Microscopy sees the light

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Clever manipulation of light is allowing researchers to image deeper into tissue to ultimately further our understanding of the brain. Abigail Williams investigates

Image: Caleb Foster/shutterstock.com

Advances in specialised camera, imaging and microscopy technologies are paving the way for a wide range of novel applications in neuroscience and live cell imaging, with AI enhancing images further.

One type of imaging commonly used in live cell imaging is fluorescence microscopy, where genetically modified proteins fluoresce when excited with light. In neuroscience, for instance, in small animals such as mice and rats, fluorescence allows researchers to visualise the position and shape of nerve fibres, synapses, and cell bodies.

Naoya Matsumoto, Associate Senior Researcher at the Hamamatsu Photonics Central Research Laboratory, explains: “Creating a neural map of the whole brain is an important topic of research. At the same time, research is also being performed to manipulate neural activity by irradiating light onto visualised cells. These research activities are helping to unravel the relationship between the brain and its functions, for example, movement and memory.”

Hamamatsu Photonics has recently developed a two-photon microscope with integrated spatial light modulators (SLMs), making it suitable for observing thick biological samples, as well as for live imaging with small animals.

Spatial light modulators are pixelated liquid crystal devices with more than a million pixels, each of which can be digitally controlled to shape the wavefront of a probe laser beam. As researchers seek to capture images deeper in tissues, aberration effects – which essentially distort the laser wavefront and limit its ability to form a sharp focus deep inside the tissue – become more pronounced. One way to counter aberration is to use SLMs to shape the laser wavefront before it enters the tissue.

“Because fluorescence is emitted only at high energy densities, the locations where fluorescence occurs are localised to the focal region at the depth of interest. The use of near-infrared light, which has high biological transparency, aids in deep tissue imaging,” says Matsumoto.

Hamamatsu SLMs were recently used to counter aberration as part of a joint research effort between the company and Hamamatsu University School of Medicine. As well as helping to increase the depth of imaging, the wavefront shaping capabilities of the new microscope also improved the resolution of images.

“This research is another example of how Hamamatsu is helping researchers pursue challenging problems by developing photonic solutions to meet their needs. Here we show that by simple modifications to a standard two-photon fluorescence microscope via the inclusion of an SLM and the use of aberration correction steps, imaging depth in tissue as well as resolution can be improved,” says Matsumoto.

The research team estimated the amount of aberration by obtaining a ray trace, and modulated the wavefront of the excitation light by SLM to perform pre-compensation – which allowed it to observe images clearly even at greater depths in scattering samples. According to Matsumoto, a further interesting illustration of the enhanced capabilities of the new device was demonstrated when the team observed a transparency-enhanced sample. Although a special objective lens is normally required in such cases, correction with the SLM function of the new microscope enabled a clear observation even with a water immersion objective lens.

As Mastumoto explains, another key advantage of the new microscope is its ability to enhance resolution. Two-photon microscopy, which uses near-infrared light as the excitation light, typically has lower resolution compared to confocal microscopy that uses visible light. To solve this problem, the Hamamatsu SLM works in combination with an azimuth polariser to enhance amplitude and phase modulation, as well as polarisation modulation. According to Matsumoto, these effects enable measurement at depths of less than 100μm inside a sample with lateral resolution 1.23 times higher than that of conventional two-photon microscopy.

“Compared to conventional two-photon microscopes, this microscope can observe deeper locations on a sample and has a resolution higher than the diffraction limit. This has the potential to observe cells in locations at previously unseen depths and to discriminate cells that could not be identified due to overlap,” says Matsumoto.

“Although we have demonstrated its potential with fixed tissue, our microscope, realised in a simple configuration, has the potential to demonstrate its properties in live imaging as well. It may make nerve activity clearer,” he adds.

Multiphoton microscope

Dr Stefanie Kiderlen, Application Scientist at microscope maker, Prospective Instruments, says that 3D imaging is also important in neuroscience research. Prospective Instruments’ MPX-1040 multiphoton microscope uses a photomultiplier tube detector to give higher penetration depth, lower photodamage, and confocal resolution in 3D, as well as sCMOS cameras for widefield fluorescence imaging.

“In future directions, 3D live cell imaging will overtake the classical 2D cell culture in many research areas,” says Kiderlen. “3D live cell imaging includes 3D cell cultures like spheroids or organoids, but also in vivo imaging of cells in their native 3D environment. The MPX delivers microscopy techniques to image any 3D sample but still allows state-of-the-art imaging of 2D cells when needed. Key applications include 3D imaging, deep tissue imaging, confocal imaging, live animal imaging, whole organ and whole slide imaging.”

According to Kiderlen, the company’s main goal in this area is to provide the research and clinical community with a flexible, modular, and highly compact multimodal imaging platform – so that high-level microscopy “should not be limited to those with technical knowledge, indoor working space, or budget”.

“Every researcher, scientist, and clinician should have access to high-quality, affordable multimodal microscopes,” she says.

Machine learning

Ultimately, Kiderlen observes that, in the field of life sciences, neuroscience and 3D cell cultures, 3D microscopy techniques are essential – particularly, for example, for time-lapse imaging of organ development in zebrafish larvae, as “one of the gold-standards in animal models”.

“Long-term imaging with low-photo impact to avoid photodamage and phototoxicity is needed to image native animal development processes. Also, deep tissue penetration of the excitation wavelength is beneficial, to image structures inside an organ or tissue, or to compensate for small movements of the animal,” she says.

Looking ahead, Kiderlen predicts that several key ongoing innovations in multiphoton microscopy will result in life science researchers, scientists and clinicians soon being able to access a wide range of capabilities in a single microscope.

She adds that enhanced AI capabilities will also be included in future devices and enable virtual tissue staining, label-free virtual staining and virtual histology.

Meanwhile, Matsumoto believes that live imaging using two-photon microscopy will allow scientists to observe the progression of disease, or the process of treatment – and lead to the development of more effective therapeutics.

“The method we have developed further enhances the features of conventional two-photon microscopy. Since it is demonstrated with a simple technique, it can be applied to live imaging. We believe that it can improve the accuracy of the position and shape of observed nerve fibres, synapses, and cell bodies,” he says.

“Machine learning can [also] be used, for example, to extract images, or to infer corrected wavefronts. 4D – 3D space plus time – image measurement with a two-photon microscope produces an enormous number of images, [and] machine learning can be used to extract meaningful information from these huge datasets,” Matsumoto adds.

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