Boston University researchers have developed a microscope specifically for imaging large groups of interacting cells in their natural environments. In a paper published today in Optica, the team showed that their new "multi-z" confocal microscopy system can image the brains of living mice at video rate and with a field of view larger than a millimeter.
Imaging large groups of cells requires capturing cellular or subcellular details at fast speeds over a large 3D volume. This is challenging because most imaging approaches come with inherent tradeoffs between speed, field of view, and resolution.
"We found a way to merge the needed imaging features in a microscopy system that is easy to build and operate," said Amaury Badon, first author of the paper. "It also provides results in real time without the need for complicated data analysis or image processing."
To acquire multiple planes simultaneously, the researchers developed a way to reuse the light for imaging cells in one plane to also image cells deeper in the sample. They used an approach called extended illumination in which the microscope's objective lens is only partially filled with the illuminating light, allowing the light to reach deeper into the sample. The full objective lens is then used to detect fluorescence, which provides high resolution. Rather than having one pinhole, like traditional confocal setups, the new microscope has a series of reflective pinholes that each capture in-focus light from a different depth within the sample.
"Our method benefits from the contrast of confocal microscopy while being able to extend to volumetric imaging without sacrificing speed," said Badon. "Although extended illumination and reflective pinholes have been used before, this is the first time they were combined in a confocal microscope setup in a light-efficient way."
The researchers also tailored the microscope for larger scale imaging than conventional confocal microscopes and designed it to image at video rate. Fast image acquisition was important because the fluorescence indicators that monitor cellular function typically operate on time scales of a few tens of milliseconds.