Andrew Williams reveals the latest developments in the use of microscopic technology for exploring human and animal brains
Over the past few months, a string of universities and research institutes around the world have announced breakthroughs in the use of microscope technology for animal and human brain imaging. So what have been some of the key recent developments? And what are the benefits of such innovative approaches to microscopy?
OPTICAL BIOPSY
One of the most interesting recent developments has been at the Barrow Neurological Institute in Arizona, USA, where a team of researchers has employed innovative confocal laser endomicroscopy (CLE) to collect real-time images from experimental brain tumours in mice, as well as from human brain tumours during their surgical removal and from human brain tumour biopsies. As part of the research, mice were injected with fluorescein sodium (FNa) before imaging and human patients received FNa intraoperatively, enabling the surgical team to view images immediately in the operating room.
As Dr Mark Preul, professor of Neurosurgery & Neuroscience and director of the Neurosurgery Research Laboratory at the Barrow Neurological Institute, and Evgenii Belykh, the lead Neurosurgery Research Fellow for the project, explain, CLE images at various depths were acquired automatically by a miniature probe-based Carl Zeiss Convivo digital optical biopsy tool equipped with a 488nm laser with either a green bandpass or a longpass filter to acquire confocal images.
“The tool acquires an ‘optical’ biopsy, that is, an image of the tissue. CLE imaging is revolutionary in neurosurgery because for the first time we have a pen-sized, hand-held portable confocal microscope system that can display tissue images in real time as the surgeon is using it ‘on-the-fly’ at the cellular level,” says Preul.
“The main goal is to apply this technology to invasive brain tumours that do not have readily definable borders to extend the resection of the tumour to find and remove invading cells or tumour tissue into what looks like normal brain, or to define locations where a positive tumour biopsy can be quickly obtained instead of searching multiple areas as is done now,” he adds.
The CLE system used also possesses other novel capabilities such as being able to focus and acquire images through a depth of tissue, yielding unique 3D image ‘blocks’. Such images are rapidly acquired, compiled and rendered into 3D volumes using Fiji software and reviewed by a neuropathologist and neurosurgeons. Some specimens were also counterstained with fluorescent dyes that cannot be used in the living human brain, such as acridine orange, acriflavine or cresyl violet and imaged on a benchtop confocal microscope to confirm what is being examined with fluorescent stains that can be used in patients.
A key conclusion of the research is that CLE imaging with 3D and Z-stack imaging of this type is a ‘unique new option’ for the live intraoperative endomicroscopy of brain tumours – particularly in view of the fact that the 3D images captured during the process foster an ‘increased spatial understanding’ of tumour cellular architecture and ‘visualisation of related structures compared with two-dimensional images.’
Looking ahead, the Arizona team concludes that fluorescence imaging of this type could help to ‘augment and expedite intraoperative decision-making and potentially aid in the identification of tumour tissue’ – and that the use of CLE microscopy as part of this fluorescence-guided procedure may lead to improved intraoperative diagnosis of tumour margins, providing a more efficient approach for achieving maximum tumour resection and, ultimately, prolonging survival time. They also speculate that the future application of specific fluorescent probes beyond FNa could benefit from this rapid in vivo imaging technology ‘for interrogation of brain tumour tissue.’
“These technologies are the wave of the future in surgery that will allow more tailored, rational and precise ‘personalised’ surgery,” says Belykh. “Importantly, as much as technologies like CLE will inform us where to operate, they will also critically tell us where to stop in a brain tumour surgery, such as when malignant tumour tissue has invaded into eloquent brain, for example areas controlling speech,” he adds.
INTRAVITAL MICROSCOPY
Elsewhere, a team of researchers at the USA’s NIH (National Institutes of Health) National Institute of Neurological Disorders and Stroke (NINDS) has pioneered the use of intravital microscopy methods to analyse the brains of mice that have developed cerebral malaria (CM).
The project aimed to understand how T cells, a population of immune cells, cause cerebral malaria. A subset of T cells known as CD8+T cells are required for the development of cerebral malaria in rodents. In the study, the team used intravital two-photon laser microscopy to image fluorescent protein-tagged CD8+T cells that were specific to the parasite and are known to cause cerebral malaria. By imaging through the skull bone of mice, they revealed that this fatal neurological disorder is caused by CD8+T cells that attack cerebral blood vessels.
“We also discovered that disease can be prevented by therapeutically displacing these T cells from cerebral blood vessels by administering antibodies that block their adhesion,” says Dr Dorian McGavern, a scientist at NINDS.
Intravital microscopy involves using a microscope to image biological processes in a living organism. In this study, the team used a Leica SP8 two-photon laser scanning microscope to image immune cells in mice infected with Plasmodium berghei, a parasite that causes cerebral malaria.
“The advantage of using intravital microscopy is that one can capture movies in the living brain of immune cells causing a disease. This helps us to better understand how diseases like cerebral malaria develop,” says McGavern.
“We use intravital microscopy on a daily basis to understand neurological disorders, including viral meningitis, viral encephalitis, cerebral malaria, traumatic brain injury, ischemia and brain tumours. This technique is used to gain real-time insights into many different biological processes that occur in the CNS during states of health and disease,” he adds.
ADAPTIVE VIEW
Also in the USA, a research team at the Howard Hughes Medical Institute (HHMI) has recently used an adaptive, multi-view light sheet microscope as part of a project to analyse embryonic development in living mice. As Dr Philipp Keller, group leader at the HHMI Janelia Research Campus in Ashburn, Virginia, explains, the study marks the first time that whole-embryo mammalian development has been imaged at the single-cell level throughout the critical period of gastrulation and early organogenesis, that is ‘throughout the critical period during which the embryo forms the early tissues and organs.’
“We were able to reach this milestone by developing a light sheet microscope that adapts itself to the complex optical properties and rapidly changing size and shape of the developing mouse embryo, thereby achieving the high spatiotemporal resolution required to systematically track cells across the embryo,” he says.
“No existing imaging methods are able to deliver such high spatiotemporal resolution in an entire, intact mouse embryo,” he adds.
The team has also developed a suite of computational tools that allows members to reconstruct embryonic development from the resulting very large and complex image data sets, running to some one million images and 10 terabytes per embryo, and convert these images into a developmental building plan of the entire embryo. By transforming these image data sets into what he describes as a ‘biologically much more interpretable form’, Keller reports that his team gained a range of biological insights into embryonic development and an ‘unprecedented view of how early tissues and organs, such as the neural tube or the heart, are formed.’
Adaptive, multi-view light-sheet microscopy is a custom microscopy method developed at Keller’s lab at Janelia. The microscope is used to illuminate a sample from multiple different directions with thin sheets of laser lights, before acquiring images of the fluorescence emitted by molecules in these thin illuminated volume sections with camera-based detection arms. The microscope itself is equipped with optical modules and a computational control framework that map the size, shape and position of the sample over time and determine how light is locally perturbed by the complex optical properties of the sample.
“The microscope then automatically corrects for these perturbations by adapting itself to the sample’s geometrical and optical properties and thereby recovers high spatial resolution throughout the sample. The microscope is built from a large number of custom-designed optical and mechanical parts, and it also includes a range of commercial components, such as commercial cameras and lasers, for example,” says Keller.
In Keller’s view, one of the main advantages of the microscope is that it provides image data of large multi-cellular specimens, such as entire brains or embryos, with ‘unprecedented spatial and temporal resolution.’ He also reveals it is capable of rapidly imaging even very large volumes, for example, the mouse embryo grows up to a diameter of 3mm in these experiments, and is ‘very gentle when imaging the sample.’
“It uses a minimal amount of light to illuminate the specimen and acquire high-quality images, which ensures that we do not perturb the physiological development and function of the sample,” he adds.
