Upgrading to Quantum Phase Imaging can sharpen the image by expanding the dynamic range.
Experts in photophysics have developed a new way to see inside living cells in more detail using current microscopy technology and without the need to add stains or fluorescent dyes.
Since individual cells are semi-transparent, microscopic cameras must detect very subtle differences in the light that passes through parts of the cell. These differences are known as the phase of light. The camera’s image sensors are limited by the amount of light phase difference they can detect, referred to as dynamic range.
“To see more details with the same image sensor, we must expand the dynamic range so that we can detect smaller phase changes in the light,” said associate professor Takuro Ideguchi from the University of Tokyo Institute of Photon Science and Technology.
The research team developed a technique to take two exposures separately to measure large and small changes in the phase of light and then seamlessly connect them to create a final, highly detailed image. They named their dynamic adaptive quantum phase imaging method (ADRIFT-QPI) and recently published their results in Light: Science and Applications.
“Our ADRIFT-QPI method does not require a special laser, nor a special microscope or image sensors;” We can use live cells, we don’t need any spots or fluorescence, and there is very little chance of phototoxicity, “said Ediguchi.
Phototoxicity refers to killing cells by light, which can become a problem with some other imaging techniques, such as fluorescence imaging.
Quantum phase imaging sends a pulse from a flat sheet of light toward the cell, and then measures the phase shift of the light waves as they pass through the cell. The computer analysis then reconstructs the image of the main structures inside the cell. Previously Ideguchi and his collaborators have devised other methods to improve quantitative microscopy.
Quantum phase imaging is a powerful tool for examining individual cells because it allows researchers to make detailed measurements, such as tracking the rate of cell growth based on the shift in light waves. However, the quantitative aspect of the technique has a low sensitivity due to the low saturation capacity of the image sensor, so tracking of nanoparticles in and around cells is not possible using the conventional approach.
The new ADRIFT-QPI method overcomes the limitations of dynamic range for quantum phase imaging. During the ADRIFT-QPI, the camera takes two exposures and produces a final image with a sensitivity seven times greater than conventional quantum microscopy images.
The first exposure is produced by conventional quantum phase imaging – a flat sheet of light is pulsed towards the sample and the phase shifts of the light are measured after it passes through the sample. A computer image analysis program develops an image of a sample based on the first exposure, then quickly designs a sculptural wavefront of light that reflects that image to the sample. Then a separate component called this ‘light sculpting’ wavefront modulator generates higher intensity light for stronger illumination and directs it towards the sample for a second exposure.
If the first exposure produces an image that is a perfect representation of the sample, the custom-sculpted light waves from the second exposure will enter the sample at various stages, pass through the sample, and then appear as a flat light sheet, causing the camera to only see a darkened image.
“Here’s the interesting thing: We kind of erase the image of the sample. We want to see almost nothing. We are canceling the larger structures so we can see the smaller ones in great detail.”
In fact, the first exposure is incomplete, so the sculpted light waves appear with slight deviations.
The second exposure reveals slight differences in the phase of light that are “faded” due to the larger differences at the first exposure. The remaining small light phase difference can be measured with increased sensitivity due to the stronger illumination used for the second exposure.
Additional computer analysis reconstructs a final image of the sample with an expanded dynamic range from the two measurement results. In proof of concept displays, researchers estimate that ADRIFT-QPI produces images with seven times greater sensitivity than conventional quantitative imaging.
Ideguchi says the real benefit of ADRIFT-QPI is its ability to see fine particles in the context of the entire living cell without the need for any stickers or spots.
“For example, small signals can be detected from nanoparticles such as viruses or particles moving in and out of the cell, allowing simultaneous monitoring of their behavior and the state of the cell,” Ediguchi said.
Reference: “Adaptive Dynamic Transformation (ADRIFT) Quantitative Phase Imaging” by K. Toda, M. Tamamitsu and T. Ideguchi, December 31, 2020 Light: Science and Applications.
DOI: 10.1038 / s41377-020-00435-z
Funding: Japan Science and Technology Agency, Japan Society for the Promotion of Science.