Microscope objective lenses determine image resolution, magnification, and numerical aperture performance in microscopy systems. This video introduces the fundamentals and applications of microscope objective lenses.
Selecting the correct microscope objective lens is essential for achieving optimal imaging performance. This video explains how magnification, numerical aperture, working distance, and application requirements influence objective lens selection.
How to Choose the Right Microscope Objective
What is a Microscope Objective Lens?
A microscope objective lens is the primary lens used to magnify and resolve objects in a microscopy system. Objective lenses determine image magnification, resolution, numerical aperture (NA), and overall optical performance.
Microscope objective lenses are widely used in biological research, semiconductor inspection, industrial imaging, and scientific analysis.
Choosing the Right Conjugate Distance
Many objective lenses are corrected for infinite conjugate distance, while others are designed for finite conjugate distance applications. Compared to infinite conjugate objectives which need a secondary lens (also called tube lens), a finite conjugate objective can generate an image of a specimen by itself.
A finite conjugate objective, as shown in Figure 1, is a good, economical choice for a simple microscopy system.
Infinity-corrected objectives are ideal for research-grade biomedical industrial applications especially when additional components (such as filters, dichroic mirrors, polarizers) are needed in the microscopy system. Adding optical plate components in the infinity space (shown in the Fig.2 labelled as “Parallel Optical Path) between the infinity-corrected objective and tube lens will not introduce spherical aberration, or change the objective’s working distance.
Choosing the Right Numerical Aperture (NA)
The most important parameter of a microscope objective is the numerical aperture (NA). NA measures the microscope objective’s ability to gather light and determines the resolution of a microscopy system.
NA is commonly expressed as
NA = n × sinθa
where θa is the maximum 1/2 acceptance angle of the objective, and n is the index of refraction of the immersion medium.
The limit of resolution of a microscope objective refers to its ability to distinguish two closely spaced Airy disks.
Resolution (r) = λ/(2NA)
Where r is resolution (the smallest resolvable distance between two objects), and λ is the imaging wavelength. The higher the NA, the better the objective resolution.
Choosing the Right Magnification
Objective lenses are used to magnify an image. In addition to numerical aperture, magnification is also an important parameter. The objective magnification typically ranges from 4X to 100X. As the image sensor size or eye observed area is fixed, the field of view of a microscopy system changes with the magnification of the objective lens. Typically a lower magnification objective lens will have a larger field of view and lower resolution, and a higher magnification objective lens will have a smaller field of view and higher resolution.
The diameter of the FOV can be calculated by using the following formula:
FOV= FN/Mag
The field number (FN) in microscopy is defined as the diameter of the area in the image plane that can be observed through the eyepiece or image sensor.
Choosing the Right Working Distance
Usually the working distance (WD) refers the distance from the front lens element of the objective to the observed object when the object is in sharp focus. Objective lenses with long working distance are needed for many scientific research applications such as atom trapping and analyzing fluid samples that require putting an object in a chamber.
The resolution of a microscopy system can be significantly affected if the observed object is not placed on the designed object plane, especially for an objective with high NA.
Choosing the Right Immersion Medium
A dry objective is designed to work with the air medium between the specimen and the objective lens, while an immersion objective requires a liquid medium to occupy the space between the object and the front element of the objective for enabling a high NA and high resolution. Figure 4 shows the oil immersion objective, which can collect more light (i.e., have a higher NA) compared to a dry objective.
The most common immersion media are air, water, oil, and silicone. Choosing the appropriate objective designed for your immersion medium will result in higher resolution images.
Frequently Asked Questions
What does a microscope objective lens do?
A microscope objective lens magnifies the specimen and determines image resolution and optical performance.
What is numerical aperture (NA)?
Numerical aperture measures the light-gathering ability and resolving power of an objective lens.
What is the difference between finite and infinity-corrected objectives?
Finite objectives form images directly, while infinity-corrected objectives require a tube lens and allow additional optical components in the optical path.
What is working distance in a microscope objective?
Working distance is the distance between the front lens element and the specimen when the image is in focus.
What immersion media are used in microscope objectives?
Common immersion media include air, water, oil, and silicone.
