEISS Airyscan works:

Read the full paper: Korobchevskaya K, Lagerholm BC, Colin-York H, and Fritzsche M, Exploring the Potential of Airyscan Microscopy for Live Cell Imaging, Photonics, 2017

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New Video Publication of Developing Arabidopsis Flowers

Live confocal imaging with ZEISS microscopes

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Watch > Live Confocal Imaging of Developing Arabidopsis Flowers by Nathanaël Prunet, Department of Biology and Biological Engineering, California Institute of Technology.

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