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Breaking the limits of optical resolution

New generation of microscopes allow super resolution imaging from the cellular level to the tissue level

TAIWAN | 13 November 2019

Exceeding the Abbe diffraction limit of optical microscopy has been one of the most challenging technological developments in life science. While several super-resolution techniques were able to overcome the Abbe diffraction limit and achieve an unparalleled nanometric resolution, such techniques require the use of objective lens with high numerical aperture. These lenses have short working distances, yielding imaging volumes that are much smaller than the size of most tissue specimens.

Recently, the collaborative efforts between Bi-Chang Chen, an Assistant Research Fellow from the Academia Sinica Applied Science Research Center, and Ann-Shyn Chiang, an Academician and Dean of the Department of Life Sciences and Brain Research Centre of Tsing Hua University have reaped unprecedented results. The resulting creation, a new generation of three-dimensional optical super-resolution microscopes, allows super resolution imaging from the cellular level to the tissue level. The microscope has such a high resolution and imaging volume that it is able to deconstruct the dopaminergic neural network (the collection of neurons in the brain that synthesize the neurotransmitter dopamine) of the whole brain of Drosophila, the common fruit fly, and allow the viewer to visualise the regeneration of memory proteins at specific neuronal synapses within the brain. The results of the research have been published in Nature Communications, an open access scientific journal on October 18 2019.

This scientific breakthrough would not have been possible without the incorporation of the specialised expertise of Bi-Chang Chen and Ann-Shyn Chiang respectively. Prior to the research, Bi-Chang Chen worked in the lab of one of the winners of the 2014 Nobel Prize in Chemistry, Dr Eric Betzig. As someone who has a background in optical technology, he had also organised a study as part of his research in Communications Biology earlier this year which made use of a lightsheet localization microscope to achieve a three-dimensional spatial resolution of less than 100nm. Using the microscope, nuclear pores could be visible to the human eye. Meanwhile, Ann-Shyn Chiang, having obtained his Ph.D. in entomology, was well-versed with the anatomy of the Drosophila, the subject of their research. He also invented a method to make biological tissues transparent within minutes, a technique he would subsequently have to employ for the study of the Drosophila brain.

Ann-Shyn Chiang hopes to leverage on Bi-Chang Chen’s technology to super-resolve the entire Drosophila brain – in other words, locate all protein molecules in the organ. This knowledge will aid him in defining the memory circuits of the Drosophila brain and subsequently allow him to delve deeper into how genes and circuits orchestrate complex behaviours.

Bi-Chang Chen noted that there were various challenges the team must first address before the technology can be applied to perform two-dimensional imaging of subcellular features in large biological tissues on three-dimensional tissue research. For example, the opaque brain of the Drosophila had low visible light transmittance; which meant that only low intensities of light could penetrate the dense tissue of the brain. Additionally, it is difficult to spread an even layer of fluorescent dye on the brain of the fly. This made it much harder for the team to utilise a lightsheet microscope to observe thin, singular layers of cells in the three-dimensional Drosophila brain.

Solving these problems would require the close cooperation of professionals in various fields of research. Ann-Shyn Chiang must first employ his method of brain tissue clearing to allow for a maximum intensity of light to pass through the brain. Such an adjustment would allow the researchers to more accurately pinpoint the exact location and number of the proteins present in thick tissue samples like the brain. Other professors who have had relevant experiences that Bi-Chang Chen and Ann-Shyn Chiang are lacking have also stepped in and offered a helping hand to the duo. For example, Academician Ting-Kuo Lee guided the team regarding image processing; while Dr Li-An Chu and Dr Chien-Han Lu carried out the experiments and analysis and closely monitored as the lightsheet localization microscope was allowed to super-resolve the distribution of protein molecules throughout the fly brain.

Previously, the process of resolving the super-resolution imaging of two-dimensional cell images would occur over a time period of approximately ten hours. However, the new microscopy and image analysis system developed by Yen-ting Lu, an undergraduate student from the national Taiwan University, allowed the researchers to analyse super-resolution image of the whole Drosophila in one day. As such, this highly-efficient system is slated to be used for the statistical analysis of the number of protein molecules in any neuron in the Drosophila brain.

The paper also discussed about the distribution of vesicular monoamine transporter (VMAT) in the dorsal paired medial neurons in the Drosophila brain. VMATs are a type of transport protein integrated into the membrane of synaptic vesicles in the axon terminal of a neuron, which help to regulate the volume of chemical neurotransmitters released to the next neuron. Memory formation occurs when synaptic connections are strengthened.Through a simple experiment which involved the memory training of one batch of fruit flies, followed by a comparison of the brains of the fruit flies with and without memory training, the researchers were able to deduce that proteins involved in memory function exists in both the cell body of the neurons and the synapses between neurons.

The research paper was published in October 2019 on Nature.

This article was contributed by Michelle Tan Min Shuen, an editorial intern at World Scientific Publishing Co. and a contributing writer for Asia-Pacific Biotech News. She is from Nanyang Girls' High School, has a keen interest in chemistry and the life sciences, and pursues taekwondo in her free time.