Why is a coverslip important

As conditions depart from these designated specifications, spherical aberration and coma increase with the numerical aperture of the objective. This occurs because the difference between the tangent and the sine of the incident angle which is responsible for departure from the sine condition needed to correct for these aberrations becomes greater with longer numerical apertures. When using oil immersion objectives, it may appear that coverslip thickness is of only limited concern, because its refractive index approximately matches that of the immersion oil.

That is true when the specimen is mounted in Canada balsam or other mounting media with refractive indexes similar to that of the coverslip. However, it is no longer true when the specimen is mounted, for example, in physiological saline or other aqueous media whose refractive index is significantly different from that of the coverslip. In these conditions, even focusing through a thin layer of water only 10 microns thick can lead to significant aberrations and a point spread function PSF that is no longer symmetrical above and below the focal plane.

This is because oil immersion objectives are designed assuming that the specimen slide is homogeneously immersed.

Unless the specimen region is adjacent to the coverslip even in an aqueous environment , optical assumptions employed to calculate the aberration corrections are no longer valid.

Investigate how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for fluctuations in coverslip thickness. Once this characteristic behavior of oil immersion objectives was recognized, microscope manufacturers began to produce well-corrected water immersion objectives around Plan Apo water immersion objectives equipped with correction collars have a numerical aperture around 1.

The latest water immersion objective designs can now focus through approximately microns of aqueous media and still retain excellent correction. It is very common to have focus drift problems with plastic slides. If you do have this problem, put a glass slide against the bottom of the plastic slide to provide a rigid backing.

Plastic slides and dishes cannot be used with polarized light techniques. Some plastic slides cannot be used with phase contrast. An alternative is to grow cells on coverslips coated with a protein that the cells like to adhere to, such as polylysine, collagen or firbronectin or maybe a synthetic compound such as PDMS. Resolution is better with most of the lenses we have when sample is directly on coverslip. Especially for confocal microscopy, where three dimensional morphology should be preserved, it is important to not squeeze the sample when mounting it.

Cammer med. Top image: Confocal microcopy single optical section through the center of a mammalian cancer cell stained for f-actin. The box shows where the cell is resampled in the Z axis. The arrows show the view in the bottom image. Bottom image: View in Z axis of narrow slice marked above in a Z series. The cell is growing on and adherent to the coverslip.

Due to being mounted too tightly, the slide is pressing on the top of the cell making it flat and forcing some of the membrane at the apical surface to fold to fit against a flat surface. A: Diagram of the cell flattened at the top. B: Diagram of the cell as it shoudl appear in its natural unsquished state. Practical issues: 1. Coverslips are made of different quality glass. If imaging by SIM, recommend high precision coverglass click here for examples.

The additional 0. Due to the improved working distance in inverted microscope objectives lenses, they are not driven to maximum resolution NA. The inverted microscope was constructed for the improved handling freedom it provides with specimens and was not created for the evaluation of resolution limits. If you wish to use a 0. Of course if you are using a slide containing liquid, turning it upside down will not be beneficial.

Next time you are preparing samples for your microscope, take a look at the objective lens and ensure you are using the proper cover slip thickness for the lens. If you have questions regarding microscope cover slips and obtaining the best image with your objective lens, contact Microscope World and we will be happy to help. Microscope Cover Slips. What type of microscope are you using? Below is a list of a variety of microscopes and their use of cover slips: Stereo Microscopes - when using a stereo microscope you do not need to use a cover slip.

The sample sits directly on the microscope stage and is not typically placed on a microscope slide at all. Inverted Biological Microscopes - Petri dishes are used with inverted microscopes in order to contain living samples in liquid. Cover slips are not used with a Petri dish, but the thickness of the Petri dish can be important. We will talk more about this below. A prepared slide that is made up of a microscope slide, specimen and a cover slip not only gives the viewer better control over the specimen, but protects the microscope as well.

The cover slip protects the ocular lens from damage by acting as a barrier between it and the specimen. A glass slide is a thin, flat, rectangular piece of glass that is used as a platform for microscopic specimen observation. A typical glass slide usually measures 25 mm wide by 75 mm, or 1 inch by 3 inches long, and is designed to fit under the stage clips on a microscope stage. Once you have the object in focus, then switch to the next higher power objective. They also affect the image quality.

Coverslips function by working with your microscope to focus light to a single point and avoiding unnecessary noise in your image.

Wet-mount Slides A wet-mount slide is when the sample is placed on the slide with a drop of water and covered with a coverslip, which holds it in place through surface tension. Advantages — This type of slide preparation allows you to view microscopic living things without them drying out.

Why is it important to put a coverslip over the drop of water when you prepare a wet mount? The water also helps the light to pass through the specimen more evenly. To protect the specimen in the water drops on the slide, you must cover it with a very thin piece of glass called a coverslip. Wet Mount. In a wet mount, a drop of water is used to suspend the specimen between the slide and cover slip.