Label-free microscopy technique films cell activity in 3D

Researchers at the University of Tokyo have developed a high-speed microscopy technique which captures images so quickly they can be used to make "videos" of the dynamic processes which occur inside living cells. The team shone mid-infrared light on samples to create a "photothermal signal" and then captured snapshots of the varied temperature-change signatures within the cell given off by different molecules. Previous similar efforts captured 0.05 images per second, but this new technique took 19.2 images per second. By taking images from multiple angles, the team could build a 3D "video" showing active biological processes in motion.

There are many ways to look inside our cells and see how they work, adapt to change and respond to treatments. One technique is label-free mid-infrared photothermal microscopy. This uses mid-infrared light to vibrate the chemical bonds within molecules, each of which has a distinctive frequency. The different frequency signatures can be used to create a map of the internal composition of a cell, which can then be seen by illuminating it with visible light. 

An advantage of this technique is that, unlike with some other methods, no dyes are used which can be toxic to live samples, and by combining shots from multiple angles, a 3D picture can be created. However, capturing images at subsecond timescales has proven challenging.

Previously, three-dimensional mid-infrared photothermal tomography was limited to less than 0.05 images per second, mainly because signal averaging and sequential measurements from different illumination angles required considerable time. However, we have developed a new technique which achieves 19.2 images per second, enabling us to acquire a complete three-dimensional image every 52 milliseconds (thousandths of a second), approximately 400 times faster than previous approaches."

Masato Fukushima, study's first author and Ph.D. student, Department of Physics

The team overcame the speed barrier by using a spatial light modulator (a device that precisely shapes the properties of light, such as its wavelike phases of crests and troughs, to steer it in different directions) to switch the illumination angle very quickly, and by maximizing the signal-to-noise ratio for each shot to make it as clear as possible. The resulting system is called video-rate mid-infrared photothermal optical diffraction tomography (MIP-ODT).

Using MIP-ODT, the team could view lipid droplets in a cell (which are responsible for regulating energy and storing lipids) and track how they moved in three-dimensional space, revealing transport behaviors which could not easily be seen through 2D imaging alone.

"Our research transforms this technique from a method primarily used to observe relatively static samples into a tool capable of quantitatively investigating fast biological processes in three dimensions," said Fukushima.

The team foresees some exciting future applications for this new technique. One use could be the study of biomolecular condensates, membraneless structures in cells which contain proteins and nucleic acids. Abnormal behavior of these condensates has been linked to neurodegenerative disorders, so investigating how they grow and change may help researchers develop new treatments.

"Our next step is to address fundamental biological questions that cannot be explored with conventional techniques. A key strength of label-free chemical imaging is its ability to provide quantitative information about the chemical composition of living cells," explained the study's senior author Professor Takuro Ideguchi from the Graduate School of Frontier Sciences. "By visualizing cellular dynamics in three dimensions and over time, we aim to advance toward a quantitative, measurement-driven understanding of life."