This document gives an overview of the basic operation of the scanning electron microscopy (SEM) and in particular, the Cryo-SEM (cryogenic scanning electron microscope). The SEM we use is the JEOL JSM-6400, which is in the MicroLab, Department of Chemical Engg., IIT-B, Powai, Mumbai - 400 076. All users of this microscope are required to know the information given in this document. Your comments are welcome. For further information, please contact Prof. Jayesh Bellare. Email: jb@iitb.ac.in

BRIEF DESCRIPTION OF APPARATUS

The JSM-6400 basic unit consist of an electron optical column mounted on a main console, a control and display system, a power supply unit and a pump box. The main console incorporates a vacuum system and the control and display system, photographic recording system (camera, film holder etc.) power supply unit is placed at the back of the control and display system.

PRINCIPLE

When a specimen is targeted with primary electron beam, the complex interactions between the specimen and the electrons give rise to a variety of signals that are detected in the SEM. The SEM derives information from scattered electrons and other signals like photons as they are reflected from the surface of the bulk samples and transmitted into the samples. Back scattered electrons are those that have undergone single or multiple scattering events in the sample and have escaped through the surface of the specimen. BSE travels in straight lines and only those electrons directly in line with detector will be used to form the image. This gives rise to useful shadowing effects particularly on fractured faces. Secondary electrons are transmitted by very low energy and are emitted from the first 5-10 nm of specimen. This shows fine structure details. A substantial proportion of electrons are transmitted if sample is thin enough. These transmitted electrons provide a mean of examining the internal structure of sample in a transmission electron microscope (TEM), but not used in an SEM. Cathodoluminescence is the phenomenon where emitted photons are collected and analysed. The JSM-6400 is associated with modern digital image processing system. This system enables image enhancement by averaging and integration and sorting of image data in frame memories. It can accommodate optical attachment for cathodoluminescence, X-rays, absorbed electrons, transmitted electrons.

APPLICATIONS:

Microscopy is a useful tool to directly get images of a sample at high magnification so that features smaller than what the unaided eye can see are revealed. SEM uses electrons so as to give high resolution and high depth of field. However, samples must be compatible with high vacuum and with the electron beam. Samples are made compatible by a process of "sample preparation." Cryo-SEM is used in special cases where usual sample preparation fails to give correct results. Cryogenic electron microscopy can be used to

• gain microstructural and morphological information of various systems that contain liquids or are of high vapour pressure, e.g., soft solids, comples liquids like liposomes and vesicles.
• study the dynamics of microprocesses by capturing instants of an evolving process
• act as a microanalyser (of electron-induced X-rays). By using an energy dispersive spectrometer allowing an accurate and efficient nondestructive elemental analysis and elemental distribution of consitituents.

Important Instructions for the use of SEM

All users of the MicroLab must follow these instructions scrictly when using the SEM:

1. Check the logbook first for any entries in the "remarks" column, and only then turn on the SEM.
2. Follow the prescribed steps in the correct order to turn on the SEM
3. When turning the SEM ON, put on the A/C and the dehumidifier 15 minutes (-0/+5 minutes) before turning on the chiller.
4. Keep the A/C, dehumidifier and chiller ON throuout your session, i.e., as long as the SEM is ON. Due to the low room temperature, you might feel cold during the session, so always keep a jacket or sweater handy.
5. Do not run a sample until you have the In-Charge's explict written permission. There are many compatibility issues that may spoil the instrument, so never run a sample without permission. There are also some legal requirements that we all have to comply with.
6. While we welcome visitors, it is only by appointment that they can see the instrument. Do not get your friends or any "tourists" to witness the instrument while you are operating. Crowds disrupt the clean environment and temperature. Special demo sessions can be organised for them. Ask the In-Charge for details.
7. Do not take out anything from the lab. Specifically, do not take out the stubs, stub holder/cryo-stage, etc.
8. Due to the nature of the entire MicroLab room environment, you are advised not to have any food or drink there. Such activity may take place in your other sitting area, e.g., Res.Schol room or Silicate lab (outer room).
9. When turning things OFF, put off the chiller 10 minutes (-0/+2 minutes) after turning the SEM OFF, then wait an additional 20 minutes (-0/+5 minutes) and then turn OFF the A/C and the dehumidifier.
10. Do not keep any tools/samples inside the SEM room. The only things you should have to take inside the SEM room are the prepared specimens.
11. At the end of the session (perhaps whilst waiting for the prescribed time limits to pass), clean up all the working areas completey.
12. All accessories of the sample preparation room must be kept only in the sample preparation room in the large thermocole box. Arrange the items neatly in the box. Keep all personal/individually issued items (like rolls, gold wire, etc.) in your locker.
13. Clean up completely after yourself: paper, samples, shoes, furniture, bits of film, etc. Ensure there are no items strewn about. Arrange all furniture neatly.
14. Keep all lN2 and freon cryocans and cylinders in designated positions and locations.
15. Take particular care in ensuring that the darkroom is clean and dry; wipe the platform dry before you leave. Ensure that spirals are washed, cleaned and dried.
16. Help in cleaning up the lab: dust/mop/vacuum once a week or more frequently.
17. If the lab is dirty when you enter it, it is your duty to note exactly what was wrong in the logbook in the remarks column. Clean up whatever you can, and claim restitution from the previous user. Otherwise, you will be held responsibe for it.
18. Before you leave, ensure that all things are in their default locations and settings.
19. Default settings of the SEM are:
    • Stage position 35, 25, 000, 222
    • Accn. voltage 5 kV
    • Filament emission to zero
    • Probe current to extreme right (16)
    • Remove samples from the stage
    • Left and right screen setting reduced using knob below the screen
    • Mag to 12x
    • Camera film to be removed; if exposures are left, forward to next exposure and leave a note on the camera.
    • Insert camera slider plate.
    • Use PF1 to blank right screen
20. Ensure that you know the purpose and function of each piece of equipment or tool that you use. You are responsible for this.
21. Sample and roll details shoulr be maintained in the format provided in the MicroLab and also online.

Various procedures for the SEM are given below.

The "Switch-On" Procedure is:

1. Check the SEM logbook for any relevant comments
2. Check the entire MicroLab for any irregularities like missing things or dirty areas.
3. Switch on both the A.C.s in the microscope room.
4. Switch on the cooler and ensure that it turns on correctly by noting that you can hear the cooler's compressor sound.
5. Wait for about half an hour.
6. Switch on the water circulation pump.
7. Open the Nitrogen gas cylinder valve and adjust/check that the pressure is 5 kg/cm2.
8. Switch on the "To Stabilizer" switch in the sample preparation room.
9. Check the voltage to be at 230 volts. If not at 230 volts, adjust to 230 volts by changing to manual mode and put it back to auto mode.
10. Switch on the stabilized supply.
11. Switch off one of the A.C. s in the microscope room.
12. Turn the "Car ignition" key to on position. Wait for five seconds and then to start position. Hold it for 3 - 4 seconds and take your hand off.
13. Wait until ready signal comes on (approximately 40 minutes).
14. The specimen is then imaged. (See below "IMAGING THE SPECIMEN")
15. Note: To the extent possible, the microscope should be kept continuously on. Turn it off if you are the last operator of the day. Frost was found to be minimum when the microscope is continuously kept on. Samples can be made even before the microscope is switched on. They can be stored under liquid nitrogen until needed.

1. The accelerating voltage is set at the desired value (generally between 5 KV to 10 KV for cryo specimens) and is switched on.
2. The Mode button is pressed until a horizontal line appears on the screen. This line is a profile of the brightness level of the image versus the gun filament current (which would be zero at this moment).
3. The filament current is slowly increased. The line profile will develop peaks and valleys (which is indicative of the specimen contrast) and will start to rise. It will go through a maximum, come down and again rise to a maximum and is saturated at this value. The filament current is set at the lowest value at which saturation occurs (this is generally between 12 and 1'o clock positions on the filament current knob).
4. Change to Imaging Mode and start at low magnification. Focus and solowly increase mag once an image is seen at low mag. Adjust Contrast and Brightness correctly.

The following steps are performed for preparing the sample.

1. A drop of the specimen is placed on a sandwich holder.
2. The sample assembly is plunged in a pool of melting freon or liquid N2 with a plunger at high speed so that amorphous ice is formed.
3. The sandwich is cryo-transferred into the SEM and held on a cryo-holder within the SEM.
4. The "frozen" sample is fractured by separating the sandwich or with the help of special knife to expose an internal surface and its embedded structures. The sample is then imaged with SEM and snaps are taken.
5. These photographs are then analysed by digital image analysing modules associated with JSM-6400.

Sometimes, Freeze-fracture replication is useful, for both SEM and TEM:

Here is an extract from the "Materials and Methods" section of a paper I wrote. Although the following paragraph is written for TEM, it is also useful for SEM and Cryo-SEM:

Samples were observed for microstructure by transmission electron microscopy using the freeze-fracture cryo-vitrified replica technique as follows. Samples for TEM were prepared by high-pressure freezing using a Bal-Tec (Liechtenstein) Model HPM-010 with gold sample holders. In this technique, a drop of sample is placed in a sample holder that sandwiches the sample between two gold sheets, one of which has a cup-shaped depression to hold a few microliters of the sample. The instrument rapidly freezes (vitrifies) the sample by application of high pressure followed by low-temeprature depressurization. The frozen sample is transferred using a cold-chain into a freeze fracture apparatus Bal-Tec (Liechtenstein) Model BAF 060 Freeze Fracture/Etch System, where the sample is kept on a liquid-nitrogen cooled stage. An internal surface of the sample is exposed by fracturing the sample by separating the sandwich and assisted by a built-in knife. The fracture surface, which has the internal microstructure of the sample exposed as topographic variations, is shadowed at 45 degree angle with a platinum-carbon film deposited by means of electron beam evaporation. Then a support layer of carbon is evaporated onto the shadowed layer, so as to give a replica that is brought up to room temperature, washed of residual sample, dried and observed by TEM. We used a FEI Technai T-12 TEM operated at 120KeV.