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High-throughput cell monitoring technique using lensfree CMOS image sensor

  • Jin, Geonsoo (진건수, 대학원 전자·정보공학과 전자·정보공학전공)

초록/요약

The cell viability test plays an important role in cell culture systems. The viability test results are directly related to the number of viable cells, which is an important part of the experiments, like toxicity assay, anabolic –activity etc. There are several cell viability methods, ranges from common trypan blue dye assay to MTT assay. All these methods are determining the viability using the stained cell samples. However, the stained cell samples cannot be reused. To address this issue, we have developed a novel imaging modality, i.e. shadow imaging technique, which is based on the cell shadow or diffraction patterns without using any reagent. For demonstrate the shadow imaging technique, we build the system that is composed of a low cost light emitting diode (LED) as a light source and a lens less complementary metal oxide semiconductor (CMOS) image sensor as an imaging system. The lights from the LED are diffracted by the cells in the sample plane to produce the diffraction patterns. These cell shadow images were captured by the CMOS sensor. The diffraction patterns convey the viability characteristics of the cells and this can be obtain by analyzing the different elements like, intensity and connectivity of the pixels in a diffraction pattern. For that we developed an image-processing algorithm, which quantitatively reports the viability by analyzing the different elements in the diffraction pattern. A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6,000 human alveolar epithelial A549 cells are monitored for up to 72 hours simultaneously and continuously with a single digital image within a cost and space effective lensfree shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lensfree shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lensfree shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lensfree shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing. We introduce a shadow-imaging platform that can determine cell viability of more than 3,000 human cancer cells immediately within a single digital image. Three types of human cancer cells (Caco2, HepG2, MCF7) are incubated in 24 well plates, and H2O2 is added to track and compare the cell viability at each concentration. According to the experimental results, we obtain high correlation indices of minimum of 0.94 between the MTT assay and the shadow-imaging platform. A custom developed image-processing algorithm quantitatively analyzes shadow patterns of the cells. This algorithm analyzes the various elements of the shadow pattern such as pixel intensity, connected pixel numbers and count the viable cells automatically such that we can easily and immediately determine the cell viability in reagent free.

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목차

ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v
CHAPTER 1 INTRODUCTION 1
1.1 Research Background 1
1.2 Research Objective 4
CHAPTER 2 Lensfree Shadow Imaging Technique 5
2.1 Shadow Imaging System 5
2.1.1 Micro Fluidic Channel Type Shadow Imaging System 5
2.1.2 Well-plate Type Shadow Imaging System 6
2.2 Materials and Methods 6
2.2.1 Quantification of Cell Shadow Images 6
2.2.2 Live Cell Count 7
2.2.3 Automated Cell Count Algorithm 8
CHAPTER 3 Continuous Cell Monitoring System Using Micro Fluidic Channel 10
3.1 Introduction 10
3.2 Materials and Methods 10
3.2.1 Cell Preparation 10
3.2.2 Cell Culture 11
3.3 Results and Discussion 13
3.3.1 Observation of Cell Division 13
3.3.2 Single Cell Viability Investigation 19
3.3.3 Live Cell Count 22
CHAPTER 4 Reagent-free Cell Viability Measurement Using Well-plate 26
4.1 Introduction 26
4.2 Materials and methods 26
4.2.1 Well-plate based Shadow Imaging Platform 26
4.2.2 Cell Preparation 29
4.2.3 MTT assay 30
4.2.4 Normalization and correlation 31
4.3 Results and discussion 31
4.3.1 Comparison between MTT assay and Shadow Imaging System 31
4.3.2 Cell Viability testing in Concentration changing 32
4.3.3 Biological Reaction between H2O2 and Human Cancer Cell 34
CHAPTER 5 Conclusion and Future Works 36
REFERENCES 37

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