Enhanced Capability of Spinning Disk Confocal Microscope

了解细胞过程需要在微观和纳米尺度上观察动态变化和分子相互作用。因此，能够以高分辨率和实时进行这些观察的成像方法的发展彻底改变了生物和生物医学科学。然而，与固定标本的观察不同，对活细胞进行成像面临着挑战，因为成像过程对细胞活力有破坏性影响，并且需要控制细胞的生理行为。旋转圆盘共聚焦（SDC）显微镜是一种最先进的荧光成像技术，是温和和快速。与传统的外显荧光或点扫描共聚焦激光显微镜相比，SDC显微镜减少了光毒性的破坏性影响，并在高速下捕获甚至微弱的荧光信号。因此，SDC显微镜是细胞生物学家和生物医学科学家不可或缺的工具。麦吉尔大学麦克唐纳校区的生命科学研究人员社区大量使用校园多尺度成像设施（MuSIF）中的SDC显微镜。该系统由Geitmann博士于2015年获得NSERC拨款，此后扩展和升级了额外的成像功能，如全内反射荧光（TIRF）探测器和高性能sCMOS相机。在本提案中，要求增加部件，以便1)进一步提高仪器的通用性，2)确保仪器的使用寿命，3)确定环境的可持续性。所要求的项目用于为长期时移成像（环境室）提供恒定的环境条件，在荧光标记方面提供更大的灵活性（额外的激光线，发射滤光片，第二个相机）和高空间分辨率（100倍物镜）。由此增强的能力将适应更广泛的生物有机体和实验设计。重要的是，通过更换10年的工作站，以及用新的LED光源取代汞基光源，仪器的预期寿命将大大延长。MuSIF的荧光成像仪器套件经常被麦克唐纳校区近20个研究团队的成员预订，这些团队来自植物科学、寄生虫学、微生物学和食品科学等不同学科。SDC显微镜的升级对于正在进行和计划中的研究项目的推进至关重要，这些项目有利于加拿大人，并有助于基础科学的发展。研究课题包括植物形态发生、病毒感染过程、纳米材料的毒性、炎症性胃肠道疾病和细胞内细菌。升级后的仪器将成为HQP技术技能培训的关键工具，并支持由NSERC发现资助的众多研究项目的活动。

Understanding cellular processes requires the observation of dynamic changes and molecular interactions at micro- and nanometric length scales. The development of imaging methodologies enabling these observations at high resolution and in real time, therefore, revolutionized the biological and biomedical sciences. However, unlike the observation of fixed specimens, imaging live cells came with challenges arising from the damaging effect of the imaging process proper on cell viability and from the necessity to control the cells' physiological behavior. Spinning disk confocal (SDC) microscopy is a state-of-the art fluorescence imaging technique that is gentle and rapid. Compared to conventional epi-fluorescence or point scanning confocal laser microscopy, SDC microscopy reduces the damaging effect of phototoxicity and captures even weak fluorescent signals at high speeds. SDC microscopy is therefore an indispensible tool for cell biologists and biomedical scientists. The community of life science researchers at McGill's Macdonald Campus makes intensive use of the SDC microscope housed in the campus' Multi-Scale Imaging Facility (MuSIF). The system was acquired by Dr. Geitmann in 2015 with an NSERC grant and has since been expanded and upgraded with additional imaging capabilities such as a Total Internal Reflection Fluorescence (TIRF) detector, and a high-performing sCMOS camera. In the present proposal, additional components are requested to 1) further enhance the versatility of the instrument, 2) ensure the instrument's longevity and 3) ascertain environmental sustainability. The requested items serve to provide constant environmental conditions for long-term time lapse imaging (environmental chamber), more flexibility in terms of fluorescent markers (additional laser line, emission filters, second camera), and high spatial resolution (100x objective). The thusly enhanced capabilities will accommodate a wider range of biological organisms and experimental designs. Importantly, the expected life time of the instrument will be significantly extended by replacing the 10-year old workstation, and by substituting the mercury-based light source by a new LED light-source. The suite of fluorescence-based imaging instruments at MuSIF is regularly booked by the members of nearly 20 research teams at Macdonald Campus from disciplines as diverse as plant science, parasitology, microbiology and food science. The upgrades to the SDC microscope are essential for the advancement of ongoing and planned research projects that benefit Canadians and contribute to the advancement of fundamental science. Among the research topics are plant morphogenesis, viral infection processes, toxicity of nanomaterials, inflammatory gastrointestinal diseases and intracellular bacteria. The upgraded instrument will be a crucial tool for HQP technical skills training and it supports the activities of numerous research programs funded through NSERC Discovery grants.