Synopsis
Adjusting numerical aperture (NA) and resolution is crucial for obtaining high-quality detailed images. When working with a microscope, you need to first determine, depending on the sample type and work, the value of the numerical aperture and then adjust your device. Sometimes, without consulting professional sources, adjusting the NA and the resolution could be a daunting task and present a significant challenge. It could potentially impact your concentration and result in low-quality output caused by superficial preparation. Today, we will discuss more on numerical aperture and resolution, and their importance. We would also try to find the optimal balance between the two for the best results.
Numerical Aperture in Digital Microscopy
When designing a device for digital microscopy, biomedical and diagnostic applications, and life science, the goal is to optimize the device for the highest resolution and the highest throughput in images per second. Microscope calculations like magnification, resolution, and numerical aperture help to determine various aspects of a microscope’s capabilities for simplification and prototyping.
What is Numerical Aperture?
Numerical aperture (NA) is the measure of the microscope’s objective to gather light and resolve fine specimen details at a fixed object distance. Image-forming light passes the specimen and enters the objective in an inverted cone. The focal length of the objective determines the angular aperture. The angle μ represents one-half of the angular aperture (A) and is related to the numerical aperture through the following equation:
where n is the refractive index of the imaging medium between the front lens of the objective and specimen cover glass. The value of n can range from 1.00 to 1.51. According to the equation, when the imaging medium has a refractive index of 1.00, then the NA is dependent only on the angle μ whose maximum value is 90°. The sin of this angle has a maximum value of 1.00. That is the theoretical maximum numerical aperture of a lens that operates with air as the imaging medium.
Numerical Aperture in Practice
In real-world practice, it is difficult to achieve an NA above 0.95 with dry objectives. The refractive index is the limiting factor. It also limits the achievement of the numerical aperture greater than 1.0. Therefore, to obtain higher numerical apertures, the refractive index between the specimen and the objective needs to be increased. There are diverse objectives available that allow working in diverse mediums such as water (refractive index= 1.33), glycerin (refractive index= 1.47), and immersion oil (refractive index= 1.51).
Numerical Aperture and Objectives
Objectives need to be handled with care as unwanted artifacts can arise when the objectives are used in different immersion mediums. It is recommended not to use microscopes designed for oil immersion with water or glycerin. Various companies are designing microscopes that can be used with multiple mediums. However, it is best to consult the respective manufacturer to determine the most suitable medium to work with.
Balancing Numerical Aperture and Magnification
Most objectives that have a magnification range between 60x and 100x are designed for work with immersion oil. Theoretically, the highest NA obtainable with immersion oil is 1.51. However, in practice, the highest numerical aperture obtainable with immersion oil is 1.4, and the most common range is from 1.0 to 1.35. The numerical aperture of the objective also depends on the correction of optical aberration. Highly corrected objectives tend to have larger numerical apertures. Most manufacturers try to have their objectives corrected as much as possible to obtain the highest NA.
Performance
Images are projected onto a detector, such as the retina, an electronic sensor, or traditional film. To optimize image quality, the detector’s resolution should closely match the microscope’s resolution. The microscope’s performance depends on the wavelength spectrum of visible light used. When the microscope is well-aligned and the objectives match the substage condenser, the NA plays a key role in determining resolution. Magnification does not directly impact resolution; only NA and wavelength are relevant factors.
The Importance of Resolution
The resolution of the objective is the smallest distance between two points of the specimen that can be observed as two separate entities. Resolution is somewhat subjective; at high magnification, an image may seem blurry even if it has reached the maximum resolution capability of the objective lens.
Resolution and Numerical Aperture
The numerical aperture determines the resolving power of the objective. The total resolution of the microscope system also depends on the NA of the substage condenser. The higher the NA of the total system, the better the resolution. To ensure maximum resolution, you need the correct alignment of the microscope optical system.Â
Resolution Capability
To achieve precise light cone formation and optimal specimen illumination, it is essential to match the substage condenser with the objective lens. The resolution capability of the microscope is influenced by the wavelength spectrum of light used. Shorter wavelengths allow for resolving finer details compared to longer wavelengths, enhancing the clarity and accuracy of the observed specimen.
Microscope’s Resolving Power
The resolving power of a microscope is crucial. It affects the pathologist’s ability to distinguish between fine details of the specimen. The primary factor in determining the resolution is the objective numerical aperture. Resolution also depends on the type of specimen, coherence of illumination, degree of aberration correction, and other factors like contrast enhancing methodology in the optical system of the microscope or in the specimen itself. Resolution is directly related to the magnification of the microscope and the perception limit of specimen detail.
Conclusion
Numerical aperture (NA) and resolution are key for successful adjustment of microscopes. The NA, described as the measure of the microscope’s objective to gather light and resolve fine specimen details, determines the resolution of the specimen. The higher the NA of the microscope, the more precise the output. The resolution, determined as the smallest distance between two points of the specimen, depends on the numerical aperture. The higher the numerical aperture, the better the resolution. To ensure high-quality and accurate results, it is crucial to optimize your device for the highest resolution. This optimization is essential for achieving the best outcomes in all your projects.
