Live-Cell Super-Resolution Imaging of Dynamic Cellular Processes

俗话说“眼见为实”，荧光显微镜通过使研究人员能够可视化细胞及其包含的结构，大大提高了我们对细胞生物学的理解。由于细胞是动态的，活细胞成像提供了更多的信息。在这方面，如果一张图片胜过千言万语，那么一段视频又值多少钱呢？最近，超分辨率成像技术的出现使科学家们能够进一步放大细胞。然而，与许多超分辨率方法相关的权衡限制了它们对活细胞视频的适用性。其中的关键是，超分辨率成像通常需要更高的激光功率和更长的曝光时间，这可能导致光漂白（信号丢失）和光毒性（细胞应力）。光漂白限制了可以拍摄的图像数量和成像的时间周期。光毒性意味着你不能在“正常”条件下成像细胞，甚至可能导致细胞死亡。此外，较长的曝光时间限制了帧速率，限制了捕获图像的频率，这意味着您可能会错过快速发生的动态细胞过程。因此，研究人员必须在图像分辨率、采集速度和成像时间之间做出妥协。这个提议是为了升级我们目前的显微镜系统，通过提高其能力和减少成像所需的激光曝光，使其能够用于活细胞超分辨率成像。具体来说，一种新的相机和改进的镜头将减少获得图像所需的激光功率和曝光时间，从而减少对电池的压力。同时，新的硬件和软件将使系统自动化，使其运行更快，并提供额外的成像模式。这些升级将增加系统的灵活性，使研究人员能够实时成像细胞过程，这是他们以前无法观察到的。我们的研究团队对基本的细胞生物学过程感兴趣，并有一个共同的需求，以尽可能高的分辨率成像活细胞。我们可以通过将现有的SD-OSR显微镜升级到最新的IXplore SpinSR系统来实现这一目标。升级后的显微镜对于提高我们nserc资助的每个项目的影响至关重要，因为这种能力在卡尔加里大学的其他地方是不可用的。此外，IXplore SpinSR系统将集成到卡尔加里大学的活细胞成像实验室，使其广泛应用于数百名研究人员，并进一步提高培训能力。这些升级将开启一种全新的成像方法，通过促进对许多研究领域的许多生物过程的更好理解，将使加拿大受益。

With respect to the adage `seeing is believing', fluorescent microscopy has greatly advanced our understanding of cell biology by enabling researchers to visualize cells and the structures they contain. As cells are dynamic, live-cell imaging provides even more information. In this regard, if a picture is worth a thousand words, how much is a video worth? Recently, the advent of super-resolution imaging has enabled scientists zoom in even further into cells. However, there are trade-offs associated with many super-resolution approaches that limit their applicability for live-cell videos. Key among them is the fact that super-resolution imaging often requires higher laser power and longer exposure times, which can cause photobleaching (loss of signal), and phototoxicity (cell stress). Photobleaching limits the number of images that can be taken and the time-period over which imaging can occur. Phototoxicity means that you are not imaging cells under `normal' conditions, and can even lead to cell death. Additionally, longer exposure times limit the frame rate, restricting how often images can be captured, which means that you can miss dynamic cellular processes that occur quickly. Thus, researchers must compromise between image resolution, acquisition speed, and length of time imaging. This proposal is for upgrades to our current microscope system that will enable its use for live-cell super-resolution imaging by improving its capabilities and reducing laser exposure required for imaging. Specifically, a new camera and improved lenses will reduce the laser power and exposure time required to obtain images, placing less stress on the cells. Meanwhile, new hardware and software will automate the system, allowing it to operate faster, and provide additional imaging modalities. These upgrades will increase the flexibility of the system, enabling researchers to image cellular processes in real time that they have previously not been able to observe. Our team of investigators is interested in fundamental cell biological processes and shares a common need to image living cells at the highest resolution possible. We can achieve this goal by upgrading our existing SD-OSR microscope to the latest IXplore SpinSR system. The upgraded microscope is critical for enhancing the impact of each of our NSERC-funded programs, as this capability is not available elsewhere at the University of Calgary. Furthermore, the IXplore SpinSR system will be integrated into the Live Cell Imaging Laboratory at the University of Calgary, making it widely available to hundreds of researchers and further enhancing training capacity. These upgrades will unlock a whole new approach of imaging and will benefit Canada by facilitating a better understanding of many biological processes across many research fields.