Pump-probe microscope accelerates 3D bioimaging
Image: Pollen grain with point-scanning (top) and pump-probe microscope (bottom).
A new pump-probe microscope developed by China-based researchers promises to produce two-photon microscopy images of a 3D sample in one second, without compromising resolution.
The new method combines a compressive sensing algorithm with a digital holographic microscope and is said to be up to five times faster than conventional point-scanning methods.
The microscope can be used to image nerve distribution of living organisms in 3D as well as the dynamics of hundreds of neurons simultaneously.
Researchers have developed a system that increases the imaging speed of two-photon microscopy by up to three to five times without compromising resolution. On the left is a CAD model of the compact, custom built, two-photon microscopy system. The free-space optics inside are pictured on the right. [The Chinese University of Hong Kong]
To accelerate point scanning, Professor Shih-Chi Chen from the Faculty of Engineering at The Chinese University of Hong Kong developed a novel multi-focus laser illumination method, based on a digital micromirror device.
As Chen points out, the DMD generates 30 points of focused laser light on randomly selected locations within a specimen.
The position and intensity of each point of light are controlled by a binary hologram that is projected onto the device.
During each measurement, the DMD switches the hologram to change the position of each focus and records the intensity of the two-photon fluorescence with a single-pixel detector.
The researchers combined the DMD multi-focus scanning with a compressive sensing algorithm to further improve the imaging speed and enable image acquisition with fewer measurements.
After conducting a simulation experiment to demonstrate the new method's performance and parameters, the researchers tested the approach with two-photon imaging experiments.
The researchers prepared two-photon microscopy images of a pollen grain by using (a) traditional point-scanning and (b) the new compressive imaging approach. The point-scanning imaging time was 2.2 seconds while the compressive imaging time required only 0.55 seconds. [The Chinese University of Hong Kong]
For example, the researchers could acquire 3D images from a pollen grain, in just 0.55 seconds.
The same images acquired with traditional point scanning took 2.2 seconds.
"This method achieved a three to five times enhancement in imaging speed without sacrificing the resolution,” says Chen. “We believe this novel approach will lead to new discoveries in biology and medicine, such as optogenetics.”
The team is now working to further improve the speed of the reconstruction algorithm and image quality, and intend to use the DMD with other advanced imaging techniques for deep tissue imaging.
Research is published in Optics Letters.