You should try protocol for preparation of suspended cells for SEM. From basic protocol for biological specimens it differs mostly by attaching cells to filter or by making pellet by centrifuging.

Basic protocol consists of fixation, dehydration in graded solutions of alcohol (30%-100%), with final drying by CPD, or, if you do not have a CPD unit, you can use HMDS instead. Final operation – coating with Au-Pd, Pt, Au, or C to make specimens conductive. Thorough specimen preparation should give you nice specimen. Specimen damage by vacuum and electron beam at magnifications below 5,000 is usually negligible.

You can find a lot of detailed protocols on the Web.

Since this is biological sample, you would have to prepare it so that it will be sustainable and stable in high vacuum. Samples containing a lot of moisture are usually problematic for FE-SEM, but there's a way to do it.

To remove the moisture from the sample, first you would have to dry the sample in vacuum, but that might influence the microstructure of your sample. Critical point drying would be the best method for retaining the fine microstructure of your samples.

Afterwards, it would be a good idea to cover it with fine thin layer of gold (few nanometers) to protect your sample, increase the conductivity and give you a nice contrast.

You can read commets about the critical point drying method at the following topic, to get more information.

https://www.researchgate.net/post/Is_there_any_method_to_view_cellulose_dispersion_by_using_field-emission_scanning_electron_microscope_FESEM

@ Marijan Marcucius but sir my question is that how to prepare the sample for critical point drying ?? as i know that in critical point drying sample are dehydrated with the ethanol ,which is distort the algal cell membrane structure, so sir please suggest me some simple protocol .

Critical point drying is actually a method specifically being used to preserve the structure of biological samples, while the simple drying method does not. Don't forget also that the substitution is being performed by CO2 at very high pressure, but how it actually operates you can read about it from my comment in the attached link above. Well, if you are aiming to see characteristic elements of cells still intact maybe you should consider other analysis methods like TEM or AFM, or confocal microscopy. One thing is for sure; electron beam from the microscope will damage the algae cell membranes significantly more with the high electron beam than the critical point drying method and gold sputtering which is performed in high vacuum as well. In your case I would give it a try and see what comes out.

Other option is to use the low vacuum environmental SEM which doesn't require so much sample preparation and operates at significantly lower vacuum than the typical high vacuum SEM.

Either way don't expect to get your samples intact after analysis.

You should try protocol for preparation of suspended cells for SEM. From basic protocol for biological specimens it differs mostly by attaching cells to filter or by making pellet by centrifuging.

Basic protocol consists of fixation, dehydration in graded solutions of alcohol (30%-100%), with final drying by CPD, or, if you do not have a CPD unit, you can use HMDS instead. Final operation – coating with Au-Pd, Pt, Au, or C to make specimens conductive. Thorough specimen preparation should give you nice specimen. Specimen damage by vacuum and electron beam at magnifications below 5,000 is usually negligible.

You can find a lot of detailed protocols on the Web.

@Vladimir & Marijan thanks for your nice suggestion but if you have refrence ,please send me the link

Today I have received new issue of “Microscopy today” with discussion on your subject.

Please, take a look here:

http://www.microscopy-today.com/jsp/search/groupsearch.jsf

Find “Specimen Preparation: algal cells for SEM”

Hope it helps.

Hi, I hope this might help you. Its general but can give you the main information. You will need to practice a bit as well

1. Carefully read and understand the maker’s instructions. Make

sure all the valves and other connection are secure. Make sure

the O-rings on the door to the CPD are undamaged and fully

functional. Make sure that the cylinder of liquid carbon dioxide

is fitted with a siphon tube with an opening at the bottom of

the inside of the cylinder to ensure delivery of liquid carbon

dioxide.

2. Make sure that the inside of the CPD is dry and at room

temperature (298 K). Check that it is connected to a cylinder

of liquid carbon dioxide (CO 2 ); to the cooling tap water; the

electrical power and that all the valves and taps are closed.

3. The dehydrated specimens that should be immersed in anhydrous

ethanol (or methanol or acetone) are quickly transferred

to the CPD sample holder, which is also filled with dry ethanol.

The loaded specimen holder quickly placed into the CPD

chamber and the door firmly and securely closed.

4. Cool the now loaded and sealed CPD chamber to 293 K. Open

the valve to the cylinder of liquid CO 2 and ensure that the

pressure is 5.53 MPa (800 psi). Slowly open the inlet valve to

the CPD chamber and fill it nearly to the top with liquid CO 2 .

The chamber pressure will rise to 5.53 MPa and the liquid–

gas meniscus of the ethanol–carbon dioxide mixture will rise

inside the chamber. This process may be observed in systems

fitted with viewing port. The inlet valve should remain open.

5. Allow the mixture to stand for a few minutes and then gently

open the vent valve to the chamber to allow fresh liquid CO 2 to

flush through the sample chamber. It is most important that

the samples remain below the meniscus to prevent exposure

to the gas phase of the CO 2 . Continue intermittent flushing

for 10–15 min to ensure the ethanol in the specimen is fully

substituted with liquid CO 2 .

6. Close both the inlet and vent valves and leave the sample to

fully infiltrate with the liquid CO 2 . This will take about an

hour with a 1-mm 3 sample, but the time will need adjusting

depending on the size, density, and permeability of the specimen.

During the infiltration stage periodically check that the

sample remains below the surface of the liquid CO 2 .

7. Re-open t he inlet valve and fill the chamber with a final flush

of fresh liquid CO 2 and slowly adjust the vent valve to ensure

the chamber fills to about 75% of its capacity. Close both the

inlet and outlet valves to the chamber and the valve on the CO 2

cylinder.

8. Turn off the cooling water and slowly heat the chamber to

308 K. The pressure will rise and the CO 2 will pass through

its critical point of 7.39 MPa and eventually reach a pressure

of 8.27 MPa (1,200 psi). This process may be observed

through the chamber window and the liquid meniscus

disappears as the CO 2 passes through the critical point.

9. Maintain the chamber temperature and very slowly open the

vent valve so that the now gaseous CO 2 from the chamber is

released. If the venting is too fast, the gas phase will momentarily

revert to the liquid phase, which will wet and damage

the specimen.

10. Once the chamber has reached atmospheric pressure, quickly

remove the now very dry specimens and immediately transfer

them to a sealed container placed in a desiccator. This transfer

should be done as quickly as possible because the now anhydrous

sample can quickly absorb atmospheric water.

11. Ensure the door to the chamber is slightly open, and then

slowly open the CO 2 inlet valve to the chamber to allow the

residual CO 2 in the line from the CO 2 cylinder to be vented.

Turn off the heater and once the chamber has reached room

temperature, replace the chamber door to ensure the inside

of the chamber remains dry.

I have been using CPD for biological samples - and if you follow all the instructions (and make the dehydration process properly), it will be ok. But as mentioned before, you will need to practice a bit. Good luck!

How do you know how long to fix the algae sample for? Some literature says 1-2 hours while some say 24-48 hours. 

Hi, did you manage to image them? If so, how did they turn out and do you have a protocol you could share please?