Researchers at the University of Tokyo have found a way to increase the sensitivity of the current quantitative phase imaging so that all the structures inside living cells, from small particles to large formations, can be viewed simultaneously. This artistic representation of the technique shows the pulses (green, top) of sculpted light that travel through the cell (center), and exits (bottom) where changes in light waves can be analyzed and converted into a more detailed image. Credit: s-ographic.co.jp, CC BY-NC-ND
An upgrade to quantitative phase imaging can increase image clarity by expanding the dynamic range.
Experts in icicle physics have developed a new way of living inside living cells in a wide range of ways, using existing microscopy techniques and without the need to add stain or fluorescent dyes.
Since individual cells are almost translucent, a microscope camera should detect very subtle differences in the light passing through parts of the cell. Those differences are known as the phase of light. Camera My image sensors, known as dynamic series, are limited by how much light phase difference they can detect.
“To see more detail using the same image sensor, we should expand the dynamic range so that we can detect small phase changes in light,” said Takuro Ideguchi, an associate professor at the Photon Science and Technology for G for Tokyo Institute.
The research team developed a technique to measure large and small changes in the light phase separately and then connect them seamlessly to create a very detailed final image. They named their method Adaptive Dynamic Range Shift Quantitative Phase Imaging (ADRIFT-QPI) and recently published their results. Light: Science and Applications.
Images of silica beads taken using conventional quantitative phase imaging (top) created using a new ADRIFT-QPI microscopy method (bottom) developed by a research team at the University of Tokyo. The photos on the left are images of the icicle phase and the images on the right show the change in the right icicle phase due to the absorption of mid-infrared (molecular niche) light by the silica structure. In the Proof-F Concept demonstration, the researchers calculated that they achieved about 7 times more sensitivity through ADRIFT-QPI than conventional QPI. Credit: Tada et al., Image by CC-BY 4.0
“Our Adrift-QPI method requires no special laser, no special microscope or image sensor; We can use living cells, we don’t need any stains or fluorescence, and the chances of phototoxicity are very low, “Ideguchi said.
Phototoxicity refers to the killing of cells with light, which can be a problem with some other imaging techniques, such as fluorescence imaging.
Quantitative phase imaging sends a pulse of a flat sheet of light to the cell, then measures the phase shift of the light waves passing through the cell. The computer rearranges the image of the main structures inside the cell after analysis. Ideguchi and his colleagues have previously introduced other methods to enhance quantitative phase microscopy.
Quantitative phase imaging is a powerful tool for examining individual cells as it allows researchers to make detailed measurements such as tracking cell growth rates based on the shift of light waves. However, due to the low saturation capacity of the image sensor, there is less sensitivity in the quantitative aspect of the technology, so tracking nanosized particles in and around cells is not possible with the traditional approach.
A standard image (top) taken using conventional quantitative phase imaging and clear image (below) using the 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 the images on the right show changes in the optical phase mainly due to the absorption of mid-infrared (nuclear specific) light by the protein. The blue arrow points to the edge of the nucleus, the white arrow points to the nucleoli (a structure inside the nucleus), and the green arrow points to other large particles. Credit: Image by Todd et al., CC-BY 4.0
The new ADRIFT-QPI method has exceeded the dynamic range of quantitative phase imaging. During ADRIFT-QPI, the camera takes two contacts and creates the final image, which is seven times more sensitive than conventional quantum phase microscopy images.
The first exposure produces conventional quantitative phase imaging – a flat sheet of light bends towards the sample and the phase shift of the light is measured after it has passed through the sample. The computer image analysis program first develops a sample image based on the exposure and then quickly designs a sculpted wavefront of light that mirrors that image of the sample. A separate component called a wavefront shaping device, then produces this “sculpture of light” with more intense light and pulses towards the sample for more light.
If an image was created in the first contact which is a perfect representation of the sample, the custom-sculpted light waves coming in the second contact will enter the sample at different stages, pass through the sample, then emerge as a flat sheet of light, causing nothing but a dark image to be seen. My camera.
“The interesting thing is: we kind of erase the image of the template. We want to see almost anything. We cancel large constructions so we can see the small large parts in detail, ”Ideguchi explained.
In reality, the first exposure is incomplete, so sculpted light waves emerge from the goo phase phase deviations.
The second exposure reveals small light phase differences that were “washed out” by large differences in the first exposure. This remaining small light phase difference can be measured with increased sensitivity due to the strong light used in the second contact.
Additional computer analysis rearranges the final image of the sample with an extended dynamic range from the results of the two measurements. In a proof-of-concept demonstration, the researchers estimated that ADRIFT-QPI produces images that are seven times more sensitive than conventional quantitative phase imaging.
Ideguchi says the real advantage of ADRIFT-QPI is its ability to see small particles in the context of an entire living cell without the need for any labels or stains.
“For example, small signals of nanoscale, such as viruses or particles moving in and out of a cell, can be detected, allowing them to simultaneously observe their behavior and the state of the cell,” Ideguchi said.
Reference: k. Tada, m. Tamamitsu and T. 31 April 2020, “Adaptive Dynamic Range Shift (ADRIFT) Quantitative Phase Imaging” by Ideguchi Light: Science and Applications.
DOI: 10.1038 / s41377-020-00435-z
Funding: Japan Science and Technology Agency, Japan Society for the Promotion of Science.
