IMAGE: Researchers at the University of Tokyo have found a way to quantify the sensitivity of existing quantitative-level images so that all structures within living cells can be seen at once, … view more
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Experts in optical physics have developed a new way to see inside living cells in more detail using existing microscope technology and without the need to add fluorescent stains or dyes.
Since individual cells are virtually motionless, microscope cameras have to detect small differences in the light passing through parts of the cell. These differences are called the light level. Camera image sensors are limited by the amount of light level difference they can detect, referred to as dynamic range.
“To see more detail using the same image sensor, we need to expand the dynamic range so that we can detect smaller light changes,” Associate Professor Takuro Ideguchi of the University of Tokyo Institute said for Photon Science and Technology.
The research team developed a technique to take two publications to measure large and small changes in light level separately and then quickly combined them to create a highly detailed final image. They named the ADRIFT-QPI and recently published their results. Light: Science & Applications.
“Our ADRIFT-QPI method does not require a special laser, or a special microscope or image sensors; we can use living cells, we do not need any stains or fluorescence, and there is little potential for phototoxicity,” Ideguchi said.
Phototoxicity refers to the killing of cells by light, which can be a problem with some other imaging techniques, such as fluorescence imaging.
Quantitative phase images send a pulse of a smooth sheet of light toward the cell, then measure the phase movement of the light waves after they pass through the cell. Computer analysis then reconstructs an image of the main structures within the cell. Ideguchi and his colleagues have pioneered other ways to strengthen quantitative-level microscopy.
Quantitative phase imaging is a powerful tool for studying individual cells because it allows researchers to make accurate measurements, such as monitoring the growth rate of a cell based on movement in light waves. However, the quantitative aspect of the method has low sensitivity due to the low absorption capacity of the image sensor, so it is not possible to monitor nanosized particles in and around cells with a conventional approach.
The new ADRIFT-QPI method has overcome the limitation of the dynamic range of quantitative-level images. During ADRIFT-QPI, the camera makes two exposures and produces a final image that has seven times the sensitivity of traditional quantitative-level microscopy images.
The first appearance is produced by standard quantitative phase images – a flat sheet of light is drawn towards the sample and the movements of the light level are measured after passing through the sample. A computer image analysis program develops an image of the sample based on the first appearance and then quickly designs a designed light face that mirrors that image of the sample. A separate component called a waveform shape device then generates this “light sculpture” with higher intensity light for stronger illumination and draws it towards the sample for a second exposure.
If the first publication produced an image that was a perfect representation of the sample, the designed light waves of the second publication would penetrate the sample at different stages, passing through the sample, and then appears as a smooth sheet of light, causing the camera to see nothing but a dark image.
“Here’s the interesting thing: We remove the image of the sample. We want to see almost nothing. We take off the large structures so that we can see the smallest ones in detail,” explained Ideguchi.
In reality, the first appearance is imperfect, so the carved light waves appear with subtle phase movements.
The second release reveals tiny light level differences that were “washed out” with larger differences in the first appearance. The small remaining light level differences can be measured with increased sensitivity due to the stronger illumination used in the second exposure.
Additional computer analysis reproduces a final image of the sample with an extended dynamic range from the two measurement results. In hypothesis demonstrations, researchers estimate that the ADRIFT-QPI produces images with seven times the sensitivity of normal quantitative-rate images.
Ideguchi states that the real advantage of ADRIFT-QPI is the ability to see tiny particles in the context of the entire living cell without the need for any labels or stains.
“For example, small traces of nanoscale grains are detected as viruses or particles moving around the inside and outside of a cell, allowing simultaneous vision of the behavior and condition of the cell,” he said. Ideguchi.
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Research Article
K. Toda, M. Tamamitsu, T. Ideguchi. November 2020. Quantitative dynamic range motion (ADRIFT) image. Light: Science & Applications. DOI: 10.1038 / s41377-020-00435-z https: /
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Associate Professor Takuro Ideguchi
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Funders
Japan Science and Technology Association (JPMJPR17G2)
Japan Society for the Advancement of Science KAKENHI (17H04852, 17K19071)
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