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What is new in the world of imaging?

In this article, Drug Target Review’s Hannah Balfour discusses three of the latest developments in imaging for disease research and drug development.

Close up of a light microscopy being used by a researcher - image shows just the metal lens and table with slide on

Imaging, in its many forms, is an essential part of life sciences research. It enables researchers to visualise what is hidden by size or many layers of membranes to unearth potential drug targets and, in some cases, explore how drugs targeting them work. In this article find out about some of the latest innovations in imaging that are driving drug target identification and drug development forward…

Two-exposure technique enhances quantitative phase imaging

Scientists have developed a new technique to visualise the insides of living cells without the need for stains or fluorescent dyes that may cause the cells to die.

As individual cells are almost translucent without stains, microscope cameras must detect extremely subtle differences in the light passing through parts of the cell. These differences are known as the phase of the light. Camera image sensors are limited by what level of light phase difference they can detect, referred to as dynamic range. To get a more detailed image, the dynamic range of the sensor must be extended so it can detect smaller phase changes.

Artistic Representation of ADRIFT-QPI. Researchers at the University of Tokyo have found a way to enhance 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) traveling through a cell (center), and exiting (bottom) where changes in the light waves can be analyzed and converted into a more detailed image [Credit: s-graphics.co.jp, CC BY-NC-ND].

Artistic Representation of ADRIFT-QPI. Researchers at the University of Tokyo have found a way to enhance 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) traveling through a cell (center), and exiting (bottom) where changes in the light waves can be analyzed and converted into a more detailed image [Credit: s-graphics.co.jp, CC BY-NC-ND].

Researchers at the University of Tokyo Institute for Photon Science and Technology, Japan, expanded the dynamic range of their microscope by taking two different exposures – one measuring large changes in light phase, the other small changes – then connecting them to create a highly detailed final image. According to the scientists, with the technique they dubbed adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI), the final image has seven times greater sensitivity than traditional quantitative phase microscopy images.

“Our ADRIFT-QPI method needs no special laser, no special microscope or image sensors; we can use live cells, we do not need any stains or fluorescence and there is very little chance of phototoxicity,” said Associate Professor Takuro Ideguchi. Phototoxicity is the killing of cells with light, which can become a problem with some other imaging techniques, such as fluorescence imaging.

Quantitative phase imaging is a powerful tool for examining individual cells, but its utility has been hampered by low sensitivity, making tracking nanosized particles in and around cells impossible.

Ideguchi said that because ADRIFT-QPI increases the sensitivity of the technique so much, it will allow researchers to track tiny particles in context of the whole living cell without requiring any labels or stains. “For example, small signals from nanoscale particles like viruses or particles moving around inside and outside a cell could be detected, which allows for simultaneous observation of their behaviour and the cell’s state,” he concluded.

The study was published in Light: Science & Applications.

Fluorescence lifetime imaging microscopy (FLIM) could revolutionise cellular imaging

One of the problems of conventional fluorescence microscopy techniques is that the results are difficult to evaluate quantitatively. This is primarily because fluorescence intensity – how brightly a cellular component/compound fluoresces – is significantly affected by both experimental conditions and the concentration of the fluorescent substance. To overcome this, scientists in Japan have been imaging based on fluorescence lifetime, how long a component fluoresces for, instead of how bright it shines.

They developed fluorescence lifetime imaging microscopy (FLIM), which measures how the fluorescence of a specific substance decays (reduces) over time. According to the team, this is unique to each substance and independent of experimental conditions, allowing them to accurately quantify fluorescent molecules and changes in their environment. However, to capture this fluorescence decay, they had to develop a new approach that would enable them to use multiple single-point photodetectors to capture the whole sample at once.

Fluorescence "lifetime" microscopy technique. 2D arrangement of 44,400 light stopwatches enables scan-less fluorescence lifetime imaging [Credit: Tokushima University].

Fluorescence “lifetime” microscopy technique. 2D arrangement of 44,400 light stopwatches enables scan-less fluorescence lifetime imaging [Credit: Tokushima University].

Professor Takeshi Yasui, from Institute of Post-LED Photonics (pLED), Tokushima University, Japan, who led the study published in Science Advances, explained that their approach allows them to simultaneously map the fluorescence decay at 44,400 points over a two-dimensional (2D) space in a single shot.

To achieve this, they used an optical frequency comb as the light source for the sample. An optical frequency comb is a light signal made up of multiple, equally spaced light beams, all repeatedly switching on and off. Due to the space between each beam, they hit the sample in spatially distinct locations. Each individually lit area acts as a ‘pixel’ in the final image.

The fluorescence emitted from the sample in response to each irradiation by the optical frequency comb is focused using a lens on a high-speed single-point photodetector. The fluorescence lifetime at each pixel is then calculated from the phase delay between the excitation signal and the measured signal. Over time, the repeated pulses of light and detections of fluorescence show how substances and organelles move within cells.

According to Yasui, FLIM will make it easier to exploit the advantages of fluorescence lifetime measurements and will be helpful in life sciences research where dynamic observations of living cells are needed.

Could imaging improve antibiotics?

Collaborative work has enabled researchers from the Francis Crick Institute, UK, and the University of Western Australia to develop a new imaging method that shows where antibiotics have reached bacteria within tissues.

According to the team, their new method could be used to help develop more effective antibiotic treatments that would limit the risk and spread of antibiotic resistance.

For antibiotics to be successful they must be able to penetrate human cells, as this is where bacteria reside during infections. Researchers believe that if they could select or develop more effective antibiotics based on which tissues they can enter, they may be able to reduce the length of treatment needed for infections and in turn reduce the risk of antibiotic resistance developing.

Mycobacterium tuberculosis infected mouse lungs. A computed tomography scan of mouse lungs infected with Mycobacterium tuberculosis. Red inclusions indicate the location of granulomas within the infected tissue. These sites were further investigated to identify the intracellular distribution of the anti-tuberculosis drug Bedaquiline and its localisation in specific immune cells [Credit: Tony Fearns].

Mycobacterium tuberculosis infected mouse lungs. A computed tomography scan of mouse lungs infected with Mycobacterium tuberculosis. Red inclusions indicate the location of granulomas within the infected tissue. These sites were further investigated to identify the intracellular distribution of the anti-tuberculosis drug Bedaquiline and its localisation in specific immune cells [Credit: Tony Fearns].

To achieve this, the researchers created a new imaging method called Correlative light, electron and ion microscopy in tissue (CLEIMiT). In the paper published in PLoS Biology, the team used their new technology to observe which cells and tissues the antibiotic bedaquiline reached in the lungs of mice infected with tuberculosis (TB).

They demonstrated using CLEIMiT that the antibiotic, commonly used to treat TB, did not reach all the infected tissues, tended to accumulate in certain types of immune cells (macrophages and polymorphonuclear cells) and entered tissues that were not infected with bacteria.

Tony Fearns, author and senior laboratory research scientist in the Host-Pathogen Interactions in Tuberculosis Laboratory at the Crick, said: “Our approach could be used to help develop new antibiotics or to re-assess current antibiotics to judge how effectively they reach their targets. The more we learn about how drugs behave in the body, for example where they collect, the better we will be able to treat bacterial diseases like TB.”

The scientists said they are continuing to adapt the method for other types of antibiotic and to image multiple antibiotics simultaneously.

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