1. Conventional SEM Specimen at high vacuum – requires sample fixation and dehydration or freezing. Charging is minimized by coating sample with metal or carbon or lowering the operating kV.

2. Variable Pressure Scanning Electron Microscope -Vacuum in the sample chamber can range from high vacuum (< 10-6 Pascals) up to 3,000 Pa. -Environmental- 3,000 Pa/ Variable – 750 Pa - Gas in the sample chamber allows uncoated and unfixed samples to be imaged. -Detectors used at higher pressures are backscatter or special secondary detectors. - Moisture on the sample can be controlled by cooling/heating stage and water injection system.

3. LV SEM – High Vacuum Mode

4. LV SEM - Low Vacuum Mode

5. SEM Electron/Specimen Interactions When the electron beam strikes a sample, both photon and electron signals are emitted. Incident Beam Specimen X-rays - composition info Auger electrons - Surface sensitive compositional Backscattered electrons - Atomic number and topographical Cathodoluminescence - Electrical Secondary electrons - Topographical Specimen Current Electrical

6. Secondary Electron Detector

7. Backscatter electron detector

9. Signal Detection in Variable Pressure Mode

10. Charging is minimized

12. Peltier stage Heats to 50 C Cools to - 25 C

13. Control of Water vapor and temperature

14. Applications Live centipede Bacteria on rock

15. Fresh moss with liquid water

16. Pattern produced in silica gel

17. Live Drosophila larva