Driving New Discoveries With Lattice Light Sheet Microscopy in an Advanced Core Imaging Facility
Introduction

Driving New Discoveries With Lattice Light Sheet Microscopy in an Advanced Core Imaging Facility

The Centre for Dynamic Imaging (CDI) at the Walter and Eliza Hall Institute of Medical Research (WEHI) in Parkville, Victoria (Australia) is both a core facility and a research lab for advanced microscopy. ZEISS Lattice Lightsheet 7 is now part of the suite of tools available at the center and has recently lead to a variety of new findings and approaches.

Dr. Niall Geoghegan, senior post doc fellow and lattice light sheet specialist at WEHI

The CDI is helping scientists use advanced imaging technology and powerful computational resources to generate detailed and real-time views of biological systems. These new insights are advancing medical research and our understanding of how diseases develop, spread and respond to treatment.

ZEISS Lattice Lightsheet 7 is now part of the suite of tools available at the center and has recently lead to a variety of new findings and approaches.

Dr. Niall Geoghegan, senior post doc fellow and lattice light sheet specialist (image above), and Dr. Kelly Rogers, laboratory head & center manager, share their experiences and provide compelling insights into their research:

Interview

How does lattice light sheet microscopy complement your existing range of equipment?

Many applications within the center require us to understand the kinetics and dynamics of sub-cellular processes. It may be the case that a researcher has a rough idea of the timing and dynamics of a particular biological event but does not know the precise order and manner in which it occurs.

The lattice light sheet microscope acts as a great platform to begin such experiments where we can take a broad view, temporally and spatially, to determine just when, and how, a particular biological process occurs.

Dr Niall Geoghegan | Centre for Dynamic Imaging

We can then leverage this information to dig deeper, whether this be through increased spatial resolution or by imaging faster, using other microscopes within the facility. One example where we applied this approach was in previous work looking at just how mitochondrial DNA escapes the mitochondria during cell death (McArthur et al. 2018 Science). Using lattice light sheet microscopy McArthur et al. were able to pinpoint the exact moment this escape occurred. They were then able to use this information to time perfectly the capture of this event using 3D-structured illumination microscopy to resolve it in greater detail. If you were to image the event from the beginning using 3D-SIM, the light dose necessary to image quickly enough would likely induce cell death itself.

Lattice light sheet microscopy was able to guide the researchers in time so they could look with greater detail to learn more about this complex process.

Dr Niall Geoghegan | Centre for Dynamic Imaging

Conversely, lattice light sheet imaging provides us with a technique perfectly suited to tracking fast subcellular processes in 3D. Processes such as protein trafficking, microtubule tip growth and pathogenic entry to host cells. It allows us to track 3D dynamics across a broad temporal range but with the confidence that the sample is not damaged by the imaging itself. There is nothing worse than setting up a 24-hour cell division experiment, only to find that phototoxic effects arrest the cell cycle process and you need to start all over again.

An engineered T-cell (green) seeking out and killing a cancerous target cell (Magenta).

The movie shows transient calcium fluxes in the T-Cell prior to its killing of the target which lights up with PI upon death. Imaged using ZEISS Lattice Lightsheet 7. Courtesy of Ms. Kylie Luong, Associate Prof Misty Jenkins.

Do you approach experiments differently now that you have access to lattice light sheet technology?

Due to the unprecedented detail that lattice light sheet microscopy affords researchers, it really is beginning to change the way in which we look at cell biology and how we design experiments to extract the most useful information from a given biological system. The data the system provides is intensely information rich and we are only beginning to scratch the surface in terms of what is possible with this type of technique. This rich data is heralding a new era of discovery-based science, where previously hidden processes are resolved with great clarity and, more importantly, confidence.

We still approach the majority of experiments in a conventional manner in terms of design, execution and eventual data analysis. However, we are finding more and more that there are often answers hidden in the data, to questions that a researcher wasn’t initially even thinking about. This is helping to drive the development of novel analysis tools which are able to extract quantified information at a scale previously unimaginable (TBs and TBs of data).

One of the most interesting aspects of this type of data is how we can leverage and quantify the rich 4D information in the data to answer questions that previously remained unanswered.

Dr Niall Geoghegan | Centre for Dynamic Imaging

Where ZEISS Lattice Lightsheet 7 really excels is in the volume of biological data it is able to produce. By this, I mean, we are now able to parallelize 4D imaging experiments that, previously, we could only perform sequentially. Coupling this to new tools for image analysis, we are entering the era of data science for 4D imaging. We are also finding ZEISS Lattice Lightsheet 7 is an incredible tool, not only for quantitative analysis, but for exploratory science. At WEHI there are numerous researchers and teams with incredible cellular models for a wide variety of diseases. Elements of these models are typically assessed in isolation, whether by biochemical or molecular methods.

With ZEISS Lattice Lightsheet 7 we can visualize the intricate interplay of various molecular markers, but in the context of the whole cell, across a large population of cells. Just the ability to see where and when a protein of interest will be, given a certain stimulus, can inform and direct entire bodies of research.

Dr Niall Geoghegan | Centre for Dynamic Imaging

Nucleus of a field of Neutrophils exploding as they undergo a process called Netosis.

The color scale is depth encoded highlighting the volumetric nature of the data. Imaged using ZEISS Lattice Lightsheet 7. Courtesy of Dr George Ashdown, Dr Anna Coussens.

What new discoveries have been possible using this new capability? Can you share some of your highlights with us?

We have had a home-built lattice light sheet microscope in house since 2017 and prior to that we had the opportunity to visit the Advanced Imaging Centre at HHMI, Janelia. We have made several discoveries with this technology. One of the first important discoveries was made in a collaboration that was led by Kate McArthur. Here we showed for the first time that mitochondrial DNA is released via BAK/BAX macropores during apoptosis. This work was published in Science (McArthur et al. 2018 Science). In another collaboration led by Andre Samson (Samson et al. 2020 Nature Communications), we used this technology to demonstrate the presence of MLKL trafficking and hotspots on the membrane during necroptosis, which is a caspase independent form of cell death.

Finally, we have used this system to image for the first time, how the malaria causing parasite, Plasmodium falciparum, invades the human red blood cell in 4-dimensions (Geoghegan et al. 2021 Nature Comms). In doing so it creates a small membrane bound home for itself within the RBC, called the parasitophorous vacuole. For decades it was debated as to whether the parasite pinched off a section of RBC membrane behind it, or it used parasite derived lipids to form this new home. Through quantifying the temporal changes in surface area of the RBC we were able to answer this question and show that the surface area loss at the RBC accounts for the newly formed vacuole. We identified an essential step of cholesterol enrichment for the formation of the parasitophorous vacuole membrane, which encloses the parasite following invasion, which serves to support the growth and development of the parasite during the blood stage of malaria. We can now image across multiple conditions to see how different genetic changes, or new therapies can affect the development and growth of the Malaria parasite. 

These studies revealed incredible new insights into a range of different research areas at WEHI. 

However, the ability to run these experiments at scale was limited due to the complicated nature of the home-built system. In the example of imaging and quantifying malaria invasion of red blood cells in 4D, we would often only be able to capture 5-10 events on a good day due to the limited field of view and complexity of the experimental setup. Now using ZEISS Lattice Lightsheet 7, we are able to get 5-10 invasion events in any given 30–40-minute experiment.

This order of magnitude increase in experimental throughput will help us drive this research further and faster, enabling new discoveries into one of the deadliest diseases in human history.

Dr Niall Geoghegan | Centre for Dynamic Imaging

WEHI Australia

About the Centre for Dynamic Imaging (CDI)

On the R&D side, the CDI focuses on the application and development of new methods, which allow to extend the scope of what is achievable with light microscopy methods. A multidisciplinary team of scientists is specialized in different areas of microscopy and image analysis. The focus of the group is the use of imaging research to gain more insight into cancer, infectious and immune diseases and developmental disorders. There are approximately 200 different users coming through the facility each year with more than 400 users over the last 5 years. A range of different types of equipment is available – including widefield, confocal, airyscan and multiphoton microscopes (e.g. ZEISS LSM 880, 980), plus several light sheet microscope platforms, including ZEISS Lightsheet Z.1 and ZEISS Lattice Lightsheet 7, as well as equipment custom-build by the team (e.g. lattice light sheet microscope).

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