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Understanding Your Objective
Match your objective to your imaging task
A microscope objective is composed of a complex set of lenses and optics, and different objectives are designed for different imaging tasks. Capturing good images relies on choosing the correct objective.
Learn the correct magnification for your experiment and how to tell if your objective can be used with air, oil, or other immersion media.
Understanding your objective
The objective is an essential part of the microscope and can greatly influence image quality. Objectives come with lots of information written on them, and most of it is written in code. But don’t worry; it’s easy to decipher.
Figure 1. Common notations found on objectives and what they mean.
Selecting the right magnification
Magnification tells you the optical magnification the objective provides
Magnification tells you the optical magnification the objective provides. The magnification you choose depends on what you want to see. The usefulness of magnification will be limited by your resolution. Making a big fuzzy blob of light even bigger won’t give you a better picture. Provided you have similar resolution at different magnifications, using higher magnification will allow you to see smaller things (such as organelles inside a cell) better. On the other hand, using a lower magnification will give you a better image of the big picture—such as a field of cells or interactions between cells.
Figure 2. Same field of cells captured at different magnifications. Each magnification can offer different information, and the best choice for your experiment will vary depending on what you want to know.
Matching immersion medium to objective
The Immersion medium is what's between the objective and the coverslip (or the bottom of the dish or flask that holds your sample).
Each objective is designed for a specific immersion medium, which is marked on the objective. The main types of immersion media are air, oil, and water. It is important that you never put air objectives in oil or other liquids. Doing this will make the person in charge of the microscope really angry! The main purpose of using different types of immersion media is to minimize the refractive index differences that are present in the space between the objective and the sample. This includes the substrate (i.e., glass coverslip) that the sample is on and the imaging medium (i.e., buffer) that the sample is in. Minimizing this difference will result in better image resolution.
Figure 3. Use of immersion media matched to the objective can minimize the refractive index differences between the objective and the sample.
Refractive index
Light will travel through different types of materials at different rates. When light travels through one material (such as air) and into another (such as water), the light is refracted. It appears bent. For instance, when you put a pencil in a glass of water and view the glass from the side, the pencil will look bent. This is because air has a different refractive index than water.
Numerical aperture and the refractive index of immersion medium
Numerical aperture is a property of the objective that indicates how good the resolution can be in the image you collect (basically how much fine detail you can see).
Lots of times, you will hear people talk about the “NA” of an objective. “NA” stands for numerical aperture and its value partly depends on the refractive index of the material that is between the objective and the glass coverslip that your sample is on. In general, objectives with higher NA give you better resolution. Higher NA objectives often have higher magnification and use some sort of immersion medium. Immersion medium is used to alter the refractive index of the space between the objective and glass coverslip so that it is closer to the refractive index of the glass coverslip itself. This minimizes refraction and loss of light, ultimately giving you a better image.
Figure 4. The pencil appears bent or broken because the refractive indexes of water and glass are different than that of air.
Indicates how many corrective lenses the objective contains for various types of aberrations which can help with resolution. Corrective lenses can help correct things like the way the light is bent as it goes through the objective so that the edges of your field are as crisp as the middle, or they may correct for the fact that different wavelengths of light will behave differently as they pass through the lenses in the objective.
Samples are often mounted on glass coverslips. The most common glass coverslip is named #1.5 and has a 0.17 mm thickness. The number for the coverglass thickness that is printed on the objective tells you the optimal thickness of glass coverslip (or any other type of substrate your sample is on, such as a plastic-bottom dish) the objective is manufactured for.
Some objectives have a long working distance, meaning that the objective can give you a good image over a large range of coverglass thicknesses. This is useful if you want to image through thicker vessels such as plastic-bottom dishes and T-75 flasks, which have thicker walls than glass bottom vessels. To give the best imaging results, the lenses in the long working distance objective are manufactured to accommodate differences in thicknesses.
Some objectives have a manual correction collar. You can turn the correction collar to a specified number that indicates the thickness of the coverslip your sample is on. Not all long–working distance objectives have correction collars, though.
The working distance is the distance between the objective and the cover glass, or between the objective and the top (or bottom) of whatever vessel you are imaging through, when your sample is in focus. When you are imaging through something thin, like a cover glass, you can use objectives with shorter working distances. But when you are imaging samples that are in a thicker vessel, such as a plastic plate or dish, you will probably need an objective that has a longer working distance. The working distance of an objective is often written on the objective. The working distance of the objective in this example is 7.4 mm. It is considered to have an ‘extra-long working distance’ and is abbreviated as ELWD on the objective.
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