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It is extremely difficult to image single cells inside living animals, but it is necessary to investigate how cells and tissues inside the body function. The use of light-based techniques enables researchers to image, identify and engineer such unique entities, but can only penetrate a certain distance through tissues.
New and improved biophonic techniques are needed to help us see further into the body. A team of Scotland researchers believes its new microscopy technique can do just that. Using a micrometer-sized laser, the team was able to track beating-induced changes in the refractive index of heart cells.
Why are Improved Biophotonic Techniques Needed?
Medical imaging techniques such as MRI and ultrasound allow researchers to see inside the body, but only a certain distance and at low resolution, making it difficult to visualize individual cells.
Fluorescence-based microscopy techniques employ a camera to record light emitted from a fluorescent dye located within cells to determine the features of individual cells. It can be used, for example, to image in vivo brain cells, but is invasive and challenging to apply as it requires replacing a piece of the skull with a porthole-like window to gain direct visual access to the tissue.
Fluorescence microscopy can also only image the outer few cell layers of a tissue – equating to around 100 μm of tissue in the heart for example, so it is not particularly useful for looking deep into tissues. “We really need a brighter light source,” states Dr Marcel Schubert of the University of St Andrews in Scotland.
New Laser-Based Microscopy Technique
Schubert and his colleagues have developed a new microscopy technique using micrometer-sized lasers placed directly inside heart cells to track their beating. Instead of placing dye molecules directly in a cell, they used micrometer-sized plastic beads infused with dye molecules; these beads are known to act as a laser.
When the bead is illuminated with light, the dye molecules begin to fluoresce. Because of its specific refractive properties, most of this fluorescent light is trapped inside the bead, and circles around its outer surface.
At certain wavelengths, this circling light “constructively interferes”, increasing in intensity; this also happens when the circling light hits an excited dye molecule and gains additional photons from stimulated emission, just like a normal laser.
Some of the circulating light escapes and produces a laser signal up to 1000 times brighter than that of conventional fluorescence-microscopy techniques. It is, therefore, easier to spot, and this is what the team was interested in.
Micrometer Lasers in Practice
When placed next to an isolated mouse heart cell, the bead was swallowed up by the cell, and when exposed to light, the wavelengths of the laser light shifted up and down whenever the cell contracted and expanded. The shifts in the color of light that the lasers emitted were only small, but clearly visible on a spectrometer said Schubert.
The colour change came as a big surprise and is believed to be caused by a previously unrecognised change in the cellular machinery of the heart muscle cells.”
Dr Marcel Schubert, Royal Society Research Fellow, School of Physics and Astronomy
These shifts are thought to occur from changes in the local refractive index of myofibrils, which are long, skinny proteins that contract each time the heart beats. The local protein density in the cell momentarily increases during contraction and falls when the protein relaxes and lengthens.
Schubert tracked the length changes of the myofibrils closest to the bead using a confocal microscope; they revealed the shortening and lengthening correlated exactly with the wavelength change.
The team conducted experiments with live zebrafish hearts and with slices taken from rat heart tissue. With the latter, they could detect beating signals from beads through 400 μm of tissue.
“Other people have tried to monitor refractive-index changes deep inside tissues without success,” Schubert said. “Here we have shown that we can really do that with our laser beads.”
The Future of Biophotonics
With techniques such as this, the future of biophotonics looks promising. The better the techniques are, the more we can learn about cells within the body without having to remove them from their natural habitat or grow them artificially in the lab.
Microlasers such as these can be readily produced in the millions and are relatively cheap compared to many modern microscopes and the associated infrastructure.
Schubert and his team are working on even smaller lasers made from a semiconductor called indium gallium phosphide. Initial works show these nanometer-sized disk lasers can track changes in a cell’s refractive index, and, because of their size, multiple lasers could be used in a single cell, enabling localized measurements within a cell.
Further experiments in lab-grown 3D heart tissues are also planned, for example, using the lasers to monitor the biomechanical properties of a heart as it develops inside embryos.
References and Further Reading
Wright, K. (2021) Lighting Up Heart Cells with Tiny Lasers, Physics: https://physics.aps.org/articles/v14/71. Accessed 25 May 2021.
University of St Andrews (2020) Feel the beat: implanted microlasers scan heart from inside, University of St Andrews: https://news.st-andrews.ac.uk/archive/feel-the-beat-implanted-microlasers-scan-heart-from-inside/. Accessed 25 May 2021.