Extreme resolution with triple-view microscope
Image: Resolution boost for immune cells and more with triple-view microscope.
US-based researchers have developed a new microscope that at least doubles the resolution of images without exposing the sample to more light or prolonging the imaging process.
By extending single-view wide-field microscopy and dual-view light-sheet microscopy to a triple-view microscopy configuration, Dr Hari Shroff from the National Institute of Biomedical Imaging and Bioengineering and colleagues more than doubled volumetric resolution, obtaining high-resolution 3D images and time-lapse image series on fixed and live cellular samples.
According to Shroff, he and colleagues developed improved acquisition, registration, and deconvolution methods for simultaneous multiview imaging.
As he highlights: "Normally unused light can be simultaneously imaged and fused with the “conventional” view normally captured in fluorescence microscopy."
"[This] improves 3D spatial resolution without compromising acquisition speed or introducing additional phototoxicity to the sample, drawbacks that currently plague other multiview approaches," he adds.
Immune cell labeled with green fluorescent protein, imaged with new triple-view microscope (left) and with double-view microscope (right).
Shroff and colleagues had previously developed dual-view selective plane illumination microscopy - diSPIM - which served as the basis for their triple-view microscope.
"diSPIM is an implementation of lightsheet microscopy that uses two 0.8 NA objectives to excite
and detect fluorescence in an alternating duty cycle, subsequently registering and deconvolving the data to achieve isotropic resolution down to ∼330 nm," explains Shroff.
But as Shroff points out, resolution was still limited by the numerical aperture of the upper lenses while the fluorescence emitted in the direction of the coverslip was not captured.
"We reasoned that, as with the triple-view wide-field microscope, simultaneously imaging this otherwise neglected signal with a higher NA lens could in principle boost the resolution of the system," he says.
The new lens images the sample from below, capturing even more light emitted from the sample, while deconvolution methods merged the three images into one to create a sharper, clearer 3D image than previously possible.
Schematic of triple-view microscope.
"The technology... collects “free” light that is neglected in conventional imaging," highlights Shroff. "Relative to state-of-the-art multiview light-sheet imaging, our method improves volumetric spatial resolution at least 4x, yet requires no custom parts and may be readily assembled with commercially
Research is published in Optica.