Selective Plane Illumination Microscope Development and Characterization
Hämäläinen, Meri (2023)
2023
Master's Programme in Science and Engineering
Tekniikan ja luonnontieteiden tiedekunta - Faculty of Engineering and Natural Sciences
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Hyväksymispäivämäärä
2023-11-29
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202311139602
https://urn.fi/URN:NBN:fi:tuni-202311139602
Tiivistelmä
The field of biomedical research includes advanced imaging modalities that enable three-dimensional (3D) imaging of cellular specimens. Potential for enhancement persists within the existing modalities in entities of high resolution and reduced photo-damage with decreased imaging duration. Optical imaging methods provide a solution and aim to achieve these benefits. Light-based imaging methods benefit from extreme accuracy and allow lower radiation exposure. Specifically optical sectioning-based imaging modalities emerge, while creating new innovative ways to produce these microscopic images.
The main topics of the thesis cover the optical fluorescence microscopy technique called selective plane illumination microscopy (SPIM). This imaging modality uses an excitation light sheet to optically section the imaged sample. The field of diverse applications of SPIM imaging technologies emerges rapidly by providing new techniques and solutions. This application field enables the 3D imaging of dynamic cellular environments, such as flow cytometry and contraction-inducing processes. Body-on-chip (BOC) and organ-on-chip (OOC) cellular environments consist of these dynamic environments for which SPIM can offer imaging solutions. When dealing with optical imaging methods, image formation and quality monitoring become important. This thesis covers these image formation processes and image reconstruction principles. Also, the principles of resolution determination criteria allow image quality assessment.
The primary objective of this thesis encompassed the development and finalization of an optical light-sheet-based microscope, an inverted selective plane illumination microscope (iSPIM). The system characterization with experimental testing allowed the determination of light sheet properties, with the resolution determination of the system. The measurements determined the lateral resolution to 1.07 µm and axial resolution to 7.31 µm. The thesis contains the acquisition of zebrafish embryo images captured using the developed equipment.
The development and characterization of the iSPIM microscope represent considerable advancement in the field of optical fluorescence microscopy. Limited documentation exists regarding the iSPIM equipment in previous scientific reports. The project led the equipment to a stage where fluorescent images can be acquired. The project will be continued in the future to implement further testing and achieve better stabilization assembly. The achievable future aim is to detect dynamic changes in live-cell culture and chip-based environments.
In the emerging field of SPIM technologies, exploration and research continues. The current state of knowledge on various SPIM technologies is partially incomplete, which creates a need for more comprehensive reporting and research in this area. This project not only enriches comprehension of SPIM imaging modalities but also underscores their role within the field of optical microscopy. Research supports future advancements and discoveries.
The main topics of the thesis cover the optical fluorescence microscopy technique called selective plane illumination microscopy (SPIM). This imaging modality uses an excitation light sheet to optically section the imaged sample. The field of diverse applications of SPIM imaging technologies emerges rapidly by providing new techniques and solutions. This application field enables the 3D imaging of dynamic cellular environments, such as flow cytometry and contraction-inducing processes. Body-on-chip (BOC) and organ-on-chip (OOC) cellular environments consist of these dynamic environments for which SPIM can offer imaging solutions. When dealing with optical imaging methods, image formation and quality monitoring become important. This thesis covers these image formation processes and image reconstruction principles. Also, the principles of resolution determination criteria allow image quality assessment.
The primary objective of this thesis encompassed the development and finalization of an optical light-sheet-based microscope, an inverted selective plane illumination microscope (iSPIM). The system characterization with experimental testing allowed the determination of light sheet properties, with the resolution determination of the system. The measurements determined the lateral resolution to 1.07 µm and axial resolution to 7.31 µm. The thesis contains the acquisition of zebrafish embryo images captured using the developed equipment.
The development and characterization of the iSPIM microscope represent considerable advancement in the field of optical fluorescence microscopy. Limited documentation exists regarding the iSPIM equipment in previous scientific reports. The project led the equipment to a stage where fluorescent images can be acquired. The project will be continued in the future to implement further testing and achieve better stabilization assembly. The achievable future aim is to detect dynamic changes in live-cell culture and chip-based environments.
In the emerging field of SPIM technologies, exploration and research continues. The current state of knowledge on various SPIM technologies is partially incomplete, which creates a need for more comprehensive reporting and research in this area. This project not only enriches comprehension of SPIM imaging modalities but also underscores their role within the field of optical microscopy. Research supports future advancements and discoveries.