How oil immersion objectives can improve your microscopy?


How oil immersion objectives can improve your microscopy?

Oil Objective

proprietary automated oil immersion objective of Hermes high content screening system

Microscopic images of biological samples at high magnification are equally beautiful as they are informative, but getting great images is challenging. Are you getting your best possible images?

Read on to find out!

Sample preparation is, of course, the first critical step in any microscopy-based study. However, once the sample is ready, several considerations about the microscope must be taken into account to obtain high-quality images that will give credit to the hard work of all the experiments needed to get to that point.

One of the most important components of an optical microscope is, without doubt, the microscope objective. It is responsible for primary image formation and enlargement, and plays a crucial role in determining the quality of the images that the microscope produces. Objectives are also essential to determine the magnification of a particular specimen and the resolution under which fine specimen detail can be visualized, identified and measured.

Magnification of an objective refers to the amount or degree it enlarges an object’s image. Magnification is measured by multiples, such as 2x, 20x and 60x, indicating that the object you are observing is enlarged to twice as big, twenty-times larger or 60 times larger, respectively. A small area of the image is captured on the microscope’s camera and the resulting image is generated. But, a larger image does not directly lead to higher resolution within the image.

The resolution relates to the ability to separate nearby objects within an image. It is dictated, initially, by the characteristics of the objective. Some objectives require water or oil or silicon, while others can simply be in the air. Each type has benefits and drawbacks, but the highest resolution is achieved using oil objectives. Examples of experimentation relying on high-quality, high magnification images are:

  • Microbiology studies and single-cell organisms
  • Intracellular structures or targeted labelling, such as FISH (Flourescence In Situ Hybridization)
  • Imaging of foci, granules or spots
  • Adjacent structures that need to be accurately resolved (for example, focal adhesion)

In this post we discuss the advantages and the challenges of using immersion oil objective for high-resolution microscopy. We then describe how our new technology can revolutionize the use of oil in automated imaging systems to increase throughput for experiments requiring high-resolution imaging.

Why you need oil for high-resolution images?

The immersion medium is defined as what is between the top of the objective and the nearest surface of the sample, such as a coverslip. As listed above, the main types of immersion media are air, water, silicon and oil. Each objective has the specification of its required immersion medium marked on it, and they should never be mixed.

But why do you need an immersion medium such as oil?

Simply stated, the oil allows the objective to collect more light to form an image due to its refractive index. A material’s refractive index, n, is a measure of how much slower light travels within a medium relative to its speed in a vacuum. For instance, air has n = 1.0003, indicating the light experiences very little speed reduction. But oil and glass have n = 1.51, so light travels about 1.51-fold slower in these mediums.

Oil Objective Figure 1

Figure 1: Representation of the numerical aperture of an objective. Adapted from Olympus.

When traveling between materials having different refractive indices, the light path will bend in a process known as refraction. Reducing the amount of refraction will transmit more of the light from a sample through the very narrow diameter of the objective lens. And it is the refractive index of the immersion medium in between the objective and the sample that determines the amount of refraction through a quantity called the Numerical Aperture (NA) of the objective.

As the word ‘aperture’ suggests, the NA indicates the size of the area is over which light can be captured, as depicted in Figure 1, and larger NA objectives collect more light. Specifically, NA is calculated by the following equation (1):

NA = n × sin⁡(μ)

In the numerical aperture equation, n represents the refractive index of the medium between the objective front lens and the specimen, and µ (sometimes denoted as α) is the one-half angular aperture of the objective. Air objectives have a maximal theoretical NA of 1.0, but in practice, the highest NA is around 0.95. Oil objectives, on the other hand, can readily be made with NA > 1.3 and even up to 1.7.

Oil Objective Figure 2

Figure 2: Use of immersion media matched to the objective can minimize the refractive index differences between the objective and the sample. Image from Molecular Probes School of Fluorescence.

Therefore, the main purpose of using immersion media like oil, which has the same refractive index as glass, is to minimize light refraction by eliminating the refractive index differences that are present in the optical system. The medium increases the numerical aperture of the objective, indicating it gathers more light and can create brighter images. (Figure 2)

A higher NA afforded by immersion oil also increases the achievable resolution out of the objective. The Rayleigh Criterion describes how higher NA increases resolution, which is the point at which two points of light imaged by the microscope can just barely be separated from one another. The resolution of the microscope is given by this equation (2):

R = 0.61 λ NA = 0.61 λ (n × sin⁡(μ))

In this equation, R is the distance between two resolvable (i.e. identifiable) point sources of light, λ is the wavelength of the light, and NA is numerical aperture. Hence, larger NA values, arising from a larger index of refraction, achieve the desirable smaller values of R to better differentiate fine biological structure. The final resolution of the digital image generated also depends on other factors in addition to the objective, notably the camera used to create the image, but the equation for R is a good estimate for the best achievable resolution.

The effect of NA on resolution is clearly seen when calculating R for a range of values, as shown in this table, where smaller R values are desirable:

R [nm]800.6533.8427.0337.1266.9246.3228.8
λ = 525 nm

When using a lower magnification objective lens such as 4x, 10x and 20x, the light refraction phenomenon is often not too noticeable, and the images still have a good resolution with low NA air objectives. However, if you use a higher magnification lens, such as 60x or 100x, the light refraction when using an air lens can become quite noticeable, and the resolution of the image may be visibly lower. For this reason, most high-quality, high-magnification images are obtained using oil immersion objectives.

When to use oil immersion lenses?

Oil is frequently used with high magnification objectives. You should use an oil immersion lens when you have a specimen that is no thicker than a few tens to one hundred micrometres. Specifically, you should use it when the structures you wish to observe are quite small, perhaps one or two micrometres in dimension or smaller. For example, oil immersion dramatically enhances the visualization of focal adhesions (Figure 3) or bacteria (Figure 4). These instances highlight the significant resolution improvement afforded by the higher NA set by the oil. Most confocal microscopy will involve the use of oil immersion lenses to get those amazing images of cells that you see on the cover of scientific journals.

Oil Objective Figure 3

Figure 3
Co-labeling of focal adhesion markers vinculin or talin with HER2 on breast cancer cells overexpressing HER2. Widefield microscope, 63x/1.40 oil objective. Adapted from Weinberg F, Han MKL, Dahmke IN, Del Campo A, de Jonge N (2020) Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane. PLOS ONE 15(6): e0234430 |

Oil Objective Figure 4

Figure 4
Microbial aggregates on the leaf surface of Phyllosphere plants. Confocal microscopy, 100x/1.4 oil. Adapted from Elena L. Peredo and Sheri L. Simmons, Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere. Front. Microbiol., 09 January 2018 |

Oil Objective Figure 5

Figure 5Depth of field schematic to compare the angular aperture of high and low NA objectives. Intepreted from Nikon.

Oil immersion objectives are not ideal for imaging deep within a sample, or with thick samples. The large NA of these objectives, and correspondingly high angular aperture (μ in Equation 1 above) result in quite short working distances, which is the distance from the top lens of the objective to the closest surface of the sample. It is related to the depth of field, which is the axial (z-axis) thickness through which a sample appears sharp and in focus, as depicted in Figure 5. Therein we observe a shorter, more open cone in orange for the high NA objective and a longer, narrower cone for the low NA objective. For this reason, high NA objectives cannot reach their focus plane far into the sample because the objective physically collides with the surface of the sample.

For this reason, it is imperative that a coverslip or the bottom of a multiwell plate be thin when using high NA, high magnification objectives. Otherwise, it is possible that the biological sample of interest could be placed so far from the objective surface that the focal plane cannot reach it. Moreover, such objectives are most often designed to be used with samples having a thickness of approximately 170 microns, which is also termed a No. 1.5 coverslip. Fancy objectives have an adjustable correction collar to compensate for other substrate thicknesses, but only within a fairly narrow range, around 0.1 – 0.25 microns. Using this thickness for your samples will ensure you get the best high-resolution, high magnification image possible.

What to do after you have used an oil immersion lens?

Clean! This is the most important thing to do once you are done imaging your samples with an oil immersion lens. Oil distorts images seen with dry lenses. So, once you place oil on a slide, you must thoroughly clean the slide before using a dry lens (i.e. air objective) to image it again. Cleaning the oil also avoids contaminating the air objective with an improper, oil immersion medium.

After cleaning oil off of the sample, you should immediately proceed with cleaning the lens. Dried oil is tough to remove from the lens and is also sticky; it can easily trap dust, dirt, fingerprints and other contaminating particles that can compromise image quality, causing optical issues like shade or blurriness. In addition, the dried oil can deteriorate the lens over time. If you clean the immersion oil off your objective soon after using it, the oil will still be clean, wet and easy to remove, and you will avoid future problems.

To clean the lens, use special lens paper for cleaning optics. Fold to create a sharp corner without touching it, then wet it with an appropriate solvent (typically anhydrous alcohol, a commercially available lens cleaning solution, or blended alcohol). Then you should take your wetted lens paper and wipe the lens from the centre to the outside in a spiral pattern to move any dirt to the periphery.

Oil immersion objectives in automated microscopy? – Now you can!

Up until now, oil immersion objectives have been off-limits for automated microscopy, and for high-content screening in particular, because of the required user intervention to add oil while scanning a sample. Additionally, just the right amount of oil must remain between the objective and the sample when automatically scanning across it. Lastly, the index of refraction matching that enhances image resolution and brightness makes autofocusing more challenging.

We at IDEA Bio-Medical recognized these challenges to automated microscopy, but also the benefits that it could bring to the life-sciences research community. Relying on our decades-long experience in many fields of ODM & OEM engineering, we have created a unique and fully automated technology that precisely adds immersion oil only when needed. Such precision prevents excess oil from collecting and dripping from the bottom of a sample, as well as a lack of oil during a scan.

Our autofocus mechanism is fully compatible with immersion oil, enabling clear and robust imaging. And cleaning is a snap, thanks to our quick-snap objective exchange technology, making it easy to take objectives out of the microscope.

Now, for the first time, an high-content imaging system can utilize oil immersion objectives for automating the above-mentioned applications. Samples in a multi-well plate format can be scanned in their entirety, unsupervised and with no user intervention. The system can accommodate a variety of multi-well plates and sample formats and offers environmental control for live cell assays.

Utilizing high NA oil objectives also enables fully automated super-resolution imaging. Such images permit clear visualization of biological structure with separation smaller than R, the Rayleigh Criteria, in equation (2). But, that is a topic for another blog post.

Get to know IDEA Bio-Medical

Whether you are performing assay development, compound screens, transfection assays or looking at a few samples in great detail, and whether you are using 3D models (such as spheroids, Organoids), Zebrafish imaging, primary cells, fixed cells or live cell imaging, WiScan® Hermes is the solution for you. Designed for easy use by beginners and satisfying experienced microscopist, Hermes easily allows you to get high-quality images from your samples. The system is intuitively operated. Its built-in applications are extremely easy to use, and are operated at the push-of-a-button.

The advantages of the fully automated Hermes platform are:

  • Unique hardware automatically adds immersion oil to objectives
  • No user intervention; No oil spilling
  • Autonomous, rapid image acquisition for full-plate scanning and time-lapse imaging of live cells
  • Optimized autofocus, X, Y, Z motion and long-duration oil capsules for easy maintenance

Examples of applications where you can get higher resolution and brighter images with WiScan® Hermes are:

  • Super-resolution radial fluctuations (SRRF) live-cell imaging
  • Fluorescence In-Situ Hybridization (FISH)
  • Microbiology, Virology & Yeast
  • Spot / Foci / Granule visualization
  • Mitochondria, Endoplasmic Reticulum, Cytoskeleton & Focal Adhesion imaging

Please get in contact with us to learn how Hermes can help you see better.


Jason Otterstrom, PhD

Application Scientist

Dr. Otterstrom is an application scientist at IDEA Bio-Medical. He has a diverse background in biophysics including microscopy, optical design, image analysis and sample labeling. His expertise is adapting biological assays to benefit from utilization of automated microscopy methods.