Light sheet microscopy allows biologists to selectively light up a thin slice of a sample. This method can drastically improve the image contrast by reducing the amount of background light, while being gentle, reducing photodamage and photobleaching within a sample. Recently, Rice University's Anna-Karin Gustavsson published a method in Nano Letters expanding the use of light sheet microscopy to include a single objective in commercially available sample chambers.
"Prior to this, light sheet microscopy typically required two objectives or specialized sample chambers," said Gustavsson, the corresponding author and an assistant professor of chemistry. "This new method allows us to use light sheet microscopy with a single objective in most commercially available sample chambers."
Gustavsson started by adapting light sheet microscopy to work in microfluidic chips, which have tiny channels where researchers can study cells under varying conditions. In that adaptation, her research team developed a single-objective approach where the light sheet was reflected into the sample inside the microfluidic chip.
"We realized we could 3D nanoprint a noncytotoxic insert to generate a mirror for light sheet reflection," said Nahima Saliba, co-first author and Rice alumna. "Then we could use this approach for single-objective light sheet imaging to reduce background fluorescence, photobleaching and photodamage directly inside the chip."
The team wanted to extend the single-objective method beyond the microfluidic chips, which can be more complicated to work with and are not compatible with all samples. The trick was to develop a pipeline for fabricating inserts that can fit snugly into commercially available sample chambers.
"With our new approach, we can now place a combined insert into most types of sample chambers, add the cells and continue on with the assay like normal, growing and treating the cells for our experiments. When we are ready to image, the mirror allows us to create and manipulate the light sheet from the same objective that we use to detect the light from the sample," said Siyang Cheng, co-first author on the paper and graduate student. "Using our single-objective light sheet reduces background light when imaging, making it easier to see the individual molecules you're interested in. It also reduces harm caused by the light and photobleaching."
Because the insert was 3D nanoprinted, inserts can easily be designed and printed for most commonly available sample chambers. In fact, Gustavsson's lab has already created and provided open access to CAD files containing designs for multiple commonly used sample chambers.
"This opens up for a more refined version of light-sheet microscopy to anyone whose system would benefit from this type of selective illumination, enabling better imaging with less damage to the sample without having to adjust sample preparation workflows," Gustavsson said.
This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM155365) and startup funds from the Cancer Prevention and Research Institute of Texas (RR200025).
