Multiresolution Autofocusing for Automated Microscopy

描述（由申请人提供）：该项目将进一步开发和完善用于自动显微镜的创新数字自动对焦技术。自动对焦对于自动显微镜成像至关重要。目前可用的技术依赖于在单个图像分辨率下的聚焦计算的各种算法，并且受到固有的性能限制，这影响了它们在临床和研究应用中的成功和利用。例如，用于荧光显微镜的自动聚焦对现有方法的所需精度、可靠性和速度提出了严重挑战，因为在这种情况下，图像具有非常低的信噪比和窄的景深，而样品暴露于荧光激发必须最小化，以避免光漂白和形成不期望的物质，如自由基和单线态氧。基于小波变换的数学理论，提出了一种新的多分辨率图像分析方法用于显微镜自动聚焦。新的方法克服了一些固有的限制，目前可用的技术，并持有承诺，使测量的显微镜聚焦功能和最佳焦点成像位置的检测相当准确，可靠，快速。这项创新技术将显著提高自动化显微镜仪器的能力和效率，用于广泛的临床和研究应用，其中需要对大量标本进行常规成像和定量分析。在第一阶段项目中，我们研究了所提出的荧光显微镜技术的可行性。我们开发了软件来实现多分辨率聚焦函数测量和聚焦成像位置搜索的算法。我们在细胞遗传学FISH标本的各种样本图像堆栈上评估了软件模拟中的新方法，并使用（1）准确性，（2）范围，（3）鲁棒性和（4）速度的标准将其与当前所有最佳显微镜自动聚焦方法进行了比较。第一阶段的结果表明，通过使用适当的基于小波的自动聚焦功能，新的多分辨率方法在上述每个性能类别中显著优于所有竞争方法，并且明显超过了第一阶段的可行性标准。在第二阶段项目中，我们将在实时操作环境中进一步开发、完善、集成和验证新技术。我们计划建立一个具有多分辨率自动聚焦功能的原型系统，用于荧光和明场显微镜成像。我们将广泛评估该系统的各种应用，包括遗传学，病理学和细胞学。我们将在常规临床实验室环境中测试新系统和技术，并在收集最终用户输入和反馈时优化技术。一旦完全开发和合格，这项新技术将获得专利，并纳入未来的IRIS自动成像细胞仪。它还将通过许可协议和合作伙伴关系向应用成像公司和其他自动显微镜仪器制造商提供商业服务。

DESCRIPTION (provided by applicant): This project will further develop and refine an innovative digital auto-focus technology for automated microscopy. Auto-focusing is essential to automated microscope imaging. Currently available techniques rely on various algorithms of focus computation at a single image resolution and suffer from inherent performance limitations, which affect their success and utilization in clinical and research applications. Auto-focusing for fluorescence microscopy, for example, represents a serious challenge to existing methods for desired accuracy, reliability and speed since in this case the images have very low signal-to-noise ratio and narrow depth-of-fields while specimen exposure to fluorescent excitation must be minimized to avoid photo-bleaching and formation of undesirable substances such as free radicals and singlet oxygen. We propose a novel multi-resolution image analysis approach to microscope auto-focusing, based on the recently developed mathematical theory of wavelet transform. The new approach overcomes a number of inherent limitations of currently available techniques, and holds the promise to make the measurement of the microscope focus function and the detection of best-focus imaging position considerably more accurate, reliable, and fast. This innovative technology will significantly increase the ability and efficacy of automated microscope instruments for a wide range of clinical and research applications where a large number of specimens need to be imaged and quantitatively analyzed on a routine basis. During the Phase 1 project we investigated the feasibility of the proposed technology for fluorescence microscopy. We developed software to implement the algorithms for multi-resolution focus function measurements and for in-focus imaging position search. We evaluated the new approach in software simulation on a variety of sample image stacks of cytogenetic FISH specimens, and compared it with all current best-performing methods for microscope auto-focusing using the criteria of (1) accuracy, (2) range, (3) robustness, and (4) speed. The Phase 1 results suggest that, by using a proper wavelet-based auto-focus function, the new multi-resolution method significantly outperforms all competing methods in each of the aforementioned performance categories, and clearly exceeds the Phase 1 feasibility criteria. In the Phase 2 project, we will further develop, refine, integrate, and validate the new technology in real-time operation environment. We plan to build a prototype system with multi-resolution auto-focusing capabilities for both fluorescence and bright-field microscope imaging. We will evaluate the system extensively for a variety of applications including genetics, pathology, and cytology. We will beta test the new system and technology in routine clinical laboratory environment and optimize the technology as end user input and feedbacks are gathered. Once fully developed and qualified, this new technology will be patented and incorporated into future IRIS automated imaging cytometry instruments. It will also be made commercially available to Applied Imaging Corporation and other manufacturers of automated microscope instruments through licensing agreements and partnerships.