Stochastic Optical Reconstruction Microscopy (STORM) is proved to be one of greatest tools for medical and biological research which can provide images with resolution beyond diffraction limit of conventional microscope. In this thesis, we developed a three color STORM system, which allows images of three different fluorescent dyes to be taken individually and simultaneously with 20 nm lateral resolution and 40 nm axial resolution. Recipe of imaging buffer is provided for the labeling fluorescent dyes CF 568, Alexa 647 and Alexa 750 to achieve stable and long-lived photo blinking. In addition, algorithms for channel alignment and de-crosstalk is applied to improve the accuracy of the system.

Because the three color STORM system makes it possible to label three different targe...[ Read more ]

Stochastic Optical Reconstruction Microscopy (STORM) is proved to be one of greatest tools for medical and biological research which can provide images with resolution beyond diffraction limit of conventional microscope. In this thesis, we developed a three color STORM system, which allows images of three different fluorescent dyes to be taken individually and simultaneously with 20 nm lateral resolution and 40 nm axial resolution. Recipe of imaging buffer is provided for the labeling fluorescent dyes CF 568, Alexa 647 and Alexa 750 to achieve stable and long-lived photo blinking. In addition, algorithms for channel alignment and de-crosstalk is applied to improve the accuracy of the system.

Because the three color STORM system makes it possible to label three different targets of interest and to be observed and imaged simultaneously, the STORM system can provide precise results for colocalization calculation and DNA optical mapping in which multiple targets are needed to be studied at same time.

In this thesis, 3D colocalization between PIP2a, PHB3 and PHB6 in Arabidopsis thaliana is calculated based on their super-resolution images by different colocalization algorithms. Besides, we explored the methodology for DNA optical imaging by applying microfluidic technology with the super-resolution microscopy.

Lambda DNAs and HeLa cell DNAs are nick labeled with Alexa 546 and the DNA backbone is labeled with YOYO-1. Super-resolution images of the DNAs which are stretched in 20-200 nm width nanochannels are taken by the three color STORM system. Recipes of imaging buffer for different devices are provided as well.

This thesis provides a design of three color stochastic optical reconstruction microscope and reveals its unique applications in colocalization analysis and DNA optical mapping to study the location and interaction of multiple targets of interest.