Related Experiment Video
Updated: Feb 28, 2026
14:09
High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
Published on: November 16, 2019
7.5K
Optical microscope illumination analysis using through-focus scanning optical microscopy.
Optics Letters
|June 15, 2017
Summary
Optical microscope aperture misalignment causes illumination asymmetry (ANILAS), leading to focus-dependent image shifts. This slant in through-focus scanning optical microscopy (TSOM) images can detect and correct ANILAS errors.
Area of Science:
- Optical microscopy
- Image analysis
- Metrology
Background:
- Microscope aperture diaphragm misalignment introduces angular illumination asymmetry (ANILAS).
- ANILAS propagates through focus, causing lateral image shifts dependent on focal position.
- Such shifts introduce significant errors in quantitative optical measurements.
Purpose of the Study:
- To investigate the impact of ANILAS on through-focus imaging.
- To establish a method for detecting and correcting ANILAS using image analysis.
- To improve the accuracy of quantitative optical microscopy techniques.
Main Methods:
- Analysis of through-focus image propagation of ANILAS.
- Correlation study between ANILAS and image slant in TSOM.
- Development of ANILAS detection and rectification methods based on TSOM image slant.
Main Results:
- ANILAS causes focus-dependent lateral image shifts.
- A direct correlation between ANILAS and TSOM image slant was identified.
- TSOM image slant serves as a reliable indicator for ANILAS.
Conclusions:
- ANILAS is a critical factor affecting quantitative accuracy in through-focus microscopy.
- TSOM image slant provides a practical means to detect, analyze, and correct ANILAS.
- This work enhances the reliability of 3D nanoparticle tracking, confocal microscopy, and TSOM.
Related Concept Videos
01:18Imaging Biological Samples with Optical Microscopy
11.9K
Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...
11.9K
01:16Confocal Fluorescence Microscopy
21.5K
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
21.5K
01:22Overview of Microscopy Techniques
17.3K
The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
17.3K