Hamamatsu boosts spatial resolution of 2-photon fluorescence microscopy

31 May 2022

Application is suited to precise observations of deep regions in biological samples.

Hamamatsu Photonics has succeeded in creating a technology “that upgrades the spatial resolution of two-photon excitation fluorescence microscopy,” the company this week stated.

Suited to high-precision observations of deep regions in biological samples, the technology will become an indispensable tool for understanding brain functions, the Japan-headquartered giant added. “The result was achieved through intensive research efforts using our spatial light modulators and Hamamatsu’s optical control technology developed over many years,” said the launch statement.

A spatial light modulator (SLM) utilizes a liquid crystal to control the wavefront of an incident light such as from lasers to adjust the wavefront shape of the reflected light. Using a spatial light modulator allows freely controlling the laser beam pattern for example to branch the incident light and correct its distortion.

Hamamatsu also believes that applying results from this research will easily enhance the resolution of two-photon excitation microscopes equipped with an SLM. This new microscopy technique is being put to practical use across a broad range of fields including neuroscience and biology.

It allows for detailed measurements of deep regions in biological samples and highly accurate observations of changes in the state of organelles that make up cells. This makes it a promising tool for applications in research on brain functions, kidney disease and other illnesses.

The results from this research were recently published in Frontiers in Neuroscience. The results were achieved in a joint research effort with Hamamatsu University School of Medicine.

Research background

In scientific fields such as neuroscience, biology and medicine, it is necessary to clearly observe deeper positions of thick biological samples such as brain tissues. Two-photon excitation fluorescence microscopy utilizes near-infrared light, which penetrates well into biological samples, reaching deeper positions compared to ordinary fluorescence microscopes using visible light.

In deep regions of a sample, however, aberrations are likely to occur depending on the lens’ characteristics and the sample itself, causing a significant loss in resolution. To cope with this problem, two-photon excitation fluorescence microscopes equipped with an SLM are being designed for everyday use. They cancel out the aberrations by feeding a hologram pattern into the SLM.

It is the growing demand from universities and research institutes for higher resolution observation that prompted us to work jointly with the Hamamatsu University School of Medicine. Together, we created this technology improving the resolution of two-photon excitation fluorescence microscopy for practical applications.

Correcting aberrations

When the laser light is emitted on the SLM, it reaches a hologram pattern for aberration correction, which is fed from a computer. It is then reflected back with its aberrations corrected. In two-photon excitation fluorescence microscopy, the sample is irradiated with the laser light with its aberrations corrected, so specific areas provide a sharper and clearer image.

In this research, Hamamatsu considered the number and shapes of hologram pattern rings based on our unique optical control technology and eventually succeeded in finding an optimal pattern that effectively improves the resolution. To further boost it, an optical component to controls the light polarization was added.

By feeding the optimal hologram pattern and adding one optical component in this way, the resolution can be enhanced by about 20% without having to make drastic changes in the microscope optics design. Hamamatsu commented, “We will continue this R&D of improving resolution to widen the scope of practical uses and applications.”