Bidirectional scattering microscope detects micro- and nanoscale structures simultaneously
A new microscope that can simultaneously measure both forward- and backward-scattered light from a sample could allow researchers to image both micro- and nanoscale objects at the same time. The device could be used to observe structures as small as individual proteins, as well as the environment in which they move, say the researchers at the University of Tokyo who developed it.
“Our technique could help us link cell structures with the motion of tiny particles inside and outside cells,” explains Kohki Horie of the University of Tokyo’s department of physics, who led this research effort. “Because it is label-free, it is gentler on cells and better for long observations. In the future, it could help quantify cell states, holding potential for drug testing and quality checks in the biotechnology and pharmaceutical industries.”
Detecting forward and backward scattered light at the same time
The new device combines two powerful imaging techniques routinely employed in biomedical applications: quantitative phase microscopy (QPM) and interferometric scattering (iSCAT).
QPM measures forward-scattered (FS) light – that is, light waves that travel in the same direction as before they were scattered. This technique is excellent at imaging structures in the Mie scattering region (greater than 100 nm, referred to as microscale in this study). This makes it ideal for visualizing complex structures such as biological cells. It falls short, however, when it comes to imaging structures in the Rayleigh scattering region (smaller than 100 nm, referred to as nanoscale in this study).
The second technique, iSCAT, detects backward-scattered (BS) light. This is light that’s reflected back towards the direction from which it came and which predominantly contains Rayleigh scattering. As such, iSCAT exhibits high sensitivity for detecting nanoscale objects. Indeed, the technique has recently been used to image single proteins, intracellular vesicles and viruses. It cannot, however, image microscale structures because of its limited ability to detect in the Mie scattering region.
The team’s new bidirectional quantitative scattering microscope (BiQSM) is able to detect both FS and BS light at the same time, thereby overcoming these previous limitations.
Cleanly separating the signals from FS and BS
The BiQSM system illuminates a sample through an objective lens from two opposite directions and detects both the FS and BS light using a single image sensor. The researchers use the spatial-frequency multiplexing method of off-axis digital holography to capture both images simultaneously. The biggest challenge, says Horie, was to cleanly separate the signals from FS and BS light in the images while keeping noise low and avoiding mixing between them.
Horie and colleagues, Keiichiro Toda, Takuma Nakamura and team leader Takuro Ideguchi, tested their technique by imaging live cells. They were able to visualize micron-sized cell structures, including the nucleus, nucleoli and lipid droplets, as well as nanoscale particles. They compared the FS and BS results using the scattering-field amplitude (SA), defined as the amplitude ratios between the scattered wave and the incident illumination wave.
“SA characterizes the light scattered in both the forward and backward directions within a unified framework,” says Horie, “so allowing for a direct comparison between FS and BS light images.”
Spurred on by their findings, which are detailed in Nature Communications, the researchers say they now plan to study even smaller particles such as exosomes and viruses.
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