Researchers at the University of Tokyo have found a way to increase the sensitivity of existing quantitative phase imaging so that all structures in living cells can be seen simultaneously, from small particles to large structures. This artistic representation of the technique shows pulses of sculpted light (green, top) moving through a cell (center) and leaving (below) where changes in the light waves can be analyzed and converted into a more detailed image. Credit: s-graphics.co.jp, CC BY-NC-ND
Upgrading to quantitative phase imaging can enhance image beauty by increasing dynamic range.
Experts in optical physics have developed a new way to see inside living cells in greater detail using existing microscopy technology and without adding stains or fluorescent dyes.
Since individual cells are almost translucent, microscope cameras have to detect extremely subtle differences in the light moving through parts of the cell. These differences are known as the phase of light. Camera image sensors are limited by the amount of light phase difference they can detect, also known as dynamic range.
“To see more detail with the same image sensor, we need to expand the dynamic range so that we can detect smaller phase changes of light,” said associate professor Takuro Ideguchi of the University of Tokyo’s Institute of Photonics and Technology.
The research team developed a technique to take two exposures to measure large and small changes in the light phase separately and connect them seamlessly to create an extremely detailed final image. They call their method adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI) and recently published their results in Light: Science and applications.
Images of silica beads taken using conventional quantitative phase imaging (above) and a clearer image developed using a new ADRIFT QPI microscopy method (below) developed by a research team at the University of Tokyo . The photos on the left are images of the optical phase and images on the right show the optical phase change due to the middle infrared (molecular specific) light absorption by the silica beads. In this proof-of-concept demonstration, researchers calculated that they achieved approximately 7 times greater sensitivity through ADRIFT-QPI than that of conventional QPI. Credit: Image by Toda et al., CC-BY 4.0
“Our ADRIFT QPI method requires no special laser, no special microscope or image sensors; we can use living cells, we do not need stains or fluorescence, and there is very little chance of phototoxicity, ”Ideguchi said.
Phototoxicity refers to the killing of cells with light, which can become a problem with other imaging techniques, such as fluorescence imaging.
Quantitative phase imaging sends a pulse from a flat sheet of skin to the cell and then measures the phase shift of the light waves as it moves through the cell. Computer analysis then reconstructs an image of the most important structures in the cell. Ideguchi and his associates have previously done pioneering work with other methods to improve quantitative phase microscopy.
Quantitative phase imaging is a powerful tool for examining individual cells, as it enables researchers to make detailed measurements, such as tracking the growth rate of a cell 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. It is therefore not possible to detect nanosize particles in and around cells using a conventional approach.
A standard image (above) taken using conventional quantitative phase imaging and a clearer image (bottom) developed using a new ADRIFT QPI microscopy method developed by a research team at the University of Tokyo . The photos on the left are images of the optical phase and images on the right show the optical phase change due to the middle infrared (molecular specific) light absorption, mainly by proteins. Blue arrow points to the edge of the nucleus, white arrow points to the nucleus (a substructure within the nucleus), and green arrows point to other large particles. Credit: Image by Toda et al., CC-BY 4.0
The new ADRIFT QPI method has overcome the dynamic scope constraint of quantitative phase imaging. During ADRIFT QPI, the camera takes two exposures and delivers a final image that is seven times more sensitive than traditional quantitative phase microscopy images.
The first exposure is produced with conventional quantitative phase imaging – a flat sheet of light is pulsed to 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 the sample based on the initial exposure and then quickly designs a shaped wavefront of light that reflects the image of the sample. A separate component called a wavefront forming device then generates this ‘sculpture’ with a higher intensity of light for stronger illumination and pulses it to the sample for a second exposure.
If the first exposure produced an image that was a perfect representation of the sample, the light waves tailored to the second exposure would penetrate the sample in different phases, pass through the sample and then appear as a flat sheet of skin, which the camera to see nothing but a dark image.
‘This is the interesting thing: we erase the image’s example. We want to see almost nothing. We cancel the large structures so that we can see the small details in detail, ”Ideguchi explained.
In fact, the first exposure is imperfect, so that the sculpted light waves with subtle phase deviations emerge.
The second exposure shows small light phase differences that were “washed out” by larger differences in the first exposure. This residual small light phase difference can be measured with increased sensitivity due to the stronger lighting used during the second exposure.
Additional computer analysis reconstructs a final image of the sample with an extended dynamic range of the two measurement results. In proof-of-concept demonstrations, researchers estimate the ADRIFT QPI delivers images with seven times greater sensitivity than conventional quantitative phase imaging.
Ideguchi says that the real advantage of ADRIFT-QPI is the ability to see small particles within the context of the entire living cell without the need for labels or stains.
“For example, small signals from nanoscale particles such as viruses or particles moving around inside and outside a cell can be detected, which can observe the behavior and the condition of the cell simultaneously,” Ideguchi said.
Reference: “Adaptive dynamic range shift (ADRIFT) quantitative phase imaging” by K. Toda, M. Tamamitsu and T. Ideguchi, 31 December 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.