Three things simultaneously were bad: focusing, coating, specimen preparation. And things you are focusing on do not look like erythrocytes. Do you have a control specimen with not damaged erythrocytes? 

 hello:

i think you should run a sample of normal red blood cells using the technique you are using and if you get bad results then as the previous colleague mentioned it could be your prep or your  focusing or coating , or something with your machine. 

i have taken my own images of RBC and cells together and the RBC's (lettr R) looks normal see fig A and i also included an image of RBC taken from a  colleague fig B and as you can see they both look like a normal RBC 

cheers

Prof K.Galil

Hello Al,

please, more details on sample preparation and SEM setting are needed as Vladimir mentioned.

But here are some hints:

1. Check your erythrocytes isolate (standard) with optical microscope for quality.

2. Fix the standard erythrocytes with buffered glutaraldehyde, intensively wash the fixed erythrocytes, mount them onto polylysine treated SPI filters of glass cover-slips by sedimentation in a wet-chamber (overnight at 4 oC). Dehydrate the samples in alcohol series and perform critical-point drying. Sputter-coat the dried samples with gold.

3. Check the performance of scanning electron microscope (astigmatisms adjustment, etc.) or ask SEM engineer for it.

4. Try to get some images of your standard erythrocytes. These should resemble to images posted by K.A. Galil.

5. If you get those images, then you can try to do SEM analysis of toxin treated erythrocytes.

Some other suggestions:

a. Perform toxin treatment of the erythrocytes and control the process under phase-contrast optical microscope.

b. After that, you should be able to specify the time table for stopping the toxin treatment process.

c. At specific times, fix the toxin treated erythrocytes with buffered glutaraldehyde and process for SEM as mentioned above.

Good luck

Oldrich

P.S.

Some time ago, we publish a paper on adenylate-cyclase toxin and erythrocytes.

DOI: 10.1002/jemt.20277

It is obvious you have the artifacts produced by CHARGING.

When that happens you have deformation of the shapes, impossibility to focus and to find correct brightness /contrast conditions.

Charging occurs when a non-conductive specimen is exposed to the electron beam, or when a conductive specimen is not correctly connected to earth.

Either your carrier is insulating, or it is not properly connected, or the specimen has not been properly coated with a conductive layer, or all of them.