Biology Dr Chunqiang Li ︱ Prism pair Femtosecond laser HWP Polarizer Mirror Novel 3D microscope provides Mirror unprecedented moving images Pickoff mirror L2 AOM L1 of biological processes 3D stage Objective DM1 L3

Dr Chunqiang Li and his team r Li and his collaborative team UNDERSTANDING 3D MICROSCOPY Sample of the University of Texas at El have achieved a breakthrough in Earlier microscopes relied broadly on Grating Paso have developed a novel Doptical microscopy for 3D imaging one of three techniques to capture DM2 three-dimensional (3D) optical without raster scanning laser beams. He 3D trajectory. The irst uses a control Camera2 microscope that uses a spectrally expects to observe interaction between mechanism called ‘feed-back’ to track AFG shaped pulse laser. Whilst living cells at the microscopic scale, in the movement in all three dimensions BP1 BP2 most prior microscopes used three dimension (3D). Dr Li’s approach will of a single particle using the scanning Imaging lens Imaging lens scanning to achieve high speed permit cellular and molecular activities like of laser beams. The second technique 2D imaging, Dr Li’s approach protein motions, subcellular signalling, involves utilising the shape change of Camera1 obtains the z-position from and viral infection to be examined and an image illuminated with a focused a technique called ‘temporal captured in real time. point of light, called the ‘point spread Schematic setup of the temporal focusing two-photon microscope. focusing’ that use ‘diffraction’ function’ to calculate the z-position. The rings and clever mathematics to The team have built on the work of inal method uses the diffraction of light infer the z-position. others in the ield who have sought of an image that has been defocused. THE TEAM AT EL PASO’S APPROACH BUILDING THE MICROSCOPE colours overlap spatially at the objective to image the trajectory, in 3D, of tiny Unlike the previous method the resultant Dr Li and his team have developed a Dr Li’s microscope uses a laser as its lens focal plane, they also overlap ‘particles’, molecules or cells for example, image contains concentric rings and the microscope that is innovative in allowing illumination source, capable of delivering temporally to achieve short laser pulses. at a precision of a nanometre, or one z-position can be determined with high the tracking of more than one particle pulses in the femtosecond range (i.e., billionth of a metre. accuracy from the radius and centre of using luorescence. Fluorescent light 10−15 or 0.000000000000001 seconds). The subjects being examined are the rings. is detected from the target ‘particles’ luorescently labelled so that they emit by an optical technique often used The ultrashort laser pulse is reduced light at certain wavelength after being in microscopes called ‘two-photon in power using optical components illuminated with the ultrafast laser pulse. 3D defocusing imaging of microbes. excitation’. In this, Dichroic beam the specimen is splitters are used labelled to emit Dr Li demonstrated the power of his to separate the light in a particular invention and was able to track the high- emission light colour when from the excitation excited by a laser speed motion of Cafeteria roenbergensis laser light. Images beam. Shaped and videos are ultrafast laser pulses as separate images that could be examined captured by illuminate the scene or combined into a 30 frames per second cameras. Different to be captured over types of subjects a large volume, in moving image can be labelled microscope terms, with difference of 100 µm, or millionths of a metre, before being compressed by passing the luorescent agents to allow multiple each side. The sample is deliberately light through a pair of prisms. A special channel imaging. moved away from the focal plane of component called grating splits the light the microscope objective lens to allow into separate colours and each colour WATCHING MARINE defocused imaging, creating concentric ray passes through a lens for collimation, ZOOPLANKTON rings caused. This approach, called or make them travel in parallel paths like The operation of Dr Li and his team’s ‘temporal focusing’ can not only obtain railway tracks. microscope was tested using a fast- 2D images of the specimen, but also moving marine zooplankton. They chose allows the z-position to be calculated A device called an ‘acousto-optic Cafeteria roenbergensis, a D-shaped from the defocused rings. Time-lapse modulator’ can manipulate each bilagella single-celled organism. The images permit the progression of the separated laser colour ray before it lagella are hair-like appendages that particles being tracked to be captured passes through two further lenses that permit the zooplankton to propel and then viewed as a video or inspected relay the laser pulse to the objective lens. themselves around the oceans. individually. When all the laser light with different

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Research Objectives Funding for Photo-medicine in Massachusetts Dr Li and his team have developed a National Science Foundation (NSF) at Harvard Medical School. Since high-speed temporal focusing two- 2010, Dr Li has worked within photon luorescence microscope for Collaborators the Department of Physics of the three-dimensional (3D) volumetric • Wei Qian (Electrical and Computer University of Texas at El Paso, leading imaging without laser beam scanning. Engineering) the Bio-photonics Laboratory. It will give scientists in biology and • Jorge Gardea-Torresdey (Chemistry) chemistry a strong background in the • Chuan Xiao (Chemistry) Contact development and use of innovative and • Kyung-An Han (Biological Sciences) Chunqiang Li, PhD contemporary instruments, signiicantly Associate Professor enhancing their ability to conduct Bio Physics Department At around 10μm across, they feed cutting edge research. This will also Dr Chunqiang Li received a Doctor of University of Texas at El Paso on bacteria and phytoplankton, are provide training for next generation Philosophy in Electrical Engineering El Paso, TX 79968 signiicant predators of marine bacteria, instrumentalists, who are currently from Princeton University in 2006 and, USA contributing to nutrient recycling, and postdoctoral researchers or graduate from 2006 until 2010, he was a Post- are important in the marine food chain. and undergraduate students. doctoral Fellow at Wellman Centre For the experiment, two colours of luorescence agents were used to label Cafeteria roenbergensis: green and red, comprising tiny beads of 100nm in Dr Li’s novel approach could lead and allows you some variability in the with another dye at another colour. diameter. Q&A design. How would this feature in a Both species are visualised with our to exciting discoveries in biology commercialised microscope? microscope, and the laser can excite The laboratory grew cultures of Cafeteria and health as it waill be possible to Your team’s approach to This acousto-optic modulator is used to both dyes simultaneously. Two CCD roenbergensis in culture medium, determining the z-position using control the spectrum of our femtosecond cameras are used, one to detect the with Escherichia coli bacteria as their examine the interaction between tiny the position of the rings in the laser. It has been used in some products irst dye, and the other one to detect food source. For the experiment, small defocused region of the image is in steering laser beams only. Our method the second dye. Therefore, there are quantities of Cafeteria roenbergensis organisms like viruses and their hosts novel. What drove you to select this explores its novel use of shaping the two detection channels, one for each were extracted from the laboratory’s mechanism? laser pulse in spectral domain. The species. The inal image/video is a mix stock and transferred to a medium tracked, denoted by green and red microscope is not an upper limit, but is Studying biological process in three incorporation of this device in future of signals from both channels. devoid of food. After four days of luorescence, to add to the other two the result of the charged-couple-device dimensions is necessary as they commercial microscope will expand starvation, the diluted luorescent dimensions ilmed. cameras he used. He has proposed happen in a 3D space. However, our ability to have better control of You mentioned the limitations beads were added to the culture. The that his team’s approach could achieve current optical microscopy can laser beams, which opens many more caused by the cameras in use in the zooplankton ingested the beads as food Dr Li demonstrated the power of his millisecond resolution, and even higher obtain 2D images/videos. The third- potential applications. microscope, particularly the frame and could be used as targets in Dr Li’s invention and was able to track the high- speed operation is a possibility for the dimension information requires extra rate. You also suggested some ways team’s microscope to track their motion. speed motion of Cafeteria roenbergensis future. effort, such as scanning the object or In your experiments, you used two around the camera limitations. What They were kept in a darkened place as separate frames that could be lens. This mechanical scanning is the frequencies of light to track two ideally would you like to achieve, to preserve the luorescence for the examined or combined into a 30 frames Dr Li’s novel approach could lead to bottle neck for achieving high speed particles simultaneously. How could and how? experiment. per second moving image. exciting discoveries in biology and health 3D imaging. Therefore, inventing a this be achieved with many particles? There is a trade-off between imaging sciences as it may be possible to examine fast way to conduct 3D imaging is In many particles, it is necessary to speed and signal-to-noise ratio in Time-lapse images were taken with IMPROVING THE MICROSCOPE the interaction between tiny organisms very useful in many research ields. develop multi-frequency tracking, i.e., detecting such week luorescence the two cameras, each camera capture The team have shown that it isn’t like viruses and their hosts but it is not Defocused imaging can provide not each particle is labelled with one-colour signals. If the luorescent signal is one of the luorescent colours, green necessary to use a scanning laser to his only achievement. The microscope only 2D information, but also the label, and our microscope can visualise strong, the imaging speed can be or red. The techniques developed by capture wide-ield 3D images on a is not Dr Li’s only achievement from third dimension information from the them simultaneously. higher. Therefore, I would like to Dr Li meant that each image showed microscopic scale, instead they use this research; he’s created complex level of defocusing. Therefore, it is my use bright and robust luorescence the position of the organism in two- two-photon luorescence as a method mathematical models, utilising modelling vision to fully utilise the information Can you explain how the microscope molecules as the label to reduce the dimensions and the defocused rings and temporal focusing to reveal the software, to generate noise reduction contained in such imaging setup. would operate if tracking the infection image acquisition time. The ideal case around each image inferred the third z-position of a target. algorithms and derive the z-position of a cell with a virus? would be to achieve sub millisecond dimension. Using a mathematical model from the temporal focusing rings with The acousto-optic modulator is an The virus is stained with one dye at time resolution to study fast molecular permitted the team to calculate the Dr Li states that the current frame high accuracy. integral part of your microscope one colour; and the cell is stained dynamics in live animals. z-position of the two zooplankton being rate of 30 frames per second of their

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