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Why 40x Microscope Objectives Should Never Be Used with Immersion Oil

As a microscope user, you’ve likely drooled over the crystal clear views promised by high-power objectives like 40x. Just think of the fine details you could resolve by combining a 40x lens with immersion oil! However, while this tempting combo may seem like a quick resolution fix, it should never be attempted.

The main reason 40x objectives are unsuitable for immersion oil is that they have a short working distance that poses a collision risk between the lens and specimen.

Understanding Microscope Resolution

To understand why 40x and oil immersion don’t mix, we first need to review some basics about microscope resolution.

Resolution refers to the minimum distance between two points that can still be distinguished as separate entities in the image. It is limited by the wavelength of visible light and the numerical aperture (NA) of the objective lens according to the formula:

Resolution = λ / (2 x NA)

Where λ is the wavelength of light (around 500 nm) and NA depends on the refractive indexes and half-angle of the light cone entering the objective.

Higher NA values equate to better resolution. Immersion oil improves resolution by increasing the NA of the objective. It fills the gaps between the specimen and lens, allowing for a wider cone of light to enter the objective.

The Short Working Distance of 40x Objectives

Now back to 40x objectives. These provide a high magnification for viewing fine details in a specimen. However, they have an extremely short working distance – the minimum distance between the front lens element and the specimen cover glass.

Typical working distances for 40x objectives are just 0.3 – 0.6 mm. In comparison, lower power objectives (10x – 20x) have working distances of multiple millimeters.

Why the Short Working Distance Occurs

The short working distance of 40x objectives arises due to the high NA (around 0.65 – 0.75) needed to deliver the high resolution expected at this magnification level.

To achieve a high NA, the cone of light entering the objective needs to be wide. This requires the front lens element to be very close to the specimen cover glass – hence the short working distance.

The Risks This Poses with Immersion Oil

The thin gap between a 40x objective and the specimen makes collisions likely if immersion oil is used.

Since oil has a different refractive index than air, it changes the optical properties between the lens and sample. The lens needs to be closer to the specimen to compensate – but there is no room for this with a 40x!

Bringing the 40x into contact with the oil risks:

  1. Damaging the fragile lens elements
  2. Breaking the specimen coverglass
  3. Crushing or contaminating your specimen

For these reasons, 40x objectives should never be used with immersion oil. The risks far outweigh any potential gains in resolution.

Ways to Boost Resolution without Using Immersion Oil

Instead of using oil immersion, here are safer ways to improve resolution if a 40x objective doesn’t provide enough magnification for your needs:

Use a 60x-100x Objective

Higher power objectives like 60x or 100x are designed for oil immersion. They provide higher NA along with a larger working distance to prevent collisions between the lens and specimen.

60x and 100x objectives allow for higher total magnification with better resolution than a 40x can achieve. The tradeoff is a smaller viewing field.

Try Digital Zoom

Another option is to use the 40x dry objective normally, and then engage digital zoom functions in the microscope camera software.

This crops and enlarges the central region of the 40x image, simulating higher magnification digitally. The resolution stays the same, but fine details are enlarged.

Digital zoom avoids the risks of physically moving the 40x objective closer with oil. Just be aware that empty magnification is increased without added resolution.

Stick to Lower Magnifications

For many specimens, a 10x or 20x dry objective provides sufficient resolution to see the necessary fine details. Higher magnifications may not be required.

Try a lower power objective first before attempting 40x observation. The resolution may already be adequate for your needs without introducing immersion oil.

Key Takeaways

  • 40x objectives have an extremely short working distance not suitable for oil immersion.
  • Collisions can damage the lens or specimen when using 40x objectives with oil.
  • Improved resolution can be obtained through 60x+ oil objectives, digital zoom, or lower power dry objectives.
  • Avoid the risks and use 40x objectives dry only.


Why can’t you just be careful with the 40x oil objective?

Even with great care, the short working distance leaves little margin for error. Small variations in cover glass thickness or slight downward drift of the stage can cause a collision. It’s best to avoid the risk entirely by not using 40x objectives with oil.

Can’t you modify the 40x lens to increase the working distance?

The working distance is difficult to change without compromising the high NA required for 40x magnification. It would essentially require redesigning the entire objective. It’s better to select a different lens designed for oil immersion.

Does oil improve resolution with 40x objectives despite the risks?

In theory, yes – filling the gap with oil boosts NA slightly to improve resolution. But in practice, the gains are marginal and not worth the high risk of collisions and damage. Lower magnification with oil or a 60x+ oil objective are safer options.

Are there any specimens for which 40x oil is safe to use?

For extremely thin samples like single-celled organisms on bare slides, the risks may be reduced, but still present. It’s better to avoid 40x oil altogether and utilize the resolution-boosting techniques mentioned earlier. Even thin samples can drift into collision over time.