Objective Lenses – Principle, Types, Specification, Uses

Objective lens is the most important lens of an optical microscope, which is kept closest to the specimen. It is the first lens that collects the light coming from the specimen (emitted or reflected). It bends the light rays and forms a real, magnified image at an intermediate image plane. This primary image is then magnified further by the eyepiece lens or recorded by an imaging sensor.

Objective lens is the most critical and complex part of an optical microscope. It is the main lens for image formation, resolution, and contrast. It is called objective because it is placed closest to the object or specimen that is being viewed.

In simple terms, the objective lens collects the light which is emitted or reflected by the specimen. This light is then bent by the lens and a real, magnified image is formed at an intermediate image plane. This primary image is then magnified further by the eyepiece or it can be captured by an imaging sensor.

Since it is the first component that light meets after leaving the specimen, the objective lens decides the final quality of the microscopic image. It mainly controls the magnification power and resolving power of the microscope image.

Purpose of Objective Lenses

• The objective lens is used to form the primary (intermediate) image of the specimen. It is the first main image formed inside the microscope.
• It is used to collect and capture the light coming from the specimen (emitted, transmitted, or reflected). This collected light is needed for image formation.
• It is used to give magnification. The light rays are bent and the specimen is enlarged so that small structures can be viewed through eyepiece or captured by camera.
• It is used to determine the resolution of the microscope. It decides how clearly fine details and two closely placed points can be separated.
• It is used to establish contrast and image quality. The objective design and aberration correction mainly decide the sharpness, clarity, and contrast of the image.
• In some microscopes (metallurgical or epi-fluorescence), it is also used to deliver illumination. It acts like a condenser by directing light to the specimen surface and then collecting the reflected light.

Specification of Objective Lens

• Manufacturer. It is the name of the company that produced the objective (Nikon, Olympus, Zeiss, Leica).
• Magnification. It is the linear magnifying power of the objective (generally 0.5x to 250x). It is written on the objective and many times it is shown with NA.
• Numerical aperture (NA). It is a dimensionless value. It decides the light acceptance angle and light gathering power. It also decides the resolving power and depth of field.
• Optical corrections. It is written as abbreviations which shows correction for chromatic and spherical aberrations (Achro or Achromat, Fluor or Fl, Apo). It may also show correction for image flatness (Plan, Pl).
• Tube length. It shows the body tube length for which the objective is designed. It can be given as a number in mm like 160 (finite tube) or as infinity symbol (∞) for infinity corrected system.
• Cover glass thickness. It shows the thickness of cover slip for which the objective is corrected. Usually it is 0.17 mm.
• Working distance (WD). It is the distance between the front lens of objective and the specimen or cover glass when the image is in sharp focus.
• Immersion medium. It shows whether the objective needs a liquid medium to achieve its NA. It can be oil (Oil, O), water (W), glycerin (Gly) or it can be dry (no code).
• Observation method / special properties. It shows if the objective is made for special techniques like phase contrast (PH), DIC, polarized light (P, Pol) or darkfield (BD).
• Color codes. It is colored rings on the barrel for quick identification. It can show magnification (yellow for 10x, white for 100x) and also immersion media (black for oil, white for water).
• Objective screw threads. It is the mechanical mounting thread used to attach the objective to the nosepiece. It can be RMS standard or larger metric threads like M25 and M32.
• Observation conditions (industrial or metallurgical). It shows if the objective is for bare surfaces without cover glass (M for metal). It can also show extended distances like LWD (long working distance).

How does an Objective Lens Works?

1. Captures light. The objective lens is placed closest to the specimen. It collects the light waves that are emitted or reflected from the object.
2. Bends light rays. The collected light passes through the glass elements of the lens. Due to change in velocity and direction, the light rays are refracted and bent.
3. Generates a magnified image. The refracted light rays are focused by the lens. A real and magnified image of the specimen is formed at an intermediate image plane. This image is later magnified again by the eyepiece.
4. Resolves fine details. The objective captures a wide cone of light. This decides the resolving power of the microscope and helps to distinguish tiny and closely spaced structures.
5. Corrects optical errors. Modern objectives have multiple lens elements arranged in a complex way. These elements work together to correct distortions like chromatic aberration (color fringing) and spherical aberration (blurring). This gives a more clear and accurate image.

Types of Objective Lenses

By level of optical correction

1. Achromatic objectives. It is the most commonly used objective. It corrects chromatic aberration in two colours (red and blue). It also corrects spherical aberration in one colour (green).
2. Fluorite (semi-apochromatic) objectives. It is made with advanced low dispersion glass. It corrects chromatic and spherical aberrations for two or three colours. It gives higher numerical aperture and more bright image.
3. Apochromatic objectives. It is the highest corrected objective lenses. It corrects chromatic aberration in three to five colours. It also corrects spherical aberration in two to four colours and gives maximum resolution.
4. Plan objectives. It is written as Plan Achromat or Plan Apochromat. It has extra lens elements to correct field curvature. It gives a flat image from center to edge.

By immersion medium

1. Dry objectives. It is used with air between the front lens and the specimen or cover slip.
2. Oil immersion objectives. It uses special oil having refractive index similar to glass (about 1.515). It reduces light refraction and increases numerical aperture and resolution.
3. Water immersion objectives. It uses water. It reduces refractive index mismatch and spherical aberration during live cell or aqueous tissue observation.
4. Glycerin and silicone immersion objectives. It is used for specific tissues or long term live imaging. It is used when water may evaporate.

By specimen application and contrast method

1. Biological objectives. It is made for transmitted light (light from below). It is corrected for light passing through 0.17 mm cover slip.
2. Metallurgical (industrial or epi) objectives. It is made for reflected light (light from above). It is used to observe opaque polished surfaces like metals and semiconductors without a cover slip.
3. Phase contrast objectives. It has a phase plate inside the objective. It produces contrast for transparent and unstained specimen.
4. DIC (differential interference contrast) objectives. It is made strain free. It prevents unwanted birefringence during polarized light use and gives high contrast 3D relief image.
5. Super-resolution objectives. It is advanced objective lenses. It is made with symmetrical point spread function. It can tolerate intense laser power and used in techniques beyond diffraction limit.

Emerging technologies

1. Metalenses. It is flat optical device. It is made of billions of sub-wavelength nanostructures. It manipulates light phases without using normal bulk glass refraction.

What are Refractive and Reflective Objectives Lenses?

Refractive objective lenses

It is made from transparent materials like glass. It works by bending (refracting) the light when it passes through the lens system and then the light is focused on the specimen. It contains many internal lens elements and these are treated with anti-reflective coatings, so that optical aberrations are corrected and good quality image is produced. It is highly used in machine vision applications for high resolution imaging of very small objects and ultra-fine details.

Reflective objective lenses

It is made by using two or more mirrors (primary and secondary mirror system). It works by reflecting the light and not by bending the light. It gives long working distance and it avoids optical aberrations seen in refractive lenses, so chromatic aberration becomes zero and resolving power is better. It is not used as much like refractive objectives, but it is useful in ultraviolet (UV) and infrared (IR) regions and it is used in applications like FTIR spectroscopy.

Factors affects the performance of a objective lens

• Numerical aperture (NA). It is the main factor for lens performance. It shows how much light the lens can gather and how much fine details can be resolved.
• Optical aberrations. The correction level for spherical aberration and chromatic aberration affects the image. If correction is poor then image becomes less sharp and clarity is reduced (color fringing and blurring).
• Field curvature. In uncorrected lenses the image field becomes curved. So when center is focused, the edges becomes blurry. Plan corrected objectives flatten the field and gives uniform sharpness from center to edge.
• Illumination wavelength. The resolution limit depends on the wavelength of light used. Shorter wavelength gives higher resolution.
• Immersion medium. The refractive index of the medium between lens and specimen (air, water, oil) should match the lens design. If not matched, light loss occurs due to scattering and refraction. Proper immersion increases NA and resolution.
• Cover glass thickness. In high NA dry objectives, small change from standard cover slip thickness (usually 0.17 mm) can create spherical aberration and image degradation.
• Working distance. The distance needed between lens front and specimen affects performance. Long working distance objectives generally have lower NA and lower resolution.
• Antireflection coatings. Multilayer coatings reduce internal reflections, flare and ghosting. It improves light transmission and image contrast.
• Glass strain and cleanliness. Internal stress or strain in glass and also scratches, dust, fingerprints on lens surface can disturb light and reduce image quality badly.

How to maintain and care objective lenses? 

Remove immersion oil immediately. After use, the front lens should be cleaned quickly. A lens cleaning tissue is used and a single sweep is done across the lens, so oil do not enter inside and damage other microscope parts.

Use clean tissues for every wipe. Each time a fresh clean tissue is taken for one pass. Wiping is continued until no oil trace is seen on the lens.

Apply approved cleaning solutions. Commercial oil removal solution can be used or a small amount of xylene is used carefully. But it should be checked with the manufacturer recommendation before putting any liquid on the objective.

Clear away dust and debris safely. Loose dust and fibers are removed by rubber balloon blowing. If wiping is needed then moist soft cotton or lens tissue is used gently, and scratching of the glass should be avoided.

Protect anti-reflection coatings. Coated optical surfaces are cleaned very carefully. If microscope is opened, internal lens elements should not be handled roughly because coatings are soft and can be damaged easily.

Avoid physical impacts. Objectives should be handled gently. Complex objectives like strain-free lens can be damaged by one jolt or mechanical stress and their optical property may be ruined.

Perform regular periodic maintenance. Regular cleaning and maintenance should be done. This protects the objective and it gives good quality image continuously.

Uses of Objective Lenses

• Biological and medical analysis. It is used to observe thin and transparent specimens like cells, tissues and microorganisms. Generally the specimen is kept under a glass cover slip for viewing.
• Industrial and metallurgical inspection. It is used to examine surface topography, grain structure and defects of opaque solid materials like metals, alloys and ceramics. In this, reflected light is used.
• Semiconductor wafer inspection. It is used to detect sub-micron defects in patterned circuits and electronic assemblies. Long working distance objectives are used so that collision with bulky parts do not happen.
• Fluorescence and super-resolution imaging. It is used to view specimens labelled with fluorescent markers. It is also used to capture molecular level details in techniques like STORM and STED.
• Deep tissue imaging. It is used in multi-photon objectives with long wavelength infrared light. It helps to penetrate and image deeper in living tissues, neural networks or whole embryos.
• Laser and spectrometry applications. It is used to focus laser light for high precision works like laser cutting, surgical procedures and spectrometry. Special UV or near-infrared (NIR) objectives are used.
• Specialized contrast imaging. It is used to view transparent unstained samples without dyes by phase contrast objectives. It is also used in polarized light to study internal stress and crystalline structures in geology and material studies.
• Initial specimen scanning. Low magnification objectives (2x to 4x) are used to scan a wide field and find the area of interest. After that high power objectives are used for detailed observation.

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