Courtesy of Avantier Inc
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The Complete Guide to Microscope Objective Lens

May 2, 2024
Courtesy of Avantier Inc
663385418289ec000995253f Screenshot 20240502 At 7

What is a Microscope Objective Lens?

Microscope objective lenses, vital optical elements in microscopy, enable precise observation of specimens. Objective lens manufacturers offer a wide range of objective designs for specific needs: high power for detailed observation, scanning for broader views, oil immersion for high-resolution imaging, and long working distance for manipulation without compromising quality. Those objectives are designed with advanced construction techniques for high performance objectives with a spring loaded retractable nose cone assembly that protects the front lens elements and the specimen from collision damage.

Adding to these features, long working distance objectives allow ample space between the lens and the specimen, facilitating the manipulation of samples without compromising image quality. Infinity correction objectives utilize infinity-corrected optical systems, providing flexibility and compatibility with various microscopy accessories.

Numerical aperture, magnification, optical tube length, degree of aberration correction, and other important characteristics are typically imprinted or engraved on the external portion of the barrel for easy reference. These specifications help researchers select the appropriate objective for their experiments, ensuring optimal performance and total magnification when combined with the ocular lens. Specifications like numerical aperture and magnification are typically labeled on the barrel for easy reference. These lenses are indispensable in scientific research providing high powered optics essential for research.

In the following content, we delve intensively into the various components and features of microscope objective lenses, exploring their construction, functionality, and specialized designs that enable researchers to gain deeper insights into the microscopic world.

Components of a microscope

A microscope is an optical device designed to magnify the image of an object, enabling details indiscernible to the human eye to be differentiated. A microscope may project the image onto the human eye or onto a camera or video device. 

Microscopes are usually complex assemblies that include an array of lenses, filters, polarizers, and beamsplitters. Illumination is arranged to provide enough light for a clear image, and sensors are used to ‘see’ the object.

Although today’s microscopes are usually far more powerful than the microscopes used historically, they are used for much the same purpose: viewing objects that would otherwise be indiscernible to the human eye.  Here we’ll start with a basic compound microscope and go on to explore the components and function of larger more complex microscopes. We’ll also take an in-depth look at one of the key parts of a microscope, the objective lens.

In many microscopes, backlight illumination is favored over traditional direct light illumination due to the latter’s tendency to over-saturate the object under inspection. One specific backlight illumination technique employed in microscopy is Koehler illumination. This method involves flooding the object with light from behind using incident light from a source like a light bulb (see Figure 2). Koehler illumination utilizes two convex lenses, the collector lens and the condenser lens(or called field lens) , to ensure even and bright illumination on both the object and image planes. This design prevents imaging the light bulb filament, a common issue with direct light illumination. Backlight illumination is also commonly referred to as brightfield illumination.
 

For brightfield illumination to be effective, there needs to be a variation in opacity across the object. Without this variation, the illumination creates a dark blur around the object, resulting in an image with relative contrast between the object’s parts and the light source. Typically, brightfield illumination allows clear visualization of each part of the object unless it is extremely transparent. In cases where transparency hinders feature distinction, darkfield illumination becomes useful.

Darkfield illumination directs light rays obliquely onto the object, avoiding direct entry into the objective. Despite this oblique angle, the rays still illuminate the object plane. The resulting darkfield illumination image achieves high contrast between the transparent object and the light source. In a darkfield setup, a light source forms an inverted cone of light that blocks central rays but allows oblique rays to illuminate the object (see Figure 3). This design effectively forces light to illuminate the object without entering the optical system, making darkfield illumination particularly suitable for transparent objects. In contrast, no rays are blocked in a brightfield illumination setup.
Epi-illumination, a third form of illumination employed in microscopy, generates light from above the objective. This setup replaces the need for a Koehler illumination configuration, as both the objective and the epi-illumination source contribute to the illumination process. The compact structure  of epi-illumination is a significant advantage, as the objective serves as a primary source for a considerable portion of the illumination. Figure 4 provides a depiction of a frequently used epi-illumination setup, particularly common in fluorescence applications.

Compound Microscope

While a magnifying glass consists of just one lens element and can magnify any element placed within its focal length, a compound lens, by definition, contains multiple lens elements. A relay lens system is used to convey the image of the object to the eye or, in some cases, to camera and video sensors.

A basic compound microscope could consist of just two elements acting in relay, the objective and the eyepiece. The objective relays a real image to the eyepiece, while magnifying that image anywhere from 4-100x.  The eyepiece magnifies the real image received typically by another 10x, and conveys a virtual image to the sensor. 

There are two major specifications for a microscope: the magnification power and the resolution. The magnification tells us how much larger the image is made to appear. The resolution tells us how far away two points must be to be distinguishable. The smaller the resolution, the larger the resolving power of the microscope. The highest resolution you can get with a light microscope is 0.2 um, but this depends on the quality of both the objective and eyepiece.

Both the objective lens and the eyepiece also contribute to the overall magnification of the system. If an objective lens magnifies the object by 10x and the eyepiece by 2x, the microscope will magnify the object by 20 times. If the microscope lens magnifies the object by 10x and the eyepiece by 10x, the microscope will magnify the object by 100x. This multiplicative relationship is the key to the power of microscopes, and the prime reason they perform so much better than simply magnifying glasses. 

In modern microscopes, neither the eyepiece nor the microscope objective is a simple lens. Instead, a combination of carefully chosen optical components work together to create a high quality magnified image. A basic compound microscope can magnify up to about 1000x. If you need higher magnification, you may wish to use an electron microscope, which can magnify up to a million times.

  • Eyepiece 
    • In the initial stages of microscope development, eyepieces are integral to the design as they provide the sole method for visually observing the object under examination. Presently, analog or digital cameras have taken on this role, projecting the object’s image onto a monitor or screen. Microscope eyepieces typically comprise a field lens and an eye lens, with various designs available, each capable of producing a broader field of view (FOV) compared to a single-element design.
  • Microscope Illumination 
    • Most microscopes rely on background illumination such as daylight or a lightbulb rather than a dedicated light source. In brightfield illumination (also known as Koehler illumination), two convex lenses, a collector lens and a condenser lens,  are placed so as to saturate the specimen with external light admitted into the microscope from behind. This provides a bright, even, steady light throughout the system. 
  • Objectives: Refractive
    • Refractive objectives have their name because the elements bend or refract light as it passes through the system. They are well suited to machine vision applications, as they can provide high resolution imaging of very small objects or ultra fine details. Each element within a refractive element is typically coated with an anti-reflective coating. A basic achromatic objective is a refractive objective that consists of just an achromatic lens and a meniscus lens, mounted within appropriate housing. The design is meant to limit the effects of chromatic and spherical aberration  as they bring two wavelengths of light to focus in the same plane. Plan Apochromat objectives can be much more complex, with up to fifteen elements. They can be quite expensive, as would be expected due to their complexity.
  • Objectives: Reflective 
    • A reflective objective works by reflecting light rather than bending it. Primary and secondary mirror systems both magnify and relay the image of the object being observed. While reflective objectives are not as widely used as refractive objectives, they offer many benefits. They can work deeper in the UV or IR spectral regions, and they are not plagued with the same aberrations as refractive objectives. As a result, they tend to offer better resolving power.

Key Concepts and Specifications

The majority of microscope objective specifications are conveniently displayed on the objective’s body, including information such as the objective design/standard, magnification, numerical aperture, working distance, lens to image distance, and cover slip thickness correction. Refer to Figure 5 for guidance on interpreting microscope objective specifications. This direct placement of specifications on the objective facilitates a clear understanding of its characteristics, a crucial aspect when integrating multiple objectives into an application. Any additional specifications, like focal length, field of view (FOV), and design wavelength, can be readily calculated or obtained from the vendor or manufacturer’s provided specifications.
 
  • Aberration Correction or Resolution: Spherical and chromatic aberrations limit the resolution of conventional microscopes. Lenses with a high degree of aberration correction result in high-resolution images over the entire field of view.
  • Conjugate Distance: Objectives are corrected for a specific projection distance. In finite conjugate optical design, light from a non-infinite source is focused down to a point. In infinity-corrected optical systems, light emitted from the specimen passes through the objective lens and enters the tube lens as an infinity parallel beam, forming a real image.
  • Numerical Aperture (NA): NA is the measure of its capability to gather light and to resolve fine specimen details at a fixed object. A lens with a high NA collects more light and can resolve finer specimen details at a fixed distance. These are the elements that determine resolution, depth of focus, and image brightness. The larger the numerical aperture, the higher the resolution and the brighter the image can be observed. The higher the magnification of the objective lens, the larger the numerical aperture.
  • Magnification: A microscope’s ability to produce larger images is referred to as magnification. High magnification objectives provide extremely detailed images of specimens. The term magnification is often confused with the term resolution, which describes the ability of an imaging system to show detail in the object that is being imaged. While high magnification without high resolution may result in very small microbes visible, it will not allow observers to distinguish between microbes or subcellular parts of a microbe. Avantier has spent years designing and manufacturing to satisfy both magnification and resolution requirements simultaneously. This is the magnification of the intermediate image (inverted real image) for the specimen. In addition to low magnification (4x to 10x), medium magnification (20x to 50x), high magnification (100x or more), extremely low magnification (2.5x) (below) etc.
  • Field of View(FOV): FOV refers to the portion of the object captured by a microscope system. The size of the FOV is dictated by the objective magnification. When employing an eyepiece-objective system, the FOV initially observed through the objective is enlarged by the eyepiece for visual examination. In a camera-objective system, this FOV is transmitted onto a rectangular camera sensor. Due to the sensor’s shape, it can only capture a segment of the complete circular FOV from the objective. In contrast, the human eye’s retina can capture a circular area, encompassing the entire FOV. Consequently, the FOV produced by a camera-microscope system tends to be slightly smaller than that of an eyepiece-microscope system.
  • Cover Glass: Objectives are usually corrected for a specific cover glass thickness, with 0.17 millimeters being the standard. The thickness of the cover glass is numerically marked on the objective lens. There are three types: one for cover glass specimens, one for non-cover specimens, and one for both cover glass specimens and no cover glass specimens.
  • Immersion Medium: The main purpose of using different types of immersion medium is to minimize the refractive index between the objective and the sample. It is crucial to use the correct medium, such as water, oil, or air/dry, as specified by the objective.
  • Working Distance: The distance between the front end of the microscope objective and the surface of the specimen at which the sharpest focus is achieved. Proper positioning is important to obtain a good image at the specified magnification. The distance from the tip of the objective lens to the specimen surface when focused. The larger the numerical aperture of the objective lens, the shorter the working distance.
  • Parfocal Length: The distance from the mounting plane of the objective to the sample plane. 
  • Working Wavelength(s): Objectives are corrected for specific wavelengths, with shorter wavelengths yielding higher resolution.
For more details, read this article

Microscope Objective Selection Guide

Microscope objectives are pivotal components in optical microscopy, especially in influencing image quality and resolution. Selecting the right objective is crucial for achieving optimal results in your microscopy applications. To guide you through the selection process, consider the following factors:

1. Working Distance

  •       Working distance (WD) is the distance from the objective to the coverglass.
  •       Inversely proportional to numerical aperture (NA); higher NA often means lower working distance.
  •       Consider long-working distance objectives for specific applications.

2. Resolution Requirement

  • Resolution is determined by NA and illumination wavelength.
  • Higher NA provides finer resolution.
  • Choose NA carefully based on your application’s resolution needs.

3. Field-of-View and Depth-of-Field

  • Evaluate field number (FN) for the diameter of the field of view.
  • Modern objectives have FNs between 22 and 26.5mm.
  • Consider depth of field, which varies with numerical aperture.

4. Specimen Size

  • Determine the size of your specimen.
  • Our microscope objectives offer a magnification range from 1.25x to 150x.
  • Consider the combined magnification with eyepieces for overall magnification.

5. Smallest Features in Specimen

  • Assess the numerical aperture (NA) of objectives.
  • Higher NA enables to gather more light, enhancing resolution and brightness.
  • NA ranges from 0.04 to 1.7; choose based on your specimen’s fine structures.

6. Fluorescence Signal Brightness

  • High NA objectives are recommended for weak fluorescence signals.
  • Avantier offers a range of objectives for fluorescence excitation across UV to NIR.

7. Multichannel Fluorescence Imaging

  • Choose objectives based on chromatic correction needs.
  • Achromat, semi-apochromat, and apochromat objectives for multichannel fluorescence.
  • Extended apochromats recommended for multichannel applications.

8. Observation Methods

  • Consider observation methods beyond brightfield.
  • Dedicated objectives for darkfield, DIC, phase contrast, and polarization.

9. Immersion Medium

  • Select objectives based on the immersion medium: air, water, oil, or silicone.
  • Immersion mediums can enhance resolution; choose accordingly.

10. Advanced Microscope Systems

  • Dedicated objectives for advanced systems like confocal microscopy.
  • Choose objectives tailored to confocal, spinning disk confocal, multi-photon excitation, and TIRF microscopy.

Custom Microscope Objectives Solutions 

Avantier is a premier manufacturer of high performance microscope objective lenses, and we produce a wide range of quality microscope objectives for applications ranging from research to industry to forensics and medical diagnostics. We carry many types of objectives in stock, including apochromat objectives, achromatic objectives,  and semi apochromat objectives.  We can also produce custom objectives designed to work as desired in your target spectral range. 

For custom microscope objective lenses, visit our Microscope Objective Lenses page. 

If you’re interested in acquiring in-stock microscope objective lenses, please visit our ‘Stock – Microscope Objective‘ page.

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