Looking Closely at Germ Cell Development

Researchers use superresolution to better understand the formation of germ granules

Germ cells are special types of stem cells that give rise to sperm and egg – the cells that combine during reproduction to create the next generation. Dr. Alexey Arkov and his lab at Murray State University (USA) work to better understand the molecules that are critical for germ cell development using the fruit fly model organism for their experiments.

In their recent publication, Dr. Arkov and his team focus on better understanding the formation of germ granules – unique molecular complexes which contain both RNA and protein and are required for germ cell development. They used the ZEISS Airyscan superresolution technology to look at specific proteins integrated into these granules to better understand how these important molecules assemble.

We interviewed Dr. Arkov to learn a bit more about his research and his use of the Airyscan superresolution technology.

Could you explain a bit more about germ granules and why they are a subject of interest for your lab and scientific discovery?

Germ granules have been found in over 80 animals, as diverse as rotifer and humans, and they are assembled from components, which are crucial for germ cell development. We would like to understand why these important components are put together in the granules to provide insights into the developmental mechanisms of germ cell specification. Furthermore, these germ granules provide a paradigm to study the assembly and function of large and dynamic RNA-protein structures, which lack the membrane, since many other membraneless RNA-protein granules are assembled in different types of cells. 

Did the Airyscan superresolution technology reveal anything novel compared to past data?

Superresolution microscopy has been used to image germ granules, however, in our work we focused on protein components of the granules, which have not been explored in the same detail by superresolution microscopy approaches in the fruit fly model as RNA components of the granules. When we looked at the protein localization in the granule with the Airyscan superresolution technology, we were surprised to find that individual proteins are not randomly distributed in the granule but rather assemble as separate clusters, which only partially overlap in the same granule.  

Imaris Snapshot
Distinct partially overlapping protein clusters assemble into germ granules. Posterior pole of early fruit fly embryo was immunostained to label two protein components of germ granules, Tudor (green) and Aubergine (red). An optical section image obtained with ZEISS Airyscan superresolution technology shows multiple individual germ granules formed from distinct Tudor and Aubergine clusters overlapping at the “interaction hubs”.

Was there anything in your publication that you are particularly excited about or that surprised you?

Proteins, described in our publication, associate with each other directly, therefore, it was really surprising to see that these proteins overlap in the granule only partially. We refer to these regions of proteins’ partial overlap as “interaction hubs”.  Overall, our data indicate that at least some protein building blocks of the granules assemble as distinct modules linked at the “interaction hubs”. Therefore, building the germ granule may be somewhat similar to building a structure from LEGO pieces or creating a mosaic art. 

3D reconstruction of individual germ granule. Multiple optical sections were used for 3D rendering of a single germ granule from fruit fly embryo’s posterior pole using Imaris software (Oxford Instruments). Tudor and Aubergine protein clusters within the granule are indicated with green and red respectively.     

Based on your findings here, where do you see your research going next?

We are in the process of studying additional components of the granules to see whether they follow the same assembly pathway as the proteins characterized in our publication. Also, we are characterizing germ granule-like structures in other cell types. We are using superresolution microscopy as well as genetics, biochemistry and structural methodology to decipher the precise assembly mechanism of these RNA-protein granules and, importantly, how this ordered and structured assembly contributes to function that these granules perform in the cell.

Learn More

Read the full article “Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development

Learn more about ZEISS Airyscan, the superresolution detector for ZEISS confocal microscopes.

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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.

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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:

More information on ZEISS LSM 980

More information on ZEISS LSM 900

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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

More information on Correlative Microscopy Solutions from ZEISS

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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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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.

More information on ZEISS Airyscan

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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Imaging Biological Samples with ZEISS LSM 800 and LSM 880

A new reference list details scientific work with ZEISS LSM 8 family of confocal microscopes with Airyscan detector.

The Confocal Laser Scanning Microscope (LSM) has become one of the most popular instruments for fluorescence imaging in biomedical research, because it affords researchers images with high contrast and a versatile optical sectioning capability to investigate three dimensional biological structures.

The optical sectioning ability of an LSM is a product of scanning a focused laser spot, across a sample to create an image one point at a time. The generated fluorescence from each point is collected by the imaging objective and results from fluorophores in the sample that reside both in the desired plane of focus and in out of focus planes. In order to segregate the fluorescence emitted from the desired focal plane, an aperture (pinhole) is positioned in the light path to block all out of focus light from reaching the detector (traditionally a PMT). The traditional principle of the LSM beampath forces the user to compromise either on resolution or sensitivity. Resolution is increased by closing the pinhole, at the same time limiting the amount of light that is allowed to pass through to the detector.

The Airyscan detector of LSM 880 and LSM 800 overcomes this challenge. The detector consists of 32 GaAsP PMT detector elements, which are arranged in a hexagonal array (Figure 1), positioned at a conjugated focal plane in the beam path the detector is functioning as the traditional LSM pinhole. This design makes it possible to collect more light (equivalent to a pinhole opened to 1.25 AU), whilst at the same time dramatically enhancing the resolution, with every detector element acting as an efficient pinhole with a diameter of only 0.2 Airy Unit (AU).

Instead of facing an either / or decision, a simultaneous enhancement of resolution by the factor of 1.7× and signal-to-noise by 4 – 8× was introduced to LSM imaging. Detailed descriptions of the theory and technology of Airyscanning can be found in separate technology note. As an area detector, Airyscan can capture spatial information that is utilized to parallelize the scanning process, collecting 4 image lines simultaneously in the Fast mode (Figure 2). This means enhancing acquisition speed by a factor of 4 while keeping high pixel dwell times to efficiently collect emitted photons. In standard mode, the focused laser beam is moved along the x-axis to acquire one image line, before it is moved in the y-axis to acquire the consecutive image line. In Fast mode imaging, four image lines are acquired at the same time when moving the laser in the x-direction. A new publication list assembles some of the scientific work that has been done with LSM 880 and 800 systems. The great variety of applications collectively profits from the light efficient beam path of the LSM systems, and the unique combination of superresolution, high sensitivity and high speed imaging provided by Airyscan.

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