Beyond Magnification: Understanding Microscope Objectives (Part II)

Read: Beyond Magnification: Understanding Microscope Objectives (Part I)

What “full-oil immersion” means

• Oil-immersion objectives are built to operate with immersion oil between the front lens and the coverslip to reach very high NA (typically 1.25–1.49).
• Oil-immersion condensers require a drop of immersion oil between the condenser top lens and the underside of the slide to deliver a high-NA illumination cone (often NA 1.2–1.4).
• When both are used together, the optical path is continuous oil from condenser → slide → coverslip → objective.

This configuration is essential for maximum resolution in high-magnification brightfield work (e.g., 100X oil objectives) and for resolving very fine structures such as bacteria, diatoms, and flagella. Often oil immersion fluid between the objective and coverglass is sufficient. The condenser can remain dry, and resolution will still meet the design specifications of most oil objectives. This is the configuration used most often in teaching, clinical, and research labs.

Objective Screw Threads

The vast majority of standard microscope objectives use the RMS (Royal Microscopical Society) thread size, however, several modern manufacturers utilize proprietary or larger metric thread sizes to accommodate advanced optics or to encourage the purchase of their objectives. Zeiss uses a larger thread size on many of their infinity microscopes. The standard Royal Microscopical Society (RMS) objective thread size is 0.800"-36. This is a Whitworth standard thread form featuring a nominal outer diameter of 0.8 inches, 36 threads per inch (TPI), and a 55-degree flank angle. The metric equivalent for the Royal Microscopical Society (RMS) objective thread is 20.32 mm in diameter with a 0.71 mm pitch (often expressed as W 0.8" × 1/36").

M25 objective refers to a microscope objective featuring a metric 25 mm diameter thread with a 0.75 mm pitch. Predominantly utilized by manufacturers like Nikon and Leica, it differs from the older, smaller 20.32 mm RMS.

M26 times 0.706 (36 tpi): Frequently used by Mitutoyo for long working distance metallurgical objectives.

M32 times 0.75 Used on some high-end, large-aperture objectives.

Four Main Types of Microscope Objectives

Objectives fall into finite and infinity-corrected optics, and within each category they are further divided based on type, optical corrections, NA, and Field Flatness.

Table 1. Four major families of objectives (finite and infinite) used for light microscopy.

Fig. 12. Left image Vitamin C crystals photographed with a 10X Achromat objective. On the right is an image photographed with a 10X Plan Achromat objective. White arrows on the left image point to out of focus edges in the image taken with an Achromat. Both images of Vitamin C crystals were taken with polarized light.

Strain-free objectives are generally used for polarizing microscopy and Differential Interference Contrast (DIC), though these objectives can also function under other illumination modes. While brightfield objectives can transmit polarized light, strain-free designs are manufactured to minimize internal glass stress which produces unwanted birefringence and light artefacts. Objectives marked PO on the barrel indicate they are specifically engineered for polarizing optics.

Phase Contrast Objectives

Light is delayed as it traverses a living cell because cellular components possess a refractive index greater than that of the surrounding medium. This produces a typical phase shift of about λ/4 (90°)—large enough to contain structural information but invisible to the eye. Phase contrast converts that invisible phase shift into intensity differences using a condenser annulus — a ring-shaped aperture that produces a hollow-cone of illumination and an objective phase ring in the objective rear focal plane that selectively retards and attenuates undeviated light - causing interference. The Objective and condenser phase rings must be matched and precisely aligned using a phase turret, a modified eyepiece. The alignment is done before viewing the cells and then the phase turret is replaced with an ordinary 10X eyepiece. When the condenser annuli are not in the light path you can use the objectives for bright field microscopy and when the condenser rings are slightly out of alignment you can obtain darkfield microscopy. Some microscope brands allow the phase rings in the condenser to be fixed in place or they use annuli that can slide into the light path below the condenser to be quickly aligned.

Undeviated light (background) passes through the condenser annulus and directly through the phase ring, where it is retarded and partially attenuated. Diffracted light (foreground detail) from the specimen bypasses the phase ring and is not retarded or attenuated. When both beams recombine at the image plane, their engineered interference produces visible bright or dark phase contrast. This interference makes cell boundaries, nuclei, membranes, and organelles appear with high contrast without staining cells and can be used on living cells. Phase contrast is useful because it reveals transparent living cells and organelles clearly. It also allows observation of motility, cell division and dynamic processes and requires no fixation or dyes, preserving natural morphology. Phase contrast microscopy was developed by Fritz Zernike in 1934, a discovery that earned him the 1953 Nobel Prize in Physics. For more information see phase contrast on Motic’s website (R. Berdan, 2019). Phase contrast can be quickly and easily tested by viewing your own cheek cells. Scrape the inside of your mouth with a clean glass slide, then smear your cheek cells onto a slide and add a cover glass, saline and view.

A principal disadvantage of phase contrast is a bright halo surrounding the cell and its organelles that can reduce resolution and obscure fine detail. Phase objectives are manufactured in positive and negative phase versions causing organelles such as nuclei to appear darker or lighter, respectively. Apodized phase objectives incorporate graded apodizing filters that suppress halos and shade-off artifacts. Apodizing phase contrast was developed by Nikon.

Phase contrast objectives require matching phase rings with those in the condenser. A complete phase-contrast system (phase condenser, several phase objectives, and phase turret is below. (Fig. 13.) A phase kit is moderate in cost and widely used by cell biologists (R. Berdan, 2021). They are available in all objective families Achromat to Apochromat.

Fig. 13. Phase contrast kit by Motic includes: condenser, phase turret and phase objectives.

Fig. 14. To determine if you have phase objectives, look at the back of the objectives for a gray ring inside the barrel as shown above. You will also need a phase condenser with matching rings.

Fig. 15. Isolated live fresh water snail neuron in culture by phase contrast microscopy.

Fig. 16. Volvox sp. found in fresh water ponds. Viewed by phase contrast microscopy.

Motic Objectives

Motic offers a variety of objectives which are engineered for various applications for education or research: Read Motic’s Objective Buyers Guide.

CCIS® EC-H Plan Achromat: Motic's standard laboratory tier. They feature multi-layer coatings for excellent color fidelity and flat-field (plan) aberration correction. Widely used in university and clinical settings. Motic ranks as a top-tier mid-range manufacturer and a premier value brand in microscopy. While it does not compete with the "Big Four" elite research brands (Zeiss, Leica, Nikon, and Evident-Olympus), its high-end infinity-corrected optics deliver exceptional optical clarity and color reproduction at a fraction of the cost.

Plan Apochromat: Motic's highest level of optical correction for chromatic and spherical aberrations. They provide incredibly crisp, high-contrast images, and come with a professional-grade price tag.

LM Plan (Metallurgical/Materials): Highly specialized, infinity-corrected non-cover-glass objectives optimized for incident light and industrial/material science applications.

Price vs. Performance: Optically and mechanically Motic is considered one of the best value microscopes and objectives for the money. Motic was founded in 1988. Manufacturing is based in China and Texas and consists of five fully-owned subsidiaries manufacturing components for the company with Motic Xiamen acting as production headquarters of the company. Motic offers exceptional value, fixed-focal length and infinity-corrected optics at a fraction of major competitor costs, and an industry-leading integration of digital microscopy tools. Most users on ResearchGate agree that Motic provides an outstanding price-to-performance, delivering clean, sharp optics that rival premium legacy brands while costing significantly less.

Objective Color Rings

Most but not all objectives have a colour band or ring around their barrels which provides information about their magnification so they can quickly be identified. The colored rings printed on objective barrels may indicate magnification, immersion fluid, or other functions. Some manufacturers may use their own color coding system. Color bands are helpful, but the printed text on the barrel is the true specification. They are useful with multi-objective nosepieces where quick switching is required.

Objective Color Coding (IS0 8578) Magnification & Immersion Fluid

Fig. 17. Color bands on objectives may differ between different brands and may be absent all together on some microscope objectives.

Objectives for metallurgy and epi-illumination

Metallurgical microscope objectives are specialized lenses designed for reflected (episcopic) illumination, meaning light travels down through the lens to illuminate opaque metal samples. These objectives are corrected for use without cover slips and include optical coatings to prevent glare. Metallurgical microscope objectives are labeled with an "M" on the barrels.

Infrared Microscopes

Infrared (IR) and μ-FTIR microscopes combine optical microscopy with FTIR spectroscopy to identify the chemical composition of microscopic samples. They use highly reflective metal-coated mirrors called Cassegrain objectives rather than glass, allowing both visible light for viewing and infrared light for precise chemical analysis. Infrared (IR) microscope objectives are specialized lenses engineered to transmit and focus light in the infrared region of the electromagnetic spectrum typically 700 nm to over 1000 nm. They allow users to visualize materials that are opaque in the visible spectrum and perform highly localized chemical analysis.

Reflective Objectives: Because standard glass heavily absorbs certain infrared wavelengths, many IR objectives utilize mirror-based (Cassegrain) reflective optics. These provide excellent chromatic and spherical aberration correction across a massive wavelength range (from UV to far-IR).

Refractive Objectives: Near-infrared (NIR) and short-wave infrared (SWIR) objectives are made from specialized crystalline materials (like Zinc Sulfide) and have anti-reflective coatings designed to maximize light transmission and minimize signal loss.

Objective Technological Advancements

Liquid Lenses: Electrically tunable fluid lenses are increasingly integrated into research-grade objectives. They allow for almost instantaneous z-axis focus adjustments, and speed up focus stacking. See white paper by Corning (2023).

All-Reflective Optics: Moving away from standard glass refraction, freeform reflective objectives are trending in semiconductor and non-linear biomedical imaging. They offer zero chromatic aberration over ultra-wide spectrums. All-reflective optics are optical systems that use only curved or flat mirrors instead of glass lenses to direct and focus light. By completely eliminating transmissive elements, these systems avoid chromatic aberration and material absorption making them standard for wide-spectrum and high-power application (Edmund Optics).

Summary

Microscope objectives are the most important components of a light microscope and fall into two families: finite and infinity-corrected. Infinity objectives are standard since the 1980s and allow for additional optical elements to be placed in the light path without introducing aberrations. Finite objectives remain lower-cost options that provide excellent images but are not designed for infinity systems. Both types of objectives are made for upright and inverted light microscopes.

Objectives are available in four correction classes—Achromat, Plan Achromat, Semi-Apochromat, and Apochromat—with Apochromats offering the highest NA, strongest aberration corrections, and greatest resolving power. Color bands indicate magnification, and total magnification equals objective × eyepiece. Most objectives range from 1× to 100×, with most high-NA designs requiring immersion media. Specialized objectives support polarizing, phase contrast, DIC, darkfield, and fluorescence. Correct coverglass thickness is essential for achieving maximum resolution. Oil immersion fluid between objective and the coverglass and between the condenser and microscope slide permit the maximum numerical aperture and resolution. According to ResearchGate, Motic offers one of the best price-to-performance ratios in microscope objectives.

Want to know which microscopes fit you best? Send us a message and our specialists are glad to help!

References

• Galbraith C. G, Galbraith J.A. Super-resolution microscopy at a glance. J Cell Sci. 2011 May 15:124 :1607-11. doi: 10.1242/jcs.080085. PMID: 21536831; PMCID: PMC3085433. J Cell Sci2011 May 15;124(Pt 10):1607-11.doi: 10.1242/jcs.080085
• Berdan, R. (2024) The Structure and function of a light Microscope. Motic America./en-ca/en-ca/blogs/articles/the-structure-and-function-of-a-light-microscope
• Volodymyr Nechyporuk-Zloy (ed) 2023 Priniciples of Light Microscopy: From Basic to Advanced. Springer pp. 1-324. Research gate. https://doi.org/10.1007/978-3-031-04477-9_14
• Spring K. R. and M.W. Davidson (2026) Infinity Optical Systems – Evident https://evidentscientific.com/en/microscope-resource/knowledge-hub/anatomy/infinityintro
• Berdan, R. (2023) The Importance of The Correct Coverglass thickness for photomicography. For Motic America. Jan. 17./en-ca/blogs/articles/the-importance-of-the-correct-coverglass-thickness-for-photomicrography
• Gill. G.W. (2013) Cytopreparation Principles and Practice. Chapt. 17: Cover Glasses. Essentials in Cytopathology Vol 12. Springer Verlag. https://catalog.nlm.nih.gov/discovery/fulldisplay/alma9916031533406676/01NLM_INST:01NLM_INT
• Daughty, D. R (2026) MIT and Broad Institute researchers break diffraction barrier in super-resolution microscopy visualize 10 nm particles. https://news.mit.edu/2026/researchers-break-diffraction-barrier-super-resolution-microscopy-0729
• Cargille, J.J (1985) Immersion oil and the Microscope 2nd edition. New York Microscopical Society Yearbook, 1964. https://www.cargille.com/wp-content/uploads/2024/07/Immersion_Oil_and_the_Microscope.pdf
• Davidson M.W Microscope Objective Specifications Nikon Microscopy Basics https://www.microscopyu.com/microscopy-basics
• Sparkle Optical Lens Cleaner manufactured by A.J. Funk & Co. and distributed by AM microscope & Amazon.com.
• (K. Thorn, 2023) Microscopy: How to Clean an Objective Lens. YouTube video https://www.youtube.com/watch?v=Tz4Dy5D6kdw
• M J. Parry-Hill and M. W. Davidson Nikon Microscopy U. Objective Correction collars https://www.microscopyu.com/tutorials/adjustment-of-objective-correction-collars
• Becktronic.com https://www.beckoptronic.com/
• R. Berdan (2021) Phase contrast by Motic. /en-ca/en-ca/blogs/articles/phase-contrast-by-motic
• Corning – liquid lenses (2023) https://www.corning.com/media/worldwide/csm/documents/WhitePaper_MicroscopyV2.pdf
• Motic Objective guide /en-ca/en-ca/pages/motic-microscopes-objectives-buyers-guide
• Spring, K.R and Davidson, M.W. Numerical Aperture Nikon Microscopy U. https://www.microscopyu.com/microscopy-basics/numerical-aperture
• Practical Guide to Lens Abberations https://www.lonelyspeck.com/a-practical-guide-to-lens-aberrations-and-the-lonely-speck-aberration-test/comment-page-6/?v=5435c69ed3bc Guide which shows example astrophotographs with and without out lens aberrations.
• Microscope aberrations and pictures https://abberior.rocks/knowledge-base/how-to-correct-for-aberrations-in-light-microscopy/
• Edmund Optics Highly reflective (HR) coatings are used to minimize light while reflecting lasers and other light sources. https://www.edmundoptics.ca/knowledge-center/application-notes/optics/highly-reflective-coatings/
• Motic objectives - /en-ca/en-ca/collections/objective