Integrated wide-field imaging and confocal microscopy with agile 3D beam scanning

描述（由申请人提供）：反射共聚焦显微镜（一种扫描激光显微镜）已显示出在皮肤恶性肿瘤的非侵入性筛查和诊断方面的早期成功，同时减少了良性病变的不必要活检，通过检测癌缘而无需活检来指导手术，并通过监测患者治疗后而无需进一步活检。由于该技术是非侵入性的和即时的，临床医生可以查看尽可能多的特征来做出诊断。这与目前的做法相反，在目前的做法中，临床医生必须猜测最好的一个或两个位置进行痛苦的，可能毁容的活检，然后等待几天从病理学家的明确诊断。由于可以避免许多不必要的活检，护理得到改善并节省了大量成本（对于皮肤癌，多达80%的活检显示正常或良性组织，在美国每年花费约50亿美元）。皮肤以外的器官的癌症也可以类似地诊断。例如，使用该技术检测口腔癌已初见成效。然而，大的仪器尺寸仍然是该技术在身体上除最易接近的部位之外的任何部位广泛使用的障碍。为了将共焦反射显微镜的诊断潜力转化为体内其他部位，我们正在开发一种名为集成扫描激光显微镜（iLSM）的新仪器，该仪器将足够小，可用于“铅笔”探头或内窥镜。该仪器将实现显示细胞细节的高分辨率、高对比度图像，并且显著地，同时提供组织表面的宽视场彩色视频图像，允许临床医生看到在病变的表面特征的背景下在哪里拍摄显微图像。这种宽视场视图对于可疑病变的准确采样至关重要，并且在以前所有尝试使用扫描激光显微镜的尝试中都缺失了。最近在两个领域的技术进步，MEMS“有源光学”和微型CMOS相机，使iLSM成为可能。该项目采用一种新颖的方法将定制的MEMS 3D激光扫描仪集成到厘米级高NA物镜透镜中。显微镜切片可以是正面的、倾斜的、垂直的或全3D的，视野为300 μm。该显微镜使用环形光瞳，将外部75%的孔径区域用于高NA成像，确保高分辨率和有效收集散射光。内部25%的光圈区域保留给一个单独的低NA、宽视场相机，也集成到透镜中，将显示几毫米的组织。该项目将开发定制的激光扫描仪，并通过对30名志愿者的皮肤进行体内成像来验证该方法。

DESCRIPTION (provided by applicant): Reflectance confocal microscopy, a type of scanning laser microscopy, has shown early success for non- invasive screening and diagnosis of skin malignancies while reducing unnecessary biopsy of benign lesions, guiding surgery by detection of cancer margins without the need for biopsies, and by monitoring patients post-treatment without requiring further biopsies. Because the technique is non-invasive and immediate, the clinician can look at as many features as necessary to arrive at a diagnosis. This is contrasted with current practice, in which the clinician must guess at the best one or two locations for a painful, potentially disfiguring biopsy, and then wait several days for a definitive diagnosis froma pathologist. Care is improved and substantial cost savings accrue, because many unnecessary biopsies can be avoided (for skin cancer, as many as 80% of all biopsies show normal or benign tissue, at a cost of some $5 billion annually in the US). Cancer of organs other than skin can be similarly diagnosed. For example, initial success is being seen using this technology for the detection of oral cancer. However, large instrument size remains a barrier toward widespread use of this technique in any but the most accessible sites on the body. To translate the diagnostic potential of confocal reflectance microscopy to other locations in the body, we are developing a new instrument called the integrated scanning laser microscope, or iLSM, that will be small enough for use in a "pencil" probe or endoscope. This instrument will achieve high resolution, high contrast images showing cellular detail, and, significantly, simultaneously provide a wide-field color video image of the tissue surface, allowing the clinician see where the microscopic images are being taken in the context of the surface features of a lesion. This wide-field view is essential for accurate sampling of a suspicious lesion, and has been missing from all previous attempts to miniaturize scanning laser microscopes. Recent technological advances in two areas, MEMS "active optics" and miniaturized CMOS cameras, have made the iLSM possible. This project takes a novel approach to integrate a custom MEMS 3D laser scanner into a centimeter-scale, high NA objective lens. Microscopy sections can be en face, oblique, vertical, or fully 3D, with a field of view of 300 μm. The microscope uses an annular pupil that devotes the outer 75% of the aperture area to high NA imaging, ensuring high resolution and efficient collection of scattered light. The inner 25% of the aperture area is reserved for a separate low NA, wide-field camera, also integrated into the lens, that will show several millimeters of tissue. This project will develop the custom laser scanner and validate the approach with in vivo imaging of the skin of 30 volunteers.