Ascending the phases to quantitative images can increase the clarity of the image by expanding the dynamic range.
Experts in optical physics have developed a new way to see more detail inside living cells using existing microscopy technology and without the need to add stains or fluorescent dyes.
Since individual cells are almost translucent, microscopic cameras must detect very subtle differences in the light that passes through parts of a cell. These differences are known as the light phase. The camera’s image sensors detect the amount of light phase differences, which is called dynamic range.
“To see more detail using the same image sensor, we need to expand the dynamic range so that smaller phase changes in light can be detected,” said Associate Professor Takuro Ideguchi of the Photon Science and Technology Institute at the University of Tokyo.
The research team developed a technique to take two exposures to measure large and small changes in the light phase separately and then to connect them to create a very accurate final accurate image. They have named their method as an image of the quantitative phases to change the range of dynamic adaptation (ADRIFT-QPI) and recently published the results Light: Science and Applications.
“The ADRIFT-QPI method does not require a special laser, it does not need a special microscope or image sensor; we can use living cells, we don’t need stains or fluorescence, and there’s 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.
The quantitative phase image sends a pulse of a flat sheet of light to the cell, and then measures the phase shift of the light waves as they pass through the cell. Computer analysis reconstructs the image of the main structures inside the cell. Ideguchi and his collaborators have previously pioneered other methods to improve quantitative phase microscopy.
Representation of quantitative phases is a powerful tool for the study of individual cells because they allow researchers to make accurate measurements, such as tracking the growth rate of a cell based on the displacement of light waves. However, the quantitative aspect of the technique has low sensitivity due to the low saturation capacity of the image sensor, so it is not possible to follow nanositized cells in and around a conventional approach.
The new ADRIFT-QPI method has exceeded the limit of the dynamic image of the quantitative phase. During ADRIFT-QPI, the camera takes two exposures and produces the final image, which is seven times more sensitive than traditional quantitative phase microscopy images.
The first exposure is generated with conventional quantitative phase images – a flat sheet of light is pulsed towards the sample and the phase phases of the light are measured after passing through the sample. A computer image analysis program develops a sample image based on the first exposure and then designs a sculpted light wave that reflects that sample image. A special component called a waveform shaping device creates this “light sculpture” with high-intensity light for stronger lighting and pushes it toward the sample for a second exposure.
If the first exposure created an image that was the perfect representation of the sample, the second perforated custom light waves would enter the sample in different phases, pass through the sample, then the camera would come out as a flat sheet of light to see only the dark image.
“This is an interesting thing: we delete the image from the sample. We don’t want to see almost anything. We cancel the large structures so that the smaller ones can be seen in more detail,” Ideguchi explained.
In fact, the first exposure is imperfect, so the sculpted light waves are generated with subtle phase deviations.
The second exposure shows small differences in light exposure phases, which were “cleared” by the larger sides in the first exposure. These small differences in the remaining light phases can be measured with greater sensitivity due to the stronger lighting used in the second exposure.
Additional computer analysis reconstructs the final image of the sample by expanding the dynamic range from the results of the two measurements. In proof-of-concept, the researchers believe that ADRIFT-QPI produces images that are seven times more sensitive than conventional quantitative phase images.
Ideguchi argues that the real benefit of ADRIFT-QPI is the ability to see tiny particles in the context of an entire living cell without the need for labeling or staining.
“For example, viruses or small nanoscale signals from particles moving in and out of a cell could be detected, which allows them to observe their behavior and the state of the cell at the same time,” Ideguchi said.
Reference: Quantitative Imaging of Adaptive Dynamic Range Shift (ADRIFT) by Ka 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 Science Promotion Association.