See the insides of living cells in more detail using the new microscopy technique


Researchers at the University of Tokyo have found a way to improve the sensitivity of existing quantitative phase imaging so that all structures inside living cells can be seen simultaneously, from tiny particles to large structures. This artistic representation of the technique shows pulses of sculpted light (green, top) passing through a cell (center) and exiting (bottom) where changes in light waves can be analyzed and converted into a more detailed image. Credit:, CC BY-NC-ND

Upgrading to quantitative phase imaging can increase image clarity by expanding dynamic range.

Experts in optical physics have developed a new way to see the insides of living cells in greater detail using existing microscopy technology and without the need to add dyes or fluorescent dyes.

Since individual cells are almost translucent, microscope cameras must detect extremely subtle differences in the light passing through parts of the cell. These differences are known as the phase of light. The camera image sensors are limited by the amount of light phase difference they can detect, called dynamic range.

“To see more detail using the same image sensor, we need to expand the dynamic range so that we can detect smaller phase changes in light,” said Associate Professor Takuro Ideguchi of the Institute of Science and photon technology from the University of Tokyo.

The research team developed a technique to take two exposures to measure large and small light phase changes separately, then connect them seamlessly to create a final, highly detailed image. They named their Adaptive Dynamic Range Shift Quantitative Phase Imaging (ADRIFT-QPI) method and recently published their results in Light: science and applications.

Extension of dynamic range by ADRIFT QPI

Images of silica beads taken using conventional quantitative phase imaging (top) and a clearer image produced using a new ADRIFT-QPI microscopy method (bottom) developed by a research team from the University of Tokyo. The photos on the left are images of the optical phase and the images on the right show the optical phase change due to absorption of mid-infrared (molecular specific) light by the silica beads. In this proof-of-concept demonstration, the researchers calculated that they achieved approximately 7 times higher sensitivity with ADRIFT-QPI than with conventional QPI. Credit: Image by Toda et al., CC-BY 4.0

“Our ADRIFT-QPI method does not require any special laser, special microscope or image sensor; we can use living cells, we don’t need any staining or fluorescence, and there’s very little chance of phototoxicity, ”said Ideguchi.

Phototoxicity refers to the destruction of cells by light, which can become a problem with certain other imaging techniques, such as fluorescence imaging.

Quantitative phase imaging sends a pulse from a flat sheet of light to the cell, then measures the phase shift of the light waves after they pass through the cell. Computer analysis then reconstructs an image of the main structures inside the cell. Ideguchi and his colleagues have already developed other methods to improve quantitative phase microscopy.

Quantitative phase imaging is a powerful tool for examining individual cells, as it allows researchers to perform detailed measurements, such as tracking a cell’s growth rate as a function of the change in light waves. However, the quantitative aspect of the technique has low sensitivity due to the low saturation capacity of the image sensor, so tracking nanoscale particles in and around cells is not possible with a conventional approach.


A standard image (top) taken using conventional quantitative phase imaging and a clearer image (bottom) produced using a new ADRIFT-QPI microscopy method developed by a research team at the University of Tokyo. The photos on the left are optical phase images and the images on the right show the optical phase change due to absorption of mid-infrared (molecular specific) light mainly by proteins. The blue arrow points to the edge of the nucleus, the white arrow points to the nucleoli (a substructure inside the nucleus), and the 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 limitation of the dynamic range of quantitative phase imaging. During ADRIFT-QPI, the camera takes two exposures and produces a final image that has a sensitivity seven times greater than traditional quantitative phase microscopy images.

The first exposure is produced with conventional quantitative phase imaging – a flat sheet of light is pulsed towards the sample and the phase shifts of the light are measured after it has passed through the sample. A computer image analysis program develops an image of the sample based on the first exposure, then quickly designs a sculpted wavefront of light that reflects that image of the sample. A separate component called a wavefront shaping device then generates this “light sculpture” with higher intensity light for stronger illumination and transmits 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-sculpted light waves from the second exposure would enter the sample at different phases, pass through the sample, and then emerge as a flat sheet of light, causing the camera to see nothing but a dark image.

“That’s the interesting thing: we kind of erase the sample image. We hardly want to see anything. We are canceling large structures so that we can see smaller ones in detail, ”Ideguchi explained.

In reality, the first exposure is imperfect, so the sculpted light waves emerge with subtle phase shifts.

The second exposure reveals tiny differences in the light phase that were “eliminated” by larger differences in the first exposure. These tiny remaining light phase differences can be measured with increased sensitivity due to the stronger lighting used in the second exposure.

Further computer analysis reconstructs a final sample image with extended dynamic range from the two measurement results. In proof-of-concept demonstrations, researchers estimate that ADRIFT-QPI produces images with a sensitivity seven times greater 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 spots.

“For example, small signals from nanoscale particles like viruses or particles moving inside and outside a cell could be detected, allowing simultaneous observation of their behavior and condition. of the cell, ”Ideguchi said.

Reference: “Adaptive dynamic range shift (ADRIFT) Quantitative Phase Imaging” by K. Toda, M. Tamamitsu and T. Ideguchi, December 31, 2020, Light: science and applications.
DOI: 10.1038 / s41377-020-00435-z

Funding: Japan Agency for Science and Technology, Japan Society for the Promotion of Science.