Researchers at Boston University have figured out how to simultaneously capture images of different depths using a standard microscope. The new method will come in handy in a variety of microscopy disciplines, making it useful for a wide range of biological and biomedical research and imaging applications. The development is reported by Optica magazine.
“Optical microscopy has been an indispensable tool for studying three-dimensional complex biological systems and processes,” explains Sheng Xiao, a research team member at Boston University. “However, our new multifocal technique allows for the observation of living cells and organisms at high speed and high contrast.”
The main distinguishing 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 clear images in a single focal plane. Although researchers have tried different strategies for simultaneously acquiring images with different depths of focus, these approaches usually require the use of multiple cameras. Or, for example, using a dedicated Diffractive Optical Separation Element (DOE) to create an image with a single camera. Both strategies are complex, and DOE is not easy at all.
Diffractive optical elements are optical substrates with amplitude and/or phase diffraction structures on one of the surfaces, calculated using a computer and manufactured by precision laser or electron-beam lithography.
The scientists used a prism with a z-beam splitter. It can be completely assembled from standard components and can be easily applied to various imaging methods. For example, with fluorescence, phase contrast microscopy, or dark-field imaging.
A prism splits the detected light to simultaneously capture multiple images in a single camera frame. Each image in the sample is focused at a different depth. The use of a high-speed camera with a large sensor area and a large number of pixels allowed researchers to distribute multiple high-resolution images on a single sensor.
The multifocal images obtained with the new technique make it possible to evaluate the defocused background of the sample much more accurately than can be done with a single image. The researchers used this information to develop an improved 3D blur removal algorithm. It eliminates defocused background light, which is often a problem with wide-angle microscopy.
“Our advanced 3D blur removal algorithm suppresses far out of focus background from sources outside of the volume of the image,” Xiao explains. “This improves both the image contrast and the signal-to-noise ratio, which makes the algorithm particularly useful for fluorescence imaging using thick samples.”
The researchers demonstrated the new technique using widely used microscopic techniques. They made 3D images with a large field of view, encompassing hundreds of neurons or whole free-moving organisms. Experts also created high-speed 3D images of rotifer cilia that move every hundredth of a second. This experiment clearly demonstrated the possibilities of a new method for obtaining high-quality three-dimensional images.
To demonstrate the capabilities of the advanced 3D blur removal algorithm, the researchers visualized various thick samples, including the brains of a living mouse. They noted significant improvements in contrast and signal-to-noise ratio. Researchers are currently working to extend this technique so that it can work with even more imaging techniques.