In microscopy, objectives are the components responsible for collecting light from a specimen and focusing the light rays to generate a real image. Objectives derive their name from the fact that they are the closest component to the observed object. A microscope’s revolving nosepiece, or objective turret, usually contains three to five objectives that allow visualization of a specimen with different magnification levels and aperture sizes.
With their multi-element design, microscope objectives are responsible for many aspects of imaging, including:
- Image formation/quality: Objectives enable primary image formation and also significantly influence the quality of image that can be achieved by a microscope.
- Magnification: A microscope’s total magnification is provided by both the eyepiece and the objective used. Most microscopes contain multiple interchangeable objectives with magnification powers ranging from 2x to 200x.
- Resolution/detail: Resolution, which is the ability of a microscope to distinguish a specimen’s detail, is affected by the numerical aperture of the objective lens.
- Correcting for chromatic and spherical aberration: Objective lenses come in specialized designs that are corrected for common visual distortions such as chromatic and spherical aberrations.
The following concepts related to microscope objectives and their imaging capabilities will be discussed in this post:
- Types of microscope objectives
- Numerical aperture
- Focal length
- Quality/image correction
- Cover slip thickness
What are the Types of Microscope Objectives?
Objectives can essentially be classified into two categories: refractive and reflective.
Refractive Microscope Objectives
In a refractive design, multiple glass elements refract the light as it passes through the system. The glass surfaces used in refractive objectives make them prone to chromatic aberrations, and their designs are often complex in an effort to counteract these optical artifacts.
Reflective Microscope Objectives
In contrast, reflective objectives use a two-mirror system to relay the image of a specimen to the eyepiece for visualization. Since the light is reflected by a metallic surface rather than refracted by a glass surface, reflective objectives experience much lower aberrations relative to refractive objectives. Furthermore, aspherical mirror surfaces enable reflective objectives to achieve substantially higher numerical apertures. These features make reflective objectives better suited than their refractive counterparts for a range of sensitive analytical applications, including:
- Ultrafast laser machining
- Fourier transform infrared (FTIR) spectroscopy
- Raman spectroscopy
- Hyperspectral imaging
Magnification refers to the degree of visual enlargement of a specimen by an optical instrument. Typically, the microscope objectives work in tandem with the eyepiece to enable magnification of an object. The total magnification can be measured by multiplying the eyepiece magnification (typically 10x) by the objective lens magnification (typically 4x, 10x, 40x or 100x). The rotatable objectives with their varying magnification powers can be interchanged as needed to deliver the appropriate level of enlargement for an object.
The objective lenses, in conjunction with the eyepiece, are essential for enlarging microscopic phenomena to a size that can be visualized. However, it is important to note that simply magnifying an image without enhancing its details is insufficient for providing a clear, accurate picture of the specimen. The resolving power of an objective lens is related to its numerical aperture.
Numerical aperture indicates the ability of a microscope objective to accept incoming light and resolve the fine structures of an object at a fixed distance. The larger the numerical aperture of a system, the narrower the focal spot and, hence, the better the resolution. The objective numerical aperture determines the brightness at which an image can be displayed, establishes a limit on spatial resolution, and directly impacts the depth of field.
The refractive index of the imaging medium, specifically dry versus immersion liquid, affects the numerical aperture of the objective.
- For dry objectives, the air between the object and the microscope objective lens acts as the imaging medium. The highest possible numerical aperture that can be achieved with a dry objective is 0.95.
- Immersion liquid.The addition of an immersion liquid with a high refractive index to the space between the specimen and the objective lens can substantially increase the numerical aperture to 1.5 or higher. Immersion objectives typically use a transparent oil with the same refractive index as glass to maximize the resolving power of the microscope.
The focal length is the required distance between the objective lens and the top of a specimen that enables in-focus image viewing. The focal length quantifies the ability of an objective lens to focus or defocus light. For a focusing objective lens that is dry (no immersion liquid), the focal length is a positive value that indicates the distance required to focus a beam of light to a single location. For a defocusing lens, the focal length value is negative and indicates the distance from the objective lens to the virtual focus.
Magnification is the ratio of the tube lens’s focal length to the objective’s focal length, so the objective magnification is changed by increasing or decreasing the focal length of the tube lens. In general, the shorter the focal length, the higher the objective magnification. Focal length also factors into numerical aperture since the numerical aperture is a function of the focal length and the diameter of the entrance pupil.
Microscope objectives come in specialized designs to counteract the occurrence of optical distortions known as aberrations. For example, certain objectives are corrected for chromatic aberrations, which are image distortions caused by the various wavelengths (colors) having different focal points. Objectives can also be corrected for spherical aberrations, which are focal discrepancies caused by the geometry of the lens. Some of the most common types of corrected objectives include:
- Achromatic objectives.These lenses are designed to bring red and blue light into focus on a common plane and are well-suited for black and white viewing.
- Fluorite or semi-apochromat objectives.These objectives are chromatically corrected for blue and red, and spherically corrected for blue and green, making them a better fit than achromatic lenses for color viewing or recording.
- Plan objectives.Unlike the curved image produced by achromatic or semi-apochromat objectives, plan objectives produce a flat image across the entire field of view.
- Infinity-corrected objectives.Whereas many microscopes are restricted to set distances, infinity-corrected lenses allow the image distance to be set to infinity.
Cover Slip Thickness
A cover slip is a thin square of glass used to cover the specimen on the glass microscope slide. Its main function is to flatten and hold the specimen in place to enable better viewing. They also decrease the specimen’s evaporation rate in both wet and dry mounted slides.
Cover slips affect the way light refracts from the specimen into the objective, so the objective must perform certain optical corrections to compensate. For this reason, most objectives indicate an optimal range of cover slide thicknesses that will allow the best image quality to be achieved. The optimal cover glass thickness for most objectives is 0.17 mm.
Microscope Objective Solutions From Optics Technology, Inc.
Microscope objectives are complex, multi-element components responsible for focusing incoming light rays to generate an image. Most optical systems feature multiple objective lenses with varying magnification levels, aperture sizes, and corrective capabilities to maximize the clarity and accuracy of an image. For any given application, careful consideration of the factors discussed here is necessary for optimizing imaging capabilities and ensuring dependable results for analytical and quantification purposes. In many cases, custom-designed objective assemblies provide the best-fit solution for meeting all of a project’s requirements.
At Optics Technology, Inc., we design and manufacture custom microscope objectives and imaging systems to support innovations in several industries, including medical, biomedical, metrology, and in vivo confocal microscopy. Taking the client’s budgetary and precision requirements into consideration, our experienced engineering team provides meticulously-crafted objective assemblies for a range of high-performance imaging and analytical applications.
Our expertise is in the engineering of limited diffraction, high numerical aperture, and miniature format optical systems. With our small-scale precision manufacturing capabilities, we are experienced in producing highly specialized and accurate lenses for in vivo imaging and research purposes. Using wavefront, resolution target, and MTF testing methods, we thoroughly inspect each of our optical devices to ensure the highest levels of quality and accuracy in everything we produce.