Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Optical two-dimensional SW-banyan network: Optical implementation, routing control, and determination of permissible permutations

A 2-D SW-banyan network is introduced by properly folding the 1-D SW-banyan network, and its corresponding optical setup is proposed by means of polarizing beamsplitters and 2-D phase spatial light modulators. Then, based on the characteristics and the proposed optical setup, the control for the routing path between any source-destination pair is given, and the method to determine whether a given permutation is permissible or not is discussed. Because the proposed optical setup consists of only optical polarization elements, it is compact in structure, its corresponding energy loss and crosstalk are low, and its corresponding available number of channels is high. (C) 1996 Society of Photo-Optical Instrumentation Engineers.

Computational microscopy: turning megapixels into gigapixels

The layout of a typical optical microscope has remained effectively unchanged over the past century. Besides the widespread adoption of digital focal plane arrays, relatively few innovations have helped improve standard imaging with bright-field microscopes. This thesis presents a new microscope imaging method, termed Fourier ptychography, which uses an LED to provide variable sample illumination and post-processing algorithms to recover useful sample information. Examples include increasing the resolution of megapixel-scale images to one gigapixel, measuring quantitative phase, achieving oil-immersion quality resolution without an immersion medium, and recovering complex three dimensional sample structure.

Discussions about FFT-based two-step phase-shifting algorithm

An FFT-based two-step phase-shifting (TPS) algorithm is described in detail and implemented by use of experimental interferograms. This algorithm has been proposed to solve the TPS problem with random phase shift except pi. By comparison with the visibility-function-based TPS algorithm, it proves that the FFT-based algorithm has obvious advantages in phase extracting. Meanwhile, we present a pi-phase-shift supplement to the TPS algorithm, which combines the two interferograms and demodulates the phase map by locating the extrema of the combined fringes after removing the respective backgrounds. So combining this method and FFT-based one, one could really implement the TPS with random phase shift. Whereafter, we systematically compare the TPS with single-interferogram analysis algorithm and conventional three-step phase-shifting one. The results demonstrate that the FFT-based TPS algorithm has a satisfactory accuracy. At last, based on the polarizing interferometry, a schematic setup of two-channel TPS interferometer with random phase shift is suggested to implement the simultaneous collection of interferograms. (c) 2007 Elsevier GrnbH. All rights reserved.

Modal analysis of deep-etched low-contrast two-port beam splitter grating

Modal analysis of a deep-etched low-contrast two-port beam splitter grating under Littrow Mounting is presented. The guideline for the design of a subwavelength transmission fused-silica phase grating as high-efficiency grating, polarizing beam splitter (PBS), and two-port beam splitter, is summarized. As an example, a polarization-independent two-port beam splitter grating is designed at wavelength of 1064 nm. We firstly analyzed the physical essence of the grating by the simplified modal method. The guideline for the grating design and the approximate grating parameters are obtained. Then using the rigorous coupled-wave analysis (RCWA) with parameters varying around the approximate ones, Optimum grating parameters can be determined. With the design guideline, the time for the rigorous calculation of the grating profile parameters can be reduced significantly. (C) 2008 Elsevier B.V. All rights reserved

四区域法消除偏振棱镜缺陷对波片相位延迟测量的影响

在起偏器待测波片检偏器系统基础上提出一种四区域测量波片相位延迟量的方法。调整待测波片和检偏器的方位角,获得相应的四组光强值,通过线性运算得到待测波片的相位延迟量,完全消除了起偏器和检偏器不完全消光带来的误差。由于测量系统中不存在标准波片或其他相位调制元件,允许测量波长仅受偏振棱镜和探测器的限制,因此四区域法可适用于很大波长范围内的波片测量。以λ/4波片为例,理论分析了测量系统利用四区域测量法后的仪器误差为σ≤±3.49065×10-3rad(约0.2°),精度比原算法提高约1个数量级。实验验证了四区域法能有效提高系统精度。

聚焦光斑磁光极克尔磁滞回线的热效应分析

实验设计并组建了一套磁光极克尔磁滞回线测量装置，该装置可以通过改变照射到样品上的激光功率来改变薄膜样品上被聚焦光斑照射的测试点的温度。同时通过计算模拟了激光照射在TbFeCo磁光薄膜上的温度分布情况，得到了薄膜的矫顽力随照射激光功率的变化关系，由此可以确定薄膜的居里温度和补偿温度。为研究磁光薄膜样品在各种温度条件下，磁光性能的变化以及多层磁光薄膜的磁耦合效应提供了有效的手段。

扫描探针显微镜系统的各种组合方式

综述了扫描探针显微镜(SPM)系统中显微镜主体部分的各种结构方式，分析其优缺点，对新型SPM的设计提供了参考。

Effects of surface treatment on sapphire substrates

The influence of mechanical polishing, chemo-mechanical polishing (CMP), as well as CMP and subsequent chemical etching on the properties of sapphire substrate surfaces has been studied. The sapphire substrates have been investigated by means of polarizing microscopy, atomic force microscopy (AFM). X-ray diffraction rocking curves (XRCs) and micro-Raman spectroscopy. The results show that CMP with subsequent chemically etching yields the best-quality sapphire substrate surfaces. (C) 2004 Elsevier B.V. All rights reserved.

晶格匹配氮化镓和氧化锌基外延薄膜衬底材料ScAlMgO4晶体生长研究

采用提拉法成功生长了氮化镓和氧化锌基外延薄膜晶格匹配的ScAlMgO4单晶衬底材料,晶体呈透明白色,尺寸为Ф30mm×59mm,表面部分沿解理面有裂纹.粉末X射线衍射（XRD）分析表明经1400℃固相反应烧结的原料基本合成了ScAlMgO4多晶相.初步的偏光显微镜观察、晶体的粉末XRD表征、透过光谱和双晶摇摆测试表明晶体具有较好的光学性质和结晶质量.研究表明晶体本身的层状结构、较大的温度梯度和热应力的不均匀性是生长过程中引起晶体开裂的几个主要原因.

ZrO2／SiO2棱镜偏振膜的膜系优化设计与制备

对以K9玻璃为基底，采用ZrO2和SiO2为高低膜料来制备棱镜偏振膜，并进行膜系优化设计。设计指标为：波长540nm处满足Tp>99％，Ts<1％。优化结果表明，膜系以H3L(HL)^13 H3LH为最佳膜系。测试结果表明，此膜系完全满足设计指标，偏振性能优良。探讨了参考波长对偏振膜工作带宽的影响。

利用等效层的消偏振宽带减反膜设计

分析了倾斜入射条件下导致光学薄膜产生偏振的原因, 针对不同偏振态的等效导纳与等效相位进行了分析, 并计算了对称膜层在45°入射条件下不同偏振态的等效折射率与等效相位厚度, 采用等效层方法设计了光学性能良好的600～900 nm波段消偏振宽带减反膜。最后利用电子束蒸发技术制备了薄膜样品, 样品的光谱性能完全能够满足使用要求。其中在600～900 nm波段范围内, 平均反射率均小于1.38%, 反射率的偏振度均低于0.89％。另外, 通过对其理论及实验光学性能、角度敏感性、膜层厚度误差敏感性等方面的分析结果可

8.0nm反射式偏振膜的设计和制备

讨论了软X射线反射式偏振膜的设计原理和方法,利用设计软件模拟设计了8.0nm处的Mo/B4C偏振膜.对影响多层膜性能的参量进行了详细的误差分析.利用磁控溅射镀膜机进行了偏振膜的制备研究,X射线小角衍射测量了多层膜的周期厚度,测量数据的拟合结果与设计值吻合很好.

4H-SiC基底Al_2O_3/SiO_2双层减反射膜的设计和制备

在4H-SiC基底上设计并制备了Al2O3/SiO2紫外双层减反射膜,通过扫描电镜(SEM)和实测反射率谱来验证理论设计的正确性。利用编程计算得到Al2O3和SiO2的最优物理膜厚分别为42.0nm和96.1nm以及参考波长λ=280nm处最小反射率为0.09%。由误差分析可知,实际镀膜时保持双层膜厚度之和与理论值一致有利于降低膜系反射率。实验中应当准确控制SiO2折射率并使Al2O3折射率接近1.715。用电子束蒸发法在4H-SiC基底上淀积Al2O3/SiO2双层膜,厚度分别为42nm和96nm。SEM截面图表明淀积的薄膜和基底间具有较强的附着力。实测反射率极小值为0.33%,对应λ=276nm,与理论结果吻合较好。与传统SiO2单层膜相比,Al2O3/SiO2双层膜具有反射率小,波长选择性好等优点,从而论证了其在4H-SiC基紫外光电器件减反射膜上具有较好的应用前景。