Researchers from Boston University have invented how to simultaneously obtain images of different depths using a standard microscope. The new method will be useful in different areas of microscopy, which makes it useful for a wide range of biological and biomedical research and imaging. Optica magazine reports about the development.
“Optical microscopy has been an indispensable tool for studying three-dimensional complex biological systems and processes,” explains Shen Xiao, a member of the research team at Boston University. “However, our new multifocal technique allows us to observe living cells and organisms at high speed and with high contrast.
The main feature of the new method is that this approach can be simply added to most existing systems and easily replicated. This will make the development available to other researchers.
Capturing multi-focus images
Standard camera-based microscopy systems produce sharp images in a single focal plane. Although researchers have tried different strategies for obtaining images at different depths of focus at the same time, these approaches usually require the use of multiple cameras. Or, for example, using a special diffractive optical separation element (DOE) to create an image with a single camera. Both strategies are complex, and DOEs are not easy at all.
Diffractive optical elements are optical substrates with amplitude and/or phase diffraction structures on one of the surfaces, calculated with a computer and produced by precision laser or electron-beam lithography.
Scientists used a prism with a z-beam splitter. It can be fully assembled from standard components and can easily be used for various imaging methods. For example, in fluorescence, phase-contrast microscopy or dark field imaging.
The prism separates the detected light to obtain several images in one camera frame at the same time. Each image in the sample is focused at a different depth. Using a high-speed camera with a large sensor area and large pixel count allowed researchers to distribute multiple high-resolution images on a single sensor.
The multifocal images obtained with this new technique allow researchers to evaluate the unfocused background of the sample much more accurately than can be done with a single image. The researchers used this information to develop an improved algorithm for removing blur in three-dimensional space. It removes out-of-focus background light, which is often a problem when using wide angle microscopy.
“Our advanced three-dimensional blur removal algorithm suppresses out-of-focus background light from sources beyond the image volume,” Xiao explains. “This improves both image contrast and signal-to-noise ratio, making the algorithm particularly useful for fluorescent imaging using thick samples.
The researchers demonstrated the new technique using widely used microscopy methods. They made three-dimensional images with a large field of view, covering hundreds of neurons or whole free-moving organisms. The experts also created high-speed three-dimensional images of rotisserie cilia that move every hundredth of a second. This experiment clearly demonstrated the possibilities of a new method of obtaining high quality three-dimensional images.
To demonstrate the possibilities of an advanced algorithm for removing blurred three-dimensional images, the researchers visualized various thick samples, including the brain of a living mouse. They noted significant improvements in contrast and signal-to-noise ratio. The researchers are now working on expanding this technique so that it can work with even more imaging techniques.