A Benthic Underwater Microscope with Pulse Amplitude Modulated Imaging Capability (BUMP)

沿海生态系统的健康和长期动态，如海藻森林、红树林、海草床，特别是珊瑚礁，在很大程度上受到发生在一毫米或更小尺度上的过程的推动。这些生态系统的许多居民是初级生产者，允许将阳光能量转化为分子能量，分子能量通过食物链流动，并将生态系统推向高潮。能量转换发生在颗粒单元中(从叶绿体到单细胞藻类，取决于环境)，这些单元的性能由其直接周围的物理化学条件决定。因此，评估每个单元的性能对于更好地了解生态系统中的适应和生产力至关重要，同时也是生物体整体健康和光合作用能力的良好指标。这些基本的微观过程引起了不同学科的科学家的兴趣，如生理学、光生物学、生态学和有机体的相互作用。尽管小规模进程很重要，但在适当的范围内研究这些进程的工具严重不足。脉冲幅度调制(PAM)技术作为一种评估生理过程的方法，已经在实验室和水下环境中得到了广泛的应用。由此产生的数据可以推断光合作用速率，提供光合作用活性的衡量标准。这种系统已经在水下进行了大量测量，但从未达到监测单个微生物所需的分辨率。在这个项目中，将创建水下凹凸显微镜(具有脉冲幅度调制成像的底栖式水下显微镜)，在其自然环境中在微观水平上测量这些过程。该系统将增强更好地了解海底海洋过程的能力，因为它能够在不干扰或将其从其自然环境中移走的情况下，对微小的光合作用生物及其生理状态进行现场测量。因此，该成像系统提供的结果将促进新的发现，以更好地了解全球海洋生物群落的结构因素。为了实现这一目标，该凸起将结合现有海底显微成像系统的一些特征，同时增加观察入射调制光诱导的动态荧光变化的能力。该系统将使用长工作距离(水中45 mm)镜头，在1.5 mm x 1.5 mm的视场内分辨率为1.5微米。一个由12个带聚焦光学的高功率宽带LED组成的斜环照明器将提供光系统饱和脉冲、光化光以及反射图像的照明。该系统将为100微秒或更短的短曝光提供足够的照明。该设计包括多个光路，允许使用2个摄像头和两个照明源进行双模成像。与控制系统的水下外壳中的iPad一起，将在两个实验室建造和测试一个全功能的微观层面的脉冲幅度调制成像系统，然后移动到野外，在各种环境中进行潜水员操作，如海藻森林和珊瑚礁。

The health and long-term dynamics of coastal ecosystems such as kelp forests, mangroves, sea grass beds, and especially coral reefs, are significantly driven by processes that occur on scales of a millimeter or less. Many inhabitants of these ecosystems are primary producers, allowing transformation of sunlight energy into molecular energy, which is what flows through food chains and drives ecosystems climax. The energy transformation takes place in particulate units (from chloroplasts to unicellular algae, depending on the context) and the performance of each of these units is dictated by their direct surrounding physical-chemical conditions. Hence, assessing the performance of each of these units is critical to better understand adaptation and productivity in the ecosystem, while also being a good proxy for the organisms overall health and ability to photosynthesize. These fundamental microscopic processes are of interest to scientists across diverse disciplines such as physiology, photobiology, ecology, and organisms' interactions. Despite the importance of small-scale processes, the availability of tools to study them at the appropriate scales has been severely lacking. As one way of assessing physiological processes, Pulse Amplitude Modulated (PAM) technology is an important method that has been implemented in both the lab and the underwater environment. The resultant data allows inference of photosynthetic rates, providing a measure of photosynthetic activity. Such systems have been implemented for bulk measurements underwater but never at the resolution needed to monitor individual microscopic organisms. In this project, the underwater BUMP microscope (Benthic Underwater Microscope with Pulse amplitude modulated imaging) will be created that will measure these processes at the microscopic level in their natural environment. The system will enhance the capacity to better understand benthic marine processes by enabling in situ measurement of microscopic photosynthetic organisms as well as their physiological status without disturbing or removing them from their natural environment. The results provided by this imaging system will thereby promote new discoveries to better understand factors that structure marine communities globally.To achieve this goal, the BUMP will incorporate a number of features of an existing system for subsea microscopic imaging while, at the same time, adding the capability to observe dynamic fluorescent changes that have been induced with incident modulated light. The system will use a long working distance (45 mm in water) lens with a resolving power of 1.5 micro-meters over a 1.5 mm x 1.5 mm field of view. An inclined ring illuminator consisting of 12 high power broadband LEDs with focusing optics will provide photosystem saturation pulses, actinic light, as well as illumination for reflectance images. The system will provide sufficient illumination for short exposures of 100 microseconds or less. The design includes multiple optical paths allowing for dual-mode imaging with 2 cameras and two illumination sources. Taken together with an IPAD in an underwater housing for controlling the system, a fully functional Pulse Amplitude Modulated imaging system at the microscopic level will be built and tested in both the lab and then moved to the field for diver operation in various environments such as kelp forests and coral reefs.