One of the main problems in light microscopy is to overcome some of the limits of optical resolution and to increase the NA of the system. In brief, the NA of an objective is the ability to gather light from a specimen whereas resolution is the ability of an objective to distinguish details in the specimen. Resolution and NA will be covered in other articles , but we will now examine immersion techniques available to microscopists which allow the imaging of specimens at high magnification whilst overcoming some of the limits of resolution.
Having an immersion liquid in place of the air gap between the front lens of an objective and the cover glass of a specimen increases the resolution of an objective. When light passes from one medium to another for example, through glass to air it refracts - in other words, it bends and scatters. Any light rays which are refracted into the air, reflected by the cover glass or actually blocked by the metal housing of the objective front lens do not contribute to the image formation.
The purpose of the immersion liquid is to decrease the amount of refraction and reflection of light from the specimen and increase the ability of the objective to capture this otherwise deviated light s. Figure 1. The physical properties of the medium through which light rays travel determines the degree to which the light will be refracted. Air has a refractive index of 1. Taking this difference into account, the purpose of the immersion liquid is to match as closely as possible the refractive index of the glass in which the specimen is mounted, therefore increasing the amount of light rays which will form the final image.
Subsequently, most immersion oils have a refractive index of 1. For common refractive indices, see Table 1. Using this system, it is possible to achieve the maximum resolution and NA. Figure 2 and Table 1.
Placing immersion liquid on the lens of the condenser is usually not necessary. If the microscope is correctly set up and aligned to achieve optimal contrast and illumination across the specimen see the article on Koehler Illumination , then the position and settings of the condenser will be optimised so as to contribute to the overall NA of the microscope system. This is simply the actual distance between the objective front lens and the surface of the cover glass when the specimen is in sharp focus s.
Figure 3. When the objective is moved to be closer to the slide, the focal plane moves further into the specimen. However, this is physically limited by the fact that the objective can only be moved until it is in contact with the cover glass. There is an inverse relationship between working distances and the magnification of each objective. For example, a 10x objective may have a working distance of 4 mm, whereas the working distance of a x oil objective will typically be in the region of mm.
A crucial factor to remember when using oil objectives is to use the correctly matched immersion oil. Only use oil which is recommended by the objective manufacturer. For many years, cedar wood oil was routinely used for immersion and is still commercially available.
Although this oil has a refractive index of 1. When planning and carrying out fluorescence microscopy, you should use a non-fluorescent oil.
Finally, no matter what imaging you are carrying out, you should always use an oil that is recommended by the manufacturer of your microscope and objectives.
Hopefully that helps and you now know why and how to use an immersion objective correctly. Have any tips on using an immersion objective? Leave them in the comments section below! Has this helped you? Then please share with your network. You must be logged in to post a comment. This site uses Akismet to reduce spam. Learn how your comment data is processed. Facebook Twitter LinkedIn More. Type F is designed to ensure the highest image quality for fluorescence applications, with maximal signal-to-noise ratio and minimal autofluorescence.
Silicone immersion oil is used for deep observations of live specimens. Silicone oil is available as a 30ml bottle glass rod is included for dispensing. Download KB. Objective Selector. Oil immersion should be used between the slide and x objective lens, this is a special oil that has the same refractive index as glass. Even when employed properly, immersion oil must be removed immediately after use to prevent its accumulation in unwanted areas of the microscope, as well as to avoid optical degradation from dried oil residue on the objective.
The disadvantageous properties of cedarwood oil are: high absorption of blue and UV light, yellowing with age, a tendency to harden on lenses due to uneven volatility, acidity, and changing viscosity diluting with solvent changes the index and dispersion. What would you observe if you forgot to use oil with the oil-immersion lens? The purpose of the oil is to contain the light rays and direct them upwards.
If oil was not used, the rays would scatter, and the image would also be less magnified and lower clarity. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. An eyepiece reticle is a small piece of glass with a ruler or grid imposed on it that fits into the microscope eyepiece. When looking through the microscope, the reticle image is imposed upon your specimen image.
Most often the reticle is used to make measurements or count particles.
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