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，如海带林、红树林、海草床，特别是珊瑚礁，在很大程度上受到发生在毫米或更小尺度上的过程的驱动。这些生态系统的许多居民都是初级生产者，他们可以将太阳能转化为分子能量，从而在食物链中流动，推动生态系统的高潮。能量转化发生在微粒单位中（从叶绿体到单细胞藻类，取决于环境），这些单位的每一个性能都是由它们周围的直接物理化学条件决定的。因此，评估这些单位的表现对于更好地了解生态系统中的适应性和生产力至关重要，同时也是生物体整体健康和光合作用能力的良好代表。这些基本的微观过程引起了生理学、光生物学、生态学和生物相互作用等不同学科的科学家的兴趣。尽管小规模过程很重要，但严重缺乏在适当规模上研究它们的工具。脉冲调幅（PAM）技术作为一种评估生理过程的方法，在实验室和水下环境中都得到了应用。由此得到的数据可以推测光合速率，提供光合活动的测量方法。这种系统已经用于水下的大量测量，但从未达到监测单个微生物所需的分辨率。在这个项目中，水下BUMP显微镜（底栖水下显微镜与脉冲振幅调制成像）将被创建，将测量这些过程在微观水平上的自然环境。该系统将提高人们更好地了解底栖海洋过程的能力，使人们能够在不干扰或将它们从自然环境中移走的情况下就地测量微观光合生物及其生理状态。该成像系统提供的结果将促进新的发现，从而更好地了解全球海洋群落结构的因素。为了实现这一目标，BUMP将整合现有海底显微成像系统的许多功能，同时增加观察入射调制光引起的动态荧光变化的能力。该系统将使用一个长工作距离（水中45毫米）的镜头，在1.5毫米× 1.5毫米的视野范围内，分辨率为1.5微米。一个由12个具有聚焦光学的高功率宽带led组成的倾斜环形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光源进行双模成像。将一个功能齐全的脉冲振幅调制成像系统与一个用于控制系统的水下住宅中的IPAD结合在一起，将在实验室中建立和测试一个微观水平的脉冲振幅调制成像系统，然后移动到现场，供潜水员在各种环境中操作，如海带森林和珊瑚礁。

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.