Imaging


For a young scientist, Hari Shroff, co-director of the Optical Microscopy and Imaging course at MBL, has seen his share of career peaks. Shroff entered the University of Washington at age 14 and graduated when many people are just starting college. After completing his doctorate in biophysics in 2006 at the University of California, Berkeley, Shroff took the MBL Physiology course. It had “a huge influence on me,” Shroff says in this interview with Prashant Prabhat of Semrock. “I was working hand-in-hand with a lot of the experts in cell biology,” Shroff recalls, and they drove home how fundamental microscopy is to their field.

That same year, Shroff heard microscope developer Eric Betzig give a talk at Berkeley. “I have always been very fascinated by the fundamental mismatch in size between what a biologist wants to see and what they actually can see,” Shroff tells Prabhat. “[Betzig] was talking a little bit about super-resolution, and I wanted to drop what I was doing and immediately work for him.” Shroff felt lucky to become one of Betzig’s first hires at his lab at Howard Hughes Medical Institute’s newly opened Janelia Research Campus.

Shroff came back to the MBL Physiology course in 2007 as a teaching assistant, along with Betzig as visiting faculty. And there was important cargo in their van when they drove to Woods Hole: the super-resolution microscope Betzig and colleagues had invented, called PALM (photoactivated localization microscopy), which Shroff had a hand in developing. The scope’s power to visualize individual molecules at nanometer resolution bowled over the Physiology course participants and soon became the talk of the MBL campus.

“Those were very heady, exciting times, but also sleepless times,” Shroff tells Prabhat. “Something very special happens [at the MBL] during the summer when you have these world-class scientists congregating for a couple of months. You end up with these collisions which are just difficult to have otherwise. People have this kind of ‘can do’ attitude about science, and it’s also a great place for microscopy because some of the world’s best microscopists usually hang out there during the summers.”

Hari Shroff of the NIH shows MBL Neurobiology course students the light-sheet microscope he built (diSPIM). Credit: Tom Kleindinst

Hari Shroff of the NIH shows MBL students the light-sheet microscope he built (diSPIM). Credit: Tom Kleindinst

Important applications of Betzig’s microscope came out of that Physiology course session, which was led by course co-director Jennifer Lippincott-Schwartz of the NIH, an early collaborator with Betzig on PALM. These included live-cell, single particle tracking (sptPALM), which Betzig says “has become one of the most useful and biologically informative applications of the technology. That idea was born while we were waiting for a ferry ride in Woods Hole.” They also figured out how to label two colors of photo-activatable probes (double-color PALM) during the course, which Shroff et al published later that year.

In 2014, Betzig won a Nobel Prize in Chemistry for his contributions to super-resolution fluorescence microscopy. Shroff, meanwhile, had become a section chief at the NIH’s National Institute of Biomedical Imaging and Engineering. He was also invited to co-direct the Optical Microscopy and Imaging course, where he shows students how to build a microscope from scratch, among other challenges. The course is a lot of work, Shroff says, but “definitely fun. I actually get some of my best ideas just from daydreaming and talking to students.”

 

If you check the MBL’s Twitter feed during the summer months, you’ll be treated to quick, highly enthusiastic, and often visually beautiful dispatches from the MBL’s Summer Courses. The students and faculty are pursuing up-to-the-minute questions in life sciences research using a wide array of high-end imaging equipment, and some of the images they produce are eye-popping. Here are just a few recent Twitter posts from MBL students and faculty:

Vincent Boudreau (@viboud), a graduate student in the Physiology Course from University of North Carolina, Chapel Hill, Tweeted out this video, which he and several students made during the course’s biochemistry bootcamp under the supervision of Sabine Petry of Princeton University and Robert Fischer of the National Institutes of Health. “This bootcamp experiment taught us students how to do the biochemical legwork involved to get these microtubules to give us such stunning images,” Boudreau says. Microtubules (red) can be seen branching off of one another, marked by the green EB1 protein at their outwardly growing extremity. Video made with a Nikon TIRF microscope.

The MBL Embryology Course, tweeting under the hashtag #embryo2015, has shared one striking image after another. This is a tardigrade (a bizarre-looking, microscopic, water-dwelling animal) imaged with light-sheet microscopy by two students in the course: Christina Zakas, a post-doc at New York University who tweets @CZakDerv, and Nick Shikuma, a post-doc at Caltech.

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Tardigrade stained with DAPI to highlight nuclei and imaged on the Zeiss lighsheet Z1. Credit: C. Zakas and N. Shikuma, MBL Embryology course

Speaking of Embryology, several students in the course are blogging about their MBL experiences at the Node, an online community resource run by The Company of Biologists.  Check out their impressions of the course — its sheer intensity, its “exquisite coordination,” and the fun that balances all the hard work.

Embryology Course Co-director Alejandro Sánchez Alvarado, an expert Tweeter, once in a while reminds the students to step back from the bench, take a deep breath, and enjoy the beauty of Woods Hole. He called this scene “the rewards of Eel Pond after a rich day of learning and experimentation.”

Eel Pond, Woods Hole. Credit: Alejandro Sánchez Alvarado of the Stowers Institute/HHMI

Eel Pond, Woods Hole. Credit: Alejandro Sánchez Alvarado of the Stowers Institute/HHMI

 

The Oosight(R) product line of microscopes, developed at the MBL  and commercialized by Cambridge Research & Instrumentation, Inc. (CRi), has been acquired by Hamilton Thorne, Ltd., a provider of precision laser devices and image analysis systems for the fertility, stem cell, and developmental biology research markets.

Widely used in fertility clinics to assess the health of unfertilized eggs (oocytes), the Oosight system provides live, high-contrast images and captures quantitative data on important oocyte structures using a patented, non-invasive, polarized-light technique. The technology was developed at the MBL by Rudolf Oldenbourg, Michael Shribak and colleagues in the 1990s and 2000s and commercialized by CRi as LC-PolScope(TM) technology. The Oosight system’s visualization capabilities have enabled breakthroughs in assisted reproductive technology, stem cell generation, and developmental biology research.

Visualization of the meiotic spindle in a rhesus monkey oocyte (egg) using the OosightTM spindle imaging system during enucleation. The spindle is near the 12 o'clock position in the egg. Credit: From Byrne, et al. 2007. Nature 450: 497-502 (Supplementary Material).

Visualization of the meiotic spindle in a rhesus monkey egg using the Oosight spindle imaging system during enucleation. The spindle is near the 12 o’clock position in the egg. Credit: Byrne et al (2007) Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450: 497-502.

“The Oosight system is a unique instrument that is complementary to our laser products in both fertility and developmental biology research labs,” remarked David Wolf, President and CEO of Hamilton Thorne. “As a long-term distributor of the Oosight system we have already completed the technical integration of the Oosight with our laser products. We believe that by leveraging our established, world-wide sales channels and investing in product marketing, we can generate incremental sales of the Oosight product.”

Additional information on the Oosight product and its multiple applications can be found at www.hamiltonthorne.com/index.php/oosight-overview.

 

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Contact: Diana Kenney, Marine Biological Laboratory
508-289-7139; dkenney@mbl.edu

WOODS HOLE, Mass.—How a brilliant-green sea slug manages to live for months at a time “feeding” on sunlight, like a plant, is clarified in a recent study published in The Biological Bulletin.

The authors present the first direct evidence that the emerald green sea slug’s chromosomes have some genes that come from the algae it eats.

These genes help sustain photosynthetic processes inside the slug that provide it with all the food it needs.

Importantly, this is one of the only known examples of functional gene transfer from one multicellular species to another, which is the goal of gene therapy to correct genetically based diseases in humans.

“Is a sea slug a good [biological model] for a human therapy? Probably not. But figuring out the mechanism of this naturally occurring gene transfer could be extremely instructive for future medical applications,” says study co-author Sidney K. Pierce, an emeritus professor at University of South Florida and at University of Maryland, College Park.

The rich green color of the photosynthesizing sea slug, Elysia chlorotica, helps to camouflage it on the ocean floor. Credit: Patrick Krug

The rich green color of the photosynthesizing sea slug, Elysia chlorotica, helps to camouflage it on the ocean floor. Credit: Patrick Krug

The team used an advanced imaging technique to confirm that a gene from the alga V. litorea is present on the E. chlorotica slug’s chromosome. This gene makes an enzyme that is critical to the function of photosynthetic “machines” called chloroplasts, which are typically found in plants and algae.

It has been known since the 1970s that E. chloritica “steals” chloroplasts from V. litorea (called “kleptoplasty”) and embeds them into its own digestive cells. Once inside the slug cells, the chloroplasts continue to photosynthesize for up to nine months—much longer than they would perform in the alga. The photosynthesis process produces carbohydrates and lipids, which nourish the slug.

How the slug manages to maintain these photosynthesizing organelles for so long has been the topic of intensive study and a good deal of controversy. “This paper confirms that one of several algal genes needed to repair damage to chloroplasts, and keep them functioning, is present on the slug chromosome,” Pierce says. “The gene is incorporated into the slug chromosome and transmitted to the next generation of slugs.” While the next generation must take up chloroplasts anew from algae, the genes to maintain the chloroplasts are already present in the slug genome, Pierce says.

“There is no way on earth that genes from an alga should work inside an animal cell,” Pierce says. “And yet here, they do. They allow the animal to rely on sunshine for its nutrition. So if something happens to their food source, they have a way of not starving to death until they find more algae to eat.”

This biological adaptation is also a mechanism of rapid evolution, Pierce says. “When a successful transfer of genes between species occurs, evolution can basically happen from one generation to the next,” he notes, rather than over an evolutionary time scale of thousands of years.

Citation:

Schwartz JA, Curtis NE, and Pierce SK (2014) FISH labeling reveals a horizontally transferred algal (Vaucheria litorea) nuclear gene on a sea slug (Elysia chlorotica) chromosome. Biol. Bull. 227: 300-312.

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The Biological Bulletin is a peer-reviewed, trans-disciplinary international journal that publishes outstanding experimental research on a wide range of organisms and biological topics, with a focus on marine models. Published since 1897 by the Marine Biological Laboratory, it is one of America’s oldest and most respected journals.


The Marine Biological Laboratory (MBL) is dedicated to scientific discovery and improving the human condition through research and education in biology, biomedicine, and environmental science. Founded in Woods Hole, Massachusetts, in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

MBL Adjunct Scientist Amy Gladfelter can now add “video producer” to her resume. Tapped to make her science “visible to the world” by Celldance Studios, a project of the American Society for Cell Biology (ASCB), Gladfelter came up with an aesthetically beautiful, simply told video about her discoveries of what goes wrong when cells form toxic aggregates, such as in Alzheimer’s disease. Her mini-movie, called “Companions in Discovery,” was filmed partly at MBL and partly at Dartmouth College, where she is an Associate Professor of Biological Sciences. It premiered for an appreciative audience in December at the ASCB annual meeting in Philadelphia.

“I like the end of the film, where members of [Gladfelter’s] lab talk briefly on camera. These young faces are the future of cell biology,” said Simon Atkinson, chairman of the ASCB’s Public Information Committee, which sponsors Celldance Studios.

Celldance Studios gave Gladfelter $1,000 to underwrite her costs, and provided video editing and post-production support. The original score is by Hollywood film composer Ted Masur, son of cell biologist Sandra Masur. More information is here.

 

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By Wallace Marshall
Co-director, MBL Physiology Course

Last month, I had a problem. I was teaching in the MBL Physiology course, using the giant, single-celled organism Stentor as a model system for students to learn quantitative approaches in cell biology. Stentor, which live in ponds, eat by creating a vortex of water that drags food into the cell’s mouth. The flow is created by thousands of cilia—tiny, hair-like cell parts that swing back and forth pushing fluid around. (Cilia are also critical for making the mucus in your airway flow away from your lungs, and patients with defects in these cilia can be really sick. So the question of how cilia make fluid flow is very important from a medical perspective. )

Stentor is a genus of large, trumpet-shaped ciliates, commonly found in freshwater ponds. Credit: EOL / micro*scope

Stentor is a genus of large, trumpet-shaped ciliates, commonly found in freshwater ponds. Credit: EOL / micro*scope

One of the students in our class, Shashank Shekhar from the CNRS Institute, France, had become interested in how the cell generates this pattern of fluid flow. Shashank started tracking the flow by putting small plastic beads into the water around the Stentor and then taking video images of the beads moving. This is a pretty standard approach in fluid dynamics called particle image velocimetry (PIV). But it’s not that commonly used in biology, and we didn’t entirely know what we were doing. The software we had been trying to use to track these particles didn’t give really nice flow lines. So this was the problem: How to use the flow of these tiny beads to figure out the pattern of flow around the cell as it feeds.

Frustrated by this problem, I decided to go get some coffee from Woods Hole Market. On the way back, I ran into my colleague Magdalena Bezanilla, an MBL Whitman Investigator from University of Massachusetts, Amherst, who works on cell biology. She thinks a lot about things moving inside cells so I figured I could get her input into our PIV challenge.

We ended up chatting about the problem in the MBL’s Waterfront Park, and while we were talking, a couple of guys emerged from the harbor in full scuba gear, carrying a huge metal bracket upon which was mounted a video camera and a laser. (This would be quite weird back home but it’s business as usual in Woods Hole.) I asked the guys what they were up to and they said they were using PIV to study the flow of fluid around ctenophores! Ctenophores or comb jellies are jellyfish-like animals that swim using cilia. So at the exact moment that we were pondering how to use PIV to track cilia-generated fluid flow in our single-celled organisms in the Physiology course, a guy walks out of the water and announces that he is doing the exact same thing, for comb jellies! (Those people who say that Woods Hole is a magical place are telling the truth.)

The guys with the scuba gear and lasers were Jack Costello of Providence College and Sean Colin of Roger Williams University, Whitman Investigators working for the summer at the MBL. Jack offered to give us advice about how to analyze our data, so I sent Shashank over to Jack’s lab in the Rowe building. With Jack’s help and expertise, Shashank was able to get beautiful flow lines from his data (see photo), which clearly reveal the pattern of cilia-generated flow around the Stentor cell while it feeds. Our big problem was solved in a single day due to a fortuitous combination of people, courses, coffee breaks, cells, beaches, marine organisms, and advanced technology. And that’s what summer at the MBL is all about.

Fluid flow around Stentor visualized through particle image velocimetry. Courtesy of Wallace Marshall.

Fluid flow around Stentor as it feeds, visualized by particle image velocimetry. Courtesy of Wallace Marshall.

Thank you to Wallace Marshall of the University of California, San Francisco, for contributing this post. All MBL scientists, students, community members, and visitors are invited to submit items for the MBL’s blog. Please contact Diana Kenney: dkenney@mbl.edu.

Adam Cohen instructing in the MBL Physiology course in 2014.
Credit: Tom Kleindinst

Adam Cohen, a faculty member and former student in the MBL’s Physiology course, is one of three winners of the inaugural Blavatnik Awards for Young Scientists. The awards, given by the Blavatnik Family Foundation and the New York Academy of Sciences, honor exceptional young U.S. scientists and engineers. Each laureate receives $250,000 – the largest unrestricted cash prize for early-career scientists. Cohen is Professor of Chemistry and Chemical Biology and Physics at Harvard University, and a Howard Hughes Medical Institute (HHMI) investigator.

Cohen was recognized for “significant breakthroughs in cellular imaging that allow for the observation of neural activity in real-time, at single-cell resolution.” Combining his expertise in chemistry, physics, and biology, Cohen uses microscopy and lasers to develop noninvasive methods of visualizing and studying the roles of cellular voltage in neurons. His novel techniques, including fluorescent voltage indicators derived from microbial rhodopsins, help to answer questions about the propagation of electrical signals and could one day lead to the design of individualized treatments for conditions such as ALS, epilepsy, and bipolar disorders.

“Cohen is recognized as one of the nation’s most promising young scientists,” said Vern Schramm, Ruth Merns Chair in Biochemistry at the Albert Einstein College of Medicine and a member of the 2014 Blavatnik Awards National Jury.

The two other 2014 Blavatnik National Laureates are Rachel Wilson, Professor of Neurobiology at Harvard University and an HHMI Investigator, who was recognized for her research on sensory processing and neural circuitry in the fruit fly; and Marin Soljačić, Professor of Physics at MIT, recognized for his discoveries of novel phenomena related to the interaction of light and matter, and his work on wireless power transfer technology.

The Blavatnik Family Foundation is headed by philanthropist Len Blavatnik, founder and chairman of Access Industries, a privately held U.S. industrial group.

By Laurel Hamers

One of the brain’s amazing abilities is self-repair: Although injury or illness may disrupt neural circuits, many connections will reform over time.

Artur Llobet, an MBL Research Awardee from the University of Barcelona, is spending his second consecutive summer in the Whitman Center for Visiting Research investigating olfactory neuron repair in Xenopus laevis, the African clawed frog.

Calcium labeling of olfactory sensory neurons' presynaptic terminals in a Xenopus laevis tadpole. Photo credit: Artur Llobet

Labelling of presynaptic terminals of olfactory sensory neurons in a Xenopus laevis tadpole using calcium green dextran. Image is pseudocolored so that yellow represents higher and blue lower calcium concentration.
Photo and caption: Artur Llobet

“One of the advantages of working with frogs is that they have fantastic regenerative capabilities,” says Llobet. Tadpoles are able to repair damaged neural circuits in a few days, making them ideal test subjects.

Llobet is working with a transgenic line of Xenopus tadpoles that express green fluorescent protein (GFP) in their neurons, allowing him to easily see the neural connections. Last year, he studied the timeframe of Xenopus neural repair by measuring how long snipped olfactory nerves took to regrow. Now, he is trying to understand in greater detail the mechanisms behind the repair process.

Neurons pass electrochemical messages between each other at junctions called synapses; when a neuron fires, the voltage change propagates along the nerve fiber (axon) and calcium increases at the presynaptic terminal, which releases neurotransmitters. By labeling the tadpoles’ synaptic terminals with calcium indicators, Llobet can visualize the functionality of the re-grown connections and determine when during the repair process the new synapses start signaling.

“In a GFP animal, we can see that the nerve has re-grown, but we don’t know if that nerve is actually working or not,” says Llobet. “So we look at the synapses and see whether the calcium concentration increases when we stimulate olfactory sensory neurons.” This calcium accumulation indicates that the new nerve is not just present, but also functional.

By examining neural repair in frogs, scientists hope to gain insight into this process in more complex systems such as the human brain.

Llobet’s research is taking place through the National Xenopus Resource (NXR) at the MBL, a center that maintains breeding stocks of frogs and provides training on advanced imaging and experimental technologies. According to Llobet, the specialized resources offered by the NXR make this research project possible. He is one of six MBL Research Awardees in 2014 to be using the animals and research services of the NXR, which is one of 28 National Institutes of Health-funded Animal Resource Centers nationwide and a cornerstone facility of the MBL’s Bell Center for Regenerative Biology and Tissue Engineering.

A whimsical, enlightening video about cuttlefish camouflage by Jacob Gindi, a senior and biology major at Brown University, appeared in The New York Times last week. Gindi had encountered live cuttlefish when he visited the MBL’s Marine Resources Center as a student in The Art and Science of Visual Perception, a Brown course co-taught by Roger Hanlon of the MBL and Mark Milloff of Rhode Island School of Design. Gindi then had a chance to make a CreatureCast video in Casey Dunn’s Invertebrate Zoology class at Brown. Inspired by Hanlon’s research, Gindi’s artful video about the cuttlefish’s amazingly adaptive skin can be enjoyed by marine biology-lovers of all ages.

“It is so gratifying to see science and art promoted at this national/international scale,” says Hanlon, an MBL senior scientist and professor in Brown’s Ecology and Evolutionary Biology Department through the Brown-MBL Partnership and Graduate Program.

CreatureCast, a collaborative blog produced by members of the Dunn Lab, is supported by a National Science Foundation grant.

 

 

Pedestrians in Edinburgh, Scotland, have been treated to a springtime display of giant photos of “glowing” or bioluminescent animals, including images of the jellyfish Aequorea aequorea captured by MBL Distinguished Scientist and 2008 Nobel Laureate Osamu Shimomura (panel behind girl on bike).

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The display, called “Living Lights,” was part of the 2014 Edinburgh International Science Festival and this week is moving to another venue in Edinburgh, Our Dynamic Earth, where it will remain through October.

Shimomura took these photos of Aequorea in 1961, when he was a young chemist at Princeton University asking, “What makes the jellyfish glow?” He captured thousands of jellyfish from the waters off Friday Harbor, Washington, and painstakingly searched for their bioluminescence molecule. The two photos on top (below) and at the one at bottom left he took in daylight, shooting directly into the clear Friday Harbor water, using a Nikon F camera and 50 mm lens. Shimomura brought one jellyfish into a darkroom and exposed it to fresh water to trigger its luminescence, allowing him to capture the phenomena on camera (bottom right).

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Shimomura did find and isolate the jellyfish’s bioluminescence protein—which he called “aequorin” –that year, and in the process he also discovered a fluorescent jellyfish protein that he called “green protein.”

Years later, in 1994, Martin Chalfie of Columbia University discovered that the jellyfish’s green fluorescent protein (GFP) could be an extremely useful tool for lighting up microscopic cells and their parts for study. GFP and other fluorescent proteins are now used in biomedical research worldwide, and they have been crucial in illuminating many processes that were previously invisible, such as the development of nerve cells or the spread of cancer. Shimomura, Chalfie, and Roger Tsien of University of California, San Diego, were awarded the 2008 Nobel Prize in Chemistry for their contributions to GFP’s discovery and applications.

The "Living Lights" photo exhibit of bioluminescent organisms in front of the Scottish National Gallery, Edinburgh.

The “Living Lights” photo exhibit of bioluminescent organisms in front of the Scottish National Gallery, Edinburgh, Spring 2014.

 

MBL Distinguished Scientist Osamu Shimomura at Friday Harbor, Washington, in 2003. Photo by Martin Chalfie

MBL Distinguished Scientist Osamu Shimomura at Friday Harbor, Washington, in 2003. Photo by Martin Chalfie

 

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