Time-Lapse Microscopy Captures Cells in the Act

Marie Freebody, Contributing Editor, marie.freebody@photonics.com

Viewing biological processes as they happen provides a fascinating insight into cell behavior and can unveil vital clues about developmental milestones that have been puzzling scientists for some time.

Time-lapse microscopy (TLM) involves repeated capture of images from a microscope at regular time intervals. The duration of the intervals determines the temporal resolution, and the resulting video sequence shows cells or organisms at work, giving scientists a first look at some important biological processes.

Demystifying embryo development

The earliest stages of human development remain largely a mystery, despite the attention given to embryonic research. Determining the reasons why some fertilized eggs go on to become healthy babies and why some stall and fail is still one of the biggest questions facing fertility experts.

Now, a company founded by a team of scientists at Stanford University School of Medicine in California is using TLM to predict with 93 percent certainty which fertilized eggs will make it to a critical developmental milestone and which will be unable to survive.

Auxogyn is a California-based company conceived by Renee Reijo Pera, director of the Stanford University Center for Human Embryonic Stem Cell Research and Education, to improve the effectiveness of in vitro fertilization (IVF).

One out of every six couples is affected by some degree of infertility, according to the International Council on Infertility Information Dissemination. With the rate increasing with maternal age and more women starting their families later in life, the demand for IVF and other assisted reproduction diagnostic tools is growing by around 10 percent each year.

The stark reality of assisted reproduction is that only one-third of human embryos will develop successfully, according to the Centers for Disease Control and Prevention in Atlanta. This often prompts the transfer of two or more embryos to increase the odds of a successful pregnancy. However, if multiple embryos implant and develop successfully, a woman and her physician may choose to selectively abort one or more to increase the odds for the remaining embryos.

This is a single frame taken from a time-lapse sequence showing embryos within the first two days after fertilization, with overlaid cell-tracking data (red circles) produced using image analysis software. Courtesy of Kevin Loewke at Auxogyn.

Identifying the embryo with the greatest potential would reduce the cost associated with multiple IVF cycles; in addition, it would substantially decrease the number of multiple births and significantly increase the success rates of IVF.

Pera and colleagues at Stanford published their findings October 2010 in Nature Biotechnology. The researchers used TLM to take a closer look at what happens during the first few days after an egg is fertilized. They followed the cells through to the development of a hollow ball called a blastocyst, which typically occurs within five to six days after fertilization and is usually an indication of a healthy embryo.

This single frame, taken from the time-lapse sequence on day five, shows embryos that have arrested and embryos that have reached the blastocyst stage. Courtesy of Kevin Loewke at Auxogyn.

“In our recent paper, we observed the developmental process of early-preimplantation human embryos during the first six days of life using time-lapse microscopy,” said Connie Wong, a co-author of the paper. “After performing a thorough analysis of the imaging data, we successfully extracted three quantitative parameters that can predict the developmental potential of an embryo before day three of life.”

The team believes that these parameters can be used as diagnostic markers in IVF clinics to aid in the selection of healthy embryos for transplantation. Currently, most IVF clinics use only time-point analysis to determine embryo health prior to transfer into patients. However, the Stanford study illustrates the importance of measuring time-resolved events.

“When compared to a traditional time-point microscopy study, in which researchers would only collect imaging data of their specimens at specific selected time points, time-lapse microscopy offers the advantage of being able to acquire data continuously during the time of the study,” Wong said. “This is especially important when time-resolved data is needed, or when the specimens tend to change frequently and unpredictably.”

Given the delicate environment required for human embryos to flourish, the team had to develop a miniature time-lapse microscope so that the embryos could be imaged inside a tissue culture incubator used to provide optimal culturing conditions.

Another crucial change in the typical TLM setup was the lighting. Most of today’s TLM studies are performed using stand-alone microscopes illuminated by standard bright-field or fluorescence illumination. Wong and colleagues, however, switched to dark-field illumination to minimize the amount of phototoxicity experienced by the embryos.

The research team at Auxogyn is now working toward commercializing the miniature time-lapse microscopes for IVF clinical use.

“We are pleased to be starting a multisite clinical trial soon that will further validate the effectiveness of the three time-resolved parameters discovered in Stanford’s recent publication,” said Lissa Goldenstein, CEO of Auxogyn. “We hope to introduce time-lapse microscopy with clinically validated parameters as a routine tool used in assisted reproduction clinics to better predict the developmental potential of embryos.”

TLM unlocks secrets of the brain

TLM is also breaking new ground in another area of human development: neuronal circuits in the brain. Dr. David Solecki at St. Jude Children’s Research Hospital in Memphis, Tenn., is using TLM to discover new details about mechanisms regulating a crucial step in brain development. The study offers insight into the origins of epilepsy, mental retardation and possibly brain tumor metastasis.

Dr. David Solecki at St. Jude Children’s Research Hospital is using TLM to reveal some of the secrets of brain development. Courtesy of St. Jude Children’s Research Hospital.

For Solecki, time-lapse imaging offers an unparalleled glimpse into those molecular and cellular mechanisms that are at the center of most biological problems.

“Many investigators feel that time-lapse imaging does not amount to much more than a set of pretty pictures that may not necessarily provide any new insights into the biology of a particular process,” he said. “Contrary to this sentiment, I’m convinced that time-lapse imaging provides an exciting opportunity to directly observe a process to reveal how it mechanistically unfolds, while at the same time directly quantitating the kinetics of that process as it normally occurs, or when we manipulate it in a rational manner.”

Time-lapse imaging tracks cell-to-cell binding for the first time. The cell borders are indicated by red and yellow lines in the first frame. Subsequent frames show cell adhesion indicated by a fluorescence signal, which intensifies upon establishment of stable contacts. Time stamp = minutes:seconds. Image has been adapted from a paper in Science, courtesy of Dr. David Solecki, St. Jude Children’s Research Hospital, Memphis, Tenn.

The process in question for Solecki and his team is neuronal migration. It’s been known for the past 15 years or so that neuronal migrations are essential for proper brain formation; in particular, neuron cells must travel to specific areas in the brain to complete necessary brain circuitry.

However, the major challenge has been to dissect the molecular and cellular mechanisms controlling when and where neurons choose to migrate during the assembly of brain circuitry. Solecki’s team suspected that how neuronal cells adhere to neighboring cells plays a part in making those migration decisions.

In a paper published in Science on Dec. 24, 2010, the team used TLM to dissect a signaling pathway that was believed to control the migration choices of maturing neurons in the developing brain. The researchers developed a fluorescent probe that, when combined with TLM, made real-time viewing of cell-to-cell binding possible for the first time. They used a spinning disk confocal, which is ideal for capturing dynamic events without overexposing the cells or brain slice.

“My laboratory has developed a variety of techniques to genetically manipulate large numbers of neurons in brain slices or primary cultures and image the cells in these preparations using a spinning disk confocal microscope,” Solecki said. “Time-lapse microscopy was integral for us to determine that the transition of one form of migration to another was related to the activation of cell adhesion in mature neurons so they were able to adhere to a new migration substrate as they move to their appropriate location in the brain.”

While he admits that TLM cannot replace the biochemical, anatomical or molecular assays that are the mainstays of modern biology, he believes that the technique can help shed light on the underlying mechanisms of particular biological processes. All you need is the imagination to make these processes amenable to imaging techniques.

“In the future, I hope to see time-lapse imaging as an alternative to the static anatomical studies that are most frequently used today in the developmental biology or neural development fields,” he said. “Given the ever-increasing number of mouse models for human developmental disease (like those for neurodevelopmental disorders or pediatric cancer), quantitative time-lapse imaging will be one tool that will be very useful to unlock the cell biology and pathology of these diseases.”