Summary | Contributions | Package/folders explanations | Storage Model | Discussion and conclusions
Depth-Extended, High-Resolution Fluorescence Microscopy: Whole-Cell Imaging with Double-Ring Phase (DRiP) Modulation
This project started from my undergradate thesis project in early 2016, which was supervised by Dr. Junhua Yuan and Dr. Shu Jia. I learnt about the basic properties of Bessel beams both theoretically and numerically and discussed about the accessability of generating a double-ring modulated Bessel beam optically. It was continued after I was matriculated by the PhD program of Biomedial Engineering Dept. at Stony Brook University in fall, 2016. At that time I had a much better understanding of Fourier Optics, microscopy and PSF engineering, and developed a systemetic approach to demonstrating the optical theories both numerically and experimentally. The project was published on Biomedical Optics Express in Jan., 2019.
We report a depth-extended, high-resolution fluorescence microscopy system based on interfering Bessel beams generated with double-ring phase (DRiP) modulation. The DRiP method effectively suppresses the Bessel side lobes, exhibiting a high resolution of the main lobe throughout a four- to five-fold improved depth of focus (DOF), compared to conventional wide-field microscopy. We showed both theoretically and experimentally the generation and propagation of a DRiP point-spread function (DRiP-PSF) of the imaging system. We further developed an approach for creating an axially-uniform DRiP-PSF and successfully demonstrated diffraction-limited, depth-extended imaging of cellular structures. We expect the DRiP method to contribute to the fast-developing field of non-diffracting-beam-enabled optical microscopy and be useful for various types of imaging modalities.
This project is mainly leaded by Xuanwen Hua with the full supervision and support from Dr. Shu Jia and Dr. Shu Jia's lab, including the contributions made by all co-authors (Changliang Guo, Jian Wang, Deborah Kim-Holzapfel, Bryce Schroeder, Wenhao Liu, Junhua Yuan, Jarrod French, and Shu Jia). We also want to thank Dr. French's lab and Deb for the collaboration and bio-sample preparation. Finally, it has to be mentioned that this project is also sponsored by the NSF-CBET Biophotonics program, the NSF-EFMA program, and the NIH-NIGMS MIRA program.
Please feel free to check here for more funding, acknowledgement and disclosure information.
- DRIM: This is a folder containing the codes for generating single-ring and double-ring masks with a blazed grating and other customized parameters. Note that the programs were written with an old-version Matlab and the deprecated GUIDE. So it is not recommended that users directly download the codes and run it before any modification. Any questions, write an email to me ([email protected])
- ShutterCAD: This folder contains the files generated by Autodesk® AutoCAD. The DWG files include all the parts of a rectangular-shaped optical shutter. The STL files are ready for 3D preview and 3D printing tasks.
- SetupDWG This folder mainly contains the 3D illustration of the PSF engineering setup drawn with Autodesk® AutoCAD.
- Programs This folder contains the Python-version, Matlab-version and Julia-version of PSF simulation, the genetic algorithm, the wide field microscopy simulation code provided by Hongting and the numerical simulation codes (PostOBJ) for the entire process of PSF engineering. For your convenience, please simply use the codes in PostOBJ folder. A future version of codes for PSF engineering Kyma is provided afterwards.
- ExpLog This folder contains some of the experiments records and progress docs.
- RepoScripts This folder contains some of the experiments reports (data analysis, usually for discussion use) and the manuscripts for the thesis project midterm report, undergraduate thesis and the final publication.
- Presentations This folder contains the ppt slides for discussion and lab meetings, and a poster for the BME Research Dat at Stony Brook.
- Experiments This folder contains the data and analysing codes used in the publication.
- PaperILL This folder contains all the figures for publication.
- Reference The folder contains part of the referred articles in my paper.
- CollegeDocs This folder contains the official notice and proposal application of my thesis project.
- Miscellaneous This folder contains any other types of files that were used at an early stage but could be useful or useless in the future. FYI.
The large files for data and results are stored on Github using Git Large File Storage(Git-LFS). Learn more about Git-LFS
In this work, we have developed a DRiP method for depth-extended, high-resolution fluorescence microscopy. Using two interfering Bessel beams, the method not only maintains the non-diffracting property of Bessel beams, but also effectively suppresses the Bessel side lobes, substantially improving image contrast and resolution. Compared to conventional wide-field microscopy, the DRiP-PSF exhibits a diffraction-limited FWHM value of ~300 nm with four- to five-fold improvement of the DOF, allowing for imaging across a significant volume of whole cells without the need for scanning. Beyond wide-field microscopy, the method can be extended to various types of imaging modalities, such as particle tracking, light-sheet microscopy and one or two-photon microscopy. Furthermore, the DRiP method can be realized by implementing a fabricated double-annulus mask to the back pupil of the objective (i.e. the Fourier plane), advancing the method as a plug-in device compatible with most commercial optical microscopes.