Upgrading to quantitative phase imaging can increase image clarity by extending the dynamic range.
Optical physics experts have developed a new way to see living cells inside in detail using existing microscopy technology and without the need to add spots or fluorescent dyes.
Because individual cells are almost translucent, microscopic cameras must detect extremely subtle differences in the light that passes through parts of the cell. These differences are known as the light phase. The camera’s image sensors are limited by the phase difference of the light they can detect, called dynamic range.
“To see more detail using the same image sensor, we need to extend the dynamic range so that we can detect smaller phase changes in light,” said Associate Professor Takuro Ideguchi of the University of Tokyo for photon science and technology.
The research team developed a technique to make two exposures to separately measure the large and small changes of the light phase and then connect them perfectly to create a very detailed final image. They called their quantitative phase imaging method (ADRIFT-QPI) and recently published their results in Light: Science and applications.
“Our ADRIFT-QPI method does not need a special laser, nor a special microscope or image sensors; we can use living cells, we don’t need spots or fluorescence and there is very little chance of phototoxicity “, said Ideguchi.
Phototoxicity refers to the killing of cells with light, which can become a problem with other imaging techniques, such as fluorescent imaging.
Quantitative phase imaging sends a pulse of a flat sheet of light to the cell, then measures the phase shift of light waves as they pass through the cell. Computer analysis then reconstructs an image of the major structures inside the cell. Ideguchi and his collaborators have previously initiated other methods to improve quantitative phase microscopy.
Quantitative phase imaging is a powerful tool for examining individual cells because it allows researchers to make detailed measurements, such as tracking a cell’s growth rate based on changing 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 nanositized particles in and around cells is not possible through a conventional approach.
The new ADRIFT-QPI method has exceeded the limitation of the dynamic range of the quantitative phase image. During ADRIFT-QPI, the camera makes two exposures and produces a final image that has a sensitivity seven times higher than traditional images with quantitative phase microscopy.
The first exposure is produced by conventional quantitative phase imaging – a flat sheet of light is pulsed towards the sample and the phase changes 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 first exposure, then quickly projects a light-sculpted wavefront that mirrors that image of the sample. A separate component called the wavefront modeling device then generates this “light sculpture” with higher intensity light for stronger illumination and propels it to the sample for a second exposure.
If the first exposure produced an image that was a perfect representation of the sample, the custom-carved light waves of the second exposure would enter the sample in different phases, pass through the sample, and then appear as a flat sheet of light. , causing the camera to see nothing but a dark image.
“This is the interesting thing: we are almost erasing the image of the sample. We don’t want to see almost anything. We cancel the large structures, so that we can see the smaller ones in detail “, Ideguchi explained.
In reality, the first exposure is imperfect, so the sculpted light waves appear with subtle phase deviations.
The second exposure reveals small differences in light phase that were “washed out” by larger differences in the first exposure. These small remaining light phase differences can be measured with increased sensitivity due to the stronger lighting used in the second exposure.
An additional computerized analysis reconstructs a final image of the sample with an extended dynamic range from the two measurement results. In demonstrations of the concept, the researchers estimate that ADRIFT-QPI produces images with a sensitivity seven times higher than conventional quantitative phase imaging.
Ideguchi says the real benefit of ADRIFT-QPI is its ability to see tiny particles in the context of the entire living cell, without the need for labels or stains.
For example, small signals from nanomatic-scale particles, such as viruses or particles moving in and out of a cell, could be detected, allowing their behavior and cell status to be observed simultaneously, he said. Ideguchi.
Reference: Adaptive dynamic range shift (ADRIFT) quantitative 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.