Researchers have developed a new fluorescence microscopy method that greatly improves the clarity of the image by using three views of the sample at the same time.

The new study in Optica examines examines a new technique in two microscopy modes in imaging several types of biological samples. Researchers expanded the microscopy configuration to a triple-view. They also improved acquisition, registration, and deconvolution methods in assembling 3D and time-lapse images.

"In our work, we captured this previously neglected fluorescence and fused it with the traditional views used in conventional microscopy," said Yicong Wu, staff scientist at the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Maryland, USA, and first author of the Optica paper. "This increases resolution without compromising either temporal resolution or adding additional light to the sample."

Using the three-view method improved the current dual-view plane illumination microscopy to a resolution of 235nm. In the wide-field mode, scanning the three objectives through the sample at the same time produced 3D views. In this mode the image was improved the axial-axis resolution to 340nm, a 45 percent increase from the dual-view.

"One helpful trick was to deconvolve each view first to increase image quality, contrast, and so forth, which then allowed accurate registration of the three views," said Wu. "In wide-field mode, we further aided registration of the images by adding fluorescent beads to the samples as point of reference." 

Funding for the study was provided by the National Institute of Biomedical Imaging and Bioengineering, National Cancer Institute and a number of other organizations.