High magnification microscopic images of biological samples are both attractive and educational, but obtaining excellent images is difficult.
Of course, the preparation of the samples is the first and most important step in any study using microscopy. Once the sample is prepared, however, there are a number of microscope considerations that must be taken into account to produce high-quality images that do justice to the labor-intensive nature of all the experiments required to get there.
Proprietary automated oil immersion objective of Hermes high content screening system. Image Credit: IDEA Bio-Medical Ltd.
The microscope objective is without a doubt one of an optical microscope’s most crucial parts. It is in charge of creating and enlarging primary images, and it is instrumental in determining the caliber of the images that the microscope generates. To determine the magnification of a specific specimen and the resolution at which fine specimen detail can be seen, recognized, and measured, objectives are also necessary.
An objective’s magnification is defined as how large or how much it enlarges the image of an object. Multiples, such as 2×, 20×, and 60×, indicate an object has been magnified to a size that is twice as big, 20 times larger, or 60 times larger, respectively. The image is created after a portion of the image is captured on the microscope’s camera. However, a larger image does not necessarily mean that the image has a higher resolution.
The ability to distinguish nearby objects within an image is related to the resolution. It is initially determined by the attributes of the objective. Some goals need silicon, water, oil, or any of these, while others only need air. Although each type has advantages and disadvantages, oil objectives provide the best resolution. Examples of research depending on excellent, magnified images include:
- Research into microbiology and single-cell organisms
- Imaging of foci, granules, or spots
- Intracellular structures or targeted labeling, like FISH (Fluorescence In Situ Hybridization).
- Neighboring structures that require accurate resolution (for example, focal adhesion)
The article will provide details on the benefits and difficulties of using an immersion oil objective for high-resolution microscopy. Then, in order to increase throughput for experiments requiring high-resolution imaging, it will explain how the new technology can revolutionize the use of oil in automated imaging systems.
Why Oil is Needed For High-Resolution Images
What lies between the top of the objective and the sample’s closest surface is known as the immersion medium, and examples include a coverslip. Air, water, silicon, and oil are the four main categories of immersion media as listed above. The required immersion medium is specified on each objective, and the two should never be combined.
Simply put, the oil’s refractive index enables the objective to gather more light for the formation of an image. The refractive index, or n, of a substance expresses how much slower light moves through that material than it does in a vacuum. For instance, the speed reduction of light in the air is very small (n = 1.0003). However, because oil and glass have n = 1.51, light moves through them 1.51 times more slowly.
Refraction is the bending of light when it passes through two materials with different refractive indices. More light from a sample will pass through the objective lens’s extremely small diameter if the amount of refraction is reduced. And the amount of refraction through an attribute known as the objective’s numerical aperture (NA) is determined by the refractive index of the immersion medium positioned between the objective and the sample.
The NA, or the “aperture,” denotes the size of the area over which light can be captured, as shown in Figure 1. Larger NA objectives gather more light. To be more precise, NA is determined by equation (1):
NA = n × sin(μ)
Figure 1. Representation of the numerical aperture of an objective. Image Credit: Adapted from Olympus
In the numerical aperture equation, n represents the refractive index of the medium between the objective front lens and the specimen, and µ (at times indicated 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.
Thus, the primary goal of using immersion media with the same refractive index as glass, such as oil, is to reduce light refraction by removing refractive index differences from the optical system. The medium makes the objective’s numerical aperture larger, which means it can capture more light and produce brighter images (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 Credit: Molecular Probes School of Fluorescence
The achievable resolution out of the objective is also increased by a higher NA provided by immersion oil. Higher NA increases resolution, which is the point at which two light points imaged by the microscope can just barely be distinguished from one another, according to the Rayleigh criterion. This equation below (2) provides the microscope’s resolution:
R = 0.61 λ ⁄ NA = 0.61 λ ⁄ (n × sin(μ))
In this equation, R stands for the separation between two point sources of light that can be distinguished or resolved, λ is the light’s wavelength and NA is the numerical aperture. To achieve the desired smaller values of R and better distinguish fine biological structure, larger NA values, which result from a larger index of refraction, are achieved.
The equation for R is a good approximation for the best achievable resolution. The final resolution of the digital image created also depends on numerous factors in addition to the objective, particularly the camera used to create the image.
When quantifying R for a range of values, as illustrated in the table, where smaller R values are preferred, the effect of NA on the resolution is clearly visible.
Source: IDEA Bio-Medical Ltd.
|
NA |
0.4 |
0.6 |
0.75 |
0.95 |
1.2 |
1.3 |
1.4 |
R [nm] |
800.6 |
533.8 |
427.0 |
337.1 |
266.9 |
246.3 |
228.8 |
λ = 525 nm |
800.6 |
|
|
|
|
|
|
The light refraction phenomenon is frequently not too noticeable when using lower magnification objective lenses like 4×, 10×, and 20×, and the images still have good resolution with low NA air objectives.
The light refraction when using an air lens, however, can become quite noticeable if you use a higher magnification lens, such as a 60× or 100×, and the resolution of the image may be visibly lower. For this reason, oil immersion objectives are used to produce the majority of high-quality, high-magnification images.
When to Use Oil Immersion Lenses
When using high magnification objectives, oil is frequently used. When there is a sample that is only a few tens to a hundred micrometer thick, an oil immersion lens is used. It is used specifically when the structures that need to be observed are very small, possibly 1 or 2 µm in size or smaller. For instance, immersion in oil significantly improves the ability to see focal adhesions (Figure 3) or bacteria (Figure 4).
Figure 3. Oil Vs. Air objectives comparison: images of Yeast cells acquired on Hermes imaging system with 60X/0.9NA air objective Vs. 60X/1.42NA oil objective. Green/Red channels same contrast levels; Acquisition: Same illumination & camera gain.
Figure 4. Microbial aggregates on the leaf surface of Phyllosphere plants. Confocal microscopy, 100x/1.4 oil. Image Credit: Adapted from Elena L. Peredo and Sheri L. Simmons, Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere. Front. Microbiol., 09 January 2018. doi.org/10.3389/fmicb.2017.02669
These examples demonstrate the notable resolution improvement made possible by the higher NA set by the oil. To obtain the stunning images of cells that are seen on the covers of scientific journals, the majority of confocal microscopy will involve the use of oil immersion lenses.
For imaging deeply within a sample or with thick samples, oil immersion objectives are not the best option. The distance between the top lens of the objective and the closest surface of the sample is called the “working distance” and is determined by the large NA of these objectives and the correspondingly high angular aperture (μ in Equation 1 above).
It has to do with the depth of field, which is shown in Figure 5 as the axial (z-axis) thickness through which a sample appears sharp and in focus. In this, an orange cone that is shorter and more open for the high NA objective is seen, and it is longer and narrower for the low NA objective. As a result of the objective’s physical collision with the sample’s surface, high NA objectives are unable to penetrate deeply enough into the sample to reach their focus plane.
Figure 5. Depth of field schematic to compare the angular aperture of high and low NA objectives. Intepreted from Nikon. Image Credit: IDEA Bio-Medical Ltd.
Therefore, when using high NA and high magnification objectives, it is essential that a coverslip or the bottom of a multiwell plate be thin. If not, it is possible that the biological sample of interest will be positioned too far from the objective surface to be visible from the focal plane. Additionally, these objectives are typically made to be used with samples that have a thickness of about 170 μm, also known as a No. 1.5 coverslip.
To account for different substrate thicknesses, fancy objectives have an adjustable correction collar, but only up to a relatively small range, roughly 0.1 to 0.25 μm. The samples will have the best high-resolution, high-magnification image possible if this thickness is used.
What to Do After Using an Oil Immersion Lens?
Cleaning is the most crucial action to take after imaging samples with an oil immersion lens. Images viewed with dry lenses are distorted by oil. So, after applying oil to a slide, it must be thoroughly cleaned before imaging it with a dry lens (an air objective). Cleaning the oil also prevents the air objective from becoming contaminated by an ineffective oil immersion medium.
The lens should be cleaned as soon as the sample has been cleaned of oil. Dried oil can easily trap dirt, dust, fingerprints, and other contaminating particles that can degrade image quality and result in optical problems like haziness or blurriness. It is difficult to remove dried oil from lenses and it is sticky.
Additionally, the lens may eventually become damaged by the dried oil. Future issues can be prevented if the immersion oil is cleaned off from the objective right away after using it. The oil will still be clean, wet, and easy to remove.
Use specialized lens cleaning paper for optics to clean the lens. Without touching it, fold it to form a sharp corner, and then wet it with the proper solvent (typically anhydrous alcohol, a commercially available lens cleaning solution, or blended alcohol). Then, using a wet lens paper, wipe the lens in a spiral motion from the center to the edge to push any dirt to the edges.
Oil Immersion Objectives in Automated Microscopy
Until now, oil immersion objectives have been off-limits for automated microscopy, and for high-content screening in particular, because of the user intervention that is required to add oil while scanning a sample.
Furthermore, when automatically scanning across the sample, precisely the right amount of oil must stay in the space between the objective and the sample. Last but not least, autofocusing becomes more difficult due to the index of refraction matching that improves image resolution and brightness.
IDEA Bio-Medical was aware of the drawbacks of automated microscopy as well as its potential advantages for the field of life sciences research. The company has developed a special, completely automated technology that accurately adds immersion oil only when necessary, drawing on decades of experience in many areas of ODM & OEM engineering.
With this level of accuracy, neither too much nor too little oil will accumulate at the bottom of a sample or leak out during a scan.
Clear and reliable imaging is made possible by the autofocus mechanism’s complete compatibility with immersion oil. Additionally, the quick-snap objective exchange technology makes it simple to remove objectives from the microscope, making cleaning a breeze.
The aforementioned applications can now be automated for the first time using oil immersion objectives with a high-content imaging system. Samples in a multi-well plate format can be completely scanned without supervision or user input. The system provides environmental control for live cell assays and is compatible with a variety of multi-well plates and sample formats.
Super-resolution imaging can also be fully automated when using high NA oil objectives. With the help of such images, the biological structure can be clearly seen with separations smaller than R, the Rayleigh Criteria, in equation (2).
Get To Know Idea Bio-Medical
Whether the user is using 3D models (like spheroids, organoids), Zebrafish imaging, primary cells, fixed cells, or live cell imaging, WiScan® Hermes is the right choice to conduct assay development, transfection assays, compound screens, or examine a few samples in great detail.
Hermes makes it simple to obtain high-quality images from the samples and is designed for both novice and expert microscopists. The system can be used easily; a single push of a button enables the use of any of its built-in applications, which are very simple to use.
The fully automated Hermes platform has the following benefits:
- Unique hardware adds immersion oil to objectives automatically
- No user intervention; no oil spilling
- Independent, fast image acquisition for full-plate scanning and time-lapse imaging of live cells
- Optimized autofocus, X, Y, and Z motion, and long-duration oil capsules for easy maintenance.
WiScan® Hermes can be used for a variety of applications that require brighter, higher resolution images, such as:
- Fluorescence In-Situ Hybridization (FISH)
- Super-Resolution Radial Fluctuations (SRRF) live-cell imaging
- Yeast, Virology, and Microbiology
- Granule, Spot, and Foci visualization
- Imaging of the cytoskeleton, mitochondria, endoplasmic reticulum, and focal adhesion
About IDEA Bio-Medical Ltd.
IDEA Bio-Medical is founded in 2007 through a partnership between YEDA (the Weizmann Institute’s commercialization arm) and IDEA Machine Development (an innovation hub).
We specialize in automated imaging systems and image analysis software, offering a broad range of biological applications based on the company’s unique algorithms library. The company is developing novel image-based screening platforms for the pharmaceutical industry and medical centers, dedicated to broadening the scope of personalized medicine.
Our WiScan Hermes system incorporates the most advanced technologies currently available in the machine vision field, integrated with engineering methodologies of high reliability and quality at the level of semi-conductors and digital printing industries, which are the specialty of our mother company, IDEA Machine Development Design and Production Ltd.
Sponsored Content Policy: AZO Life Sciences publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of AZO Life Sciences which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.