Coverslips, chambers and working distance.

Microscopy imaging with an objective lens from 1X through 40X at lower resolutions is extremely flexible and can be performed on a very wide variety of samples. However, imaging at higher resolutions, from 40X through 100+X objectives, must be imaged through a very thin layer of glass no thicker than a standard coverslip. For imaging of living samples, special chambers must be used.

Every microscope lens is designed for imaging through a specific type of coverslip, chamber, or at a sample directly. It is important to use the correct coverslip or chamber with the lens you are using to get the best possible image.

A common mistake in biological microscopy is using the wrong coverslip. In general, you should always use a 1.5 coverslip. All the high resolution (a.k.a. high numerical aperture) objective lenses we use are corrected for #1.5 coverslips.

Again, don't use 0, 1, or 2. Use 1.5. If you have #1 coverslips, just throw them out and buy #1.5 instead. Really.

For TIRF (Total Internal Reflectance Microscopy) the material must be glass and the mounting media must be aqueous.

I like 18 mm or 22 mm square coverslips because they are easy to handle. Unless you are using extremely small amounts of precious reagents, I advise staying away from the tiny round coverslips, but this merely a matter of your comfort; as long as they are #1.5 the image quality will be the same.

Imaging cells at magnifications up to a 40X objective lens is routine for samples prepared in many different ways. With dry (air) objectives at lower magnifications or with LWD (long working distance) objectives, imaging is possible in most chambers:

Many of the LWD lenses have adjustable collars to optimize the imaging quality for chambers composed of different thicknesses and materials.

High resolution imaging, meaning imaging with a 40X, 60X or 100X oil immersion objective at a numerical aperture greater than 1.0, must be performed through a number 1 1/2 coverslip. For optimal imaging, the cells or tissue should be very flat and sandwiched between a standard glass slide and the coverslip as illustrated.  For oil immersion objectives, the coverslip must be fixed in place either by a mounting medium that polymerizes or by nail polish or glue around the edges.

This method of sample preparation is useful only for fixed and stained material or for some limited applications where the space between the coverslip and slide may be an open chamber. For instance, some people use a small diamond drill bit to make holes inthe slide which may be used for fluid exchange.

A better method for live cell imaging is to grow the cells in special chambers. These chambers have a coverslip at the bottom:


Other examples are:

 

The following links are example products. (Dislclaimer: we have no commerical interest in any of these and there may be other solutions not listed here.)

Remember, #1.5 coverglass.

Fisher now (as of 2017) sells dishes & chambers by Greiner such as Bio-One CELLview™ Sterile Cell Culture Dishes With Glass Bottom and Cellview slides which really shouldn't be named "slide" since they have coverglass bottoms.

MatTek glass bottom dishes

Ibidi glass bottom dishes and plastic bottom dishes that also work with our lenses (but not for TIRF or Airyscan)

Chambered Coverglass System 1.5 Borosilicate Glass - ThermoFisher N.b. The wells at the end may not be fully accessible to the microscope lens.

Nunc® Lab-Tek® II chambered coverglass, 8 wells, polystyrene chambers, 1.5 borosilicate coverglass, 0.7 cm2/well N.b. The wells at the end may not be fully accessible to the microscope lens.

Bioptechs has a number of solutions (but have to be careful of metal rings that block lens access).

(last updated 20170407)

 


Mounting cells grown on filters

It's a tough balance between squishing the cells and having too much space.

The filter must be sandwiched bewteen the slide and the coverslip with the cells on the coverslip side.  One way to play this safe is to sandwich the filter between two 22 X 60 mm coverslips so that the cells may be imaged from either side with a high numerical aperture objective.

The filter needs to be squished enough to lie flat and to present the cells near the coverslip but not be squished so much that the cells are too flat.


Other Problems to Avoid

Plastic slides should be avoided.  It is extremely difficult to mount glass coverslips on flexible plastic slides.  For proper imaging the clear surface must be flat and uniform; even with aqueous mounting media, plastic slides tend to curve.  Plastic slides warp with non-aqueous mounting media as the media is absorbed by the slide. 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 such as cross polarization or Nomarki/DIC.  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.

Do Not Squish Samples When Mounting.  Especially for confocal microscopy, where three dimensional morphology should be preserved, it is important to not squeeze the sample when mounting it. 

MTLn3_flattop.jpg (17569 bytes)
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.
MTLn3_flattopdiagram.jpg (2721 bytes)
A:  Diagram of the cell flattened at the top.
B:  Diagram of the cell as it shoudl appear in its natural unsquished state.

 

A checklist for slides:

  • #1.5 coverslip
  • Clean coverslips.
      Dry.
      No salt crystals.
      No oil from another microscope.
  • If liquid media, sealed neatly.
      Coverslips immobile.
      No leaking mounting media.

 

 

 


 

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This web page was modified from http://cammer.net/historical/aif/instructions/coverslips/coverslips.htm

comments, questions, suggestions: Michael.Cammer@med.nyu.edu
The author of this webpage has no financial interest in any of the products mentioned on this page.