UNIST's Tunable TIRF Microscope Images Cell Protrusions and Deep Vesicles in One Frame

Inside a living cell, two things happen at once at very different depths. Thin, antenna-like protrusions called filopodia reach out from the surface to probe the surroundings, while small sac-like vesicles carrying membrane components and proteins travel closer to the substrate. A team at Ulsan National Institute of Science and Technology (UNIST) in South Korea has built a microscope that can show both in the same frame, each in sharp focus.

The device is a tunable total internal reflection fluorescence microscope, or Tunable TIRF, developed by Professor Park Jung-hoon's group in UNIST's Department of Biomedical Engineering. The work was published in the October 22 issue of the optics journal Laser & Photonics Reviews, and UNIST unveiled it on the 25th. Professors Kang Ju-heon and Kim Ha-jin of the same department took part as co-authors, along with Dr. Lee Sang-gyu of the Institute for Basic Science (IBS) Center for Memory and Glioscience.

The problem it addresses is a built-in limit of conventional microscopes: they apply a single illumination depth across the whole field at any moment. Vascular endothelial cells and certain cancer cells use filopodia to sense their environment and move, and vesicles travel along in the process. To see both the vesicles near the substrate and the protrusions rising above it, researchers had to switch illumination modes and image one depth after another. That slowed imaging and caused the fluorescent labels to fade from repeated exposure.

The team's workaround was to add a digital micromirror device (DMD) and a spatial filter to the illumination path of a standard fluorescence microscope. The DMD carries many tiny mirrors arranged like a checkerboard, and tilting them changes the direction and depth of the incoming light. Applying different patterns to different regions means one area can be lit only in a thin layer by the substrate while another is lit deep into the cell. The spatial filter lets through only light travelling in the needed direction.

The system offers six illumination depths: four total internal reflection steps in the 137 to 271 nanometer range, plus deeper illumination. Compared with changing depths one after another, recording is twice as fast when alternating between two depths and three times as fast with three. The DMD can switch its stripe patterns up to 1,440 times per second, and by alternating illumination from several directions it also reduces bright lines and speckles caused by scattering.

The researchers tested it on living cells. In human umbilical vein endothelial cells, they lit the cell edges through the full cell height and the center with shallow light near the substrate, capturing the undulating membrane and nearby vesicle movement in one video. In HeLa cervical cancer cells, they imaged the swaying of upward-reaching protrusions and the movement of vesicles near the membrane together at 20 frames per second.

Operation is meant to be simple. A researcher draws regions with a pen on the cell image shown on a tablet and assigns a depth to each, and the microscope illuminates accordingly. Because the design only adds a DMD and spatial filter to an existing fluorescence microscope, it avoids the need for costly new equipment.

Park said the work overturns the long-held premise that a microscope can use only one illumination method across the whole observation target, and that it could help researchers understand processes such as cell migration in wound healing, cancer metastasis, membrane transport and bacterial infection. Less light exposure also means living cells can be watched longer, which suits long-term tracking experiments. The announcement, however, gave no commercialization timeline, no pricing, and no estimate of how long therapeutic development might take, so how widely the tool is adopted remains to be seen.