"Optical microscopy has been an indispensable tool for studying three-dimensional complex biological
The main distinguishing feature of the new method isthat this approach can simply be 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 systemsobtain clear images in one focal plane. Although researchers have tried different strategies for simultaneously acquiring images with different depths of focus, these approaches usually require 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.
Diffraction optical elements are optical substrates with amplitude and/or phase diffraction structures on one of the surfaces, calculated with the help of a computer and manufactured by precision laser or electron-beam lithography.
The scientists used a prism with a z-beam divider, which can be completely assembled from standard components and easily applied to a variety of imaging techniques.For example, fluorescence , phase-contrast microscopy or darkfield imaging.
The prism separates the detected light forsimultaneous acquisition of several images in one 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 high pixel count allowed the researchers to distribute multiple high-resolution images on a single sensor.
Multifocal images obtained withnew techniques make it possible to estimate the defocused background of a 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 blur removal algorithm3D image suppresses far out of focus background from sources outside of the image volume, explains Xiao. "This improves both image contrast and signal-to-noise ratio, which makes the algorithm particularly useful for fluorescence imaging using thick samples."
Proven versatility
Researchers have demonstrated a new technique withusing widely used microscopy methods. 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, which 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 advanced3D blur removal algorithm, the researchers visualized various thick specimens, including the brain 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.
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