TITLE: SEM-EDS Protocol V1
AUTHORS: Saylen Sofia Paz, David Bahamon-Pinzon, Dvanega

[DESCRIPTION]
This protocol outlines the methodology for conducting Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDS) analysis of laser-inscribed graphene (LIG) electrodes using a SU5000 microscope. The procedure details the preparation of LIG electrodes, their imaging under SEM, and subsequent elemental analysis via EDS. By combining SEM imaging for morphology assessment with EDS for elemental composition identification, this protocol facilitates a comprehensive characterization of LIG electrodes, crucial for understanding their structural features and properties in various applications. The fusion of SEM and EDS techniques offers insights into the intricate structure-property relationships of LIG electrodes, advancing their potential in diverse fields such as energy storage, sensing, and electronics.




[STEPS]
SECTION: Procedure
2. Step 1) Turn on the computer and equipment screens
Login from any device with internet access to iLab's website (iLabFacilityScheduling (clemson.edu))
Log into your account
Click on the start button 
Note: Automatically, the screens of the equipment will turn on (Figure 2)

 

SECTION: Procedure
3. Step 2) Preparation of the sample
Take a piece of carbon tape and put it in a 51 mm holder
Remove the layer that is covering the tape
Place the samples over the tape using the tweezers (Fig 3A)
Put the bottom part of the holder (Fig 3B)
Verify that the height of the holder and the sample is within the range of the scale
(Fig 3C)
Note: Try to organize the samples with something that you can recognize each sample

 

SECTION: Procedure
4. Step 3) Set up the equipment 
The equipment is always under high vacuum. Letting air in the equipment is
necessary to open the chamber and place the sample. To do this, click Air in the
Specimen/vacuum setting (Figure 4)
The equipment should start beeping. Wait for the process to be completed. It beeps
three times
Click Specimen settings in the popup window (See Figure 4)

 

SECTION: Procedure
4.1. 3.1 Select stub and Height Adjustment
Select the diameter of the holder or stub (51 mm)
Click Next
Choose the height of the holder, including the sample
Click Next (See Figure 5)

 

SECTION: Procedure
4.2. 3.2 CameraNavi setting (optional)
Turn the light of the camera on with the switch
Put the holder on the camera. The holder must reach the top (Fig 3)
Click movie
Push the buttons to zoom in or out
Click capture
The holder should fit the circle in the screen. Move with the mouse.
The triangle and circle in the screen should fit the holder
Click set
Green should show up
Click next (Figure 6)

 

SECTION: Procedure
4.3. 3.3 Placeholder in the chamber
Pull the gate of the chamber all the way out until you hear a switch (Fig 7)
Click stage move
Note: It starts beeping as it moves up the stage
When it stops beeping, put a sample. The holder must reach the top. The top is gate located
next to the wall of the room 
Close the chamber slowly and verify the height of the holder. The sample should be under
the plate 
If the sample is too tall, pull the equipment out again until you hear the click and click
down as needed.
Close the door entirely until hearing a slight beep.
Click Next (See Figure 7)

 

SECTION: Procedure
4.4. 3.4 Evacuation 
Choose Conductive or non-conductive samples
Choose High or Low vacuum 
Click Evac
It says Pumping in the current vacuum
The line from the central cross indicates the direction of the sample (Figure 8)

 


SECTION: Procedure
5. Step 4) Set settings for SEM
Set the following parameters in “Electron beam”:
o  Accelerate voltage: 5 – 15 kV (for imaging).
o  Imaging: any value
o  Light elm
o  Spot int: 40. Scale 0-100. This is the current
Click start (See Figure 9)

 

SECTION: Procedure
6. Step 5) Insert detector
Verify that Detector BSE-All is selected in the top right
Check that BSE can load in the field specimen
BSE (Back-scattered Electron Detector) able to load
Insert the detector by turning slowly in the “IN” direction until it stops (Figure 10)
Click OK in the pop-up window

 

SECTION: Procedure
7. Step 6)Calibrate the beam (brightness, contrast, focus)
Note: There are several parameters that can be adjusted from the box of the equipment
Brightturn knob inc
Contrast
Speeds
o  1. Fast, 2. Slow, 3. Red (reduced). Click 2 twice to change to 2.1.
Focus 
Coarse
Stigma(stism)
Click mode to change to alignment
Loo for something to focus
When finding something, click 1. Bum aligment
Center beam by adjusting X and Y knobs
If nothing happens when clicking auto bright, check alignment
Click mode
Set an appropriate picture for alignment. The focus should not move after this
Aperture alignment
Moving alignment
 Move x. Then it doesn’t move.
Click mode
Stigma alignment for X
Click mode. I f it looks fine, push mode again.
Note: Always focus at high magnification (e.g. more than 25X)
VP: low vacuum (vacuum pressure)
60 Pa
Click start
SECTION: Procedure
8. Step 7) Set capture settings
Go to capture settings in the bottom right corner of the screen
Save settings Select the folder "Vanegas"
Note: If is necessary, create the folder 
Click to capture settings
In capture settings, select:
o  Speed: slow
o  Image size: 1280x960 (adequate for papers, not
for posters or magazines. For posters, use 5120x3840)
o  Time: 16 s. This corresponds approximately to Slow 3
SECTION: Procedure
9. Step 8) Take picture
In the box, press Capt to capture pictures.
Note:  After each capture, the software changes to Freeze. Press 4  in the box of commands to return to Run.
SECTION: Procedure
10. Step 9) Set settings for EDS
Change voltage to 15 kV. 5 kV is proper for carbon but not for platinum or copper.
Note: The working distance should be 10 mm. For example, If the distance in the screen is 6.2 mm, we need 4 mm more. We need to add 4 mm to the value of Z (in
Specimen/Vacuum settings). If Z was 5 at the beginning, set a Z of 9. 
7.4-10=2.5
SECTION: Procedure
11. Step 10) Open the software for EDS
In the second screen, open the software Aztec. Open project LIG (create a new folder or  select the folder “Vanegas”)
Process time: 6. 
Note: The process time of 1 is the slowest, and 6 is the highest.
Element distribution

SECTION: Procedure
12. Step 11) Final parameters and start
Verify that the dead time is around 40%
Select EDS-SEM in the top left area of the screen.
Set Analyze: overall. 
Select Point 81D (scan specific position). Other options include color map, line graph
Click New specimen
Click New sample
Uncheck box Specimen is coated
Click Scan image
Click Settings
Select a Scan size of 1024 
Dwell t (time/pixel): 5 μs
In the “sun” at the bottom right corner, the brightness can be adjusted
Click Start
SECTION: Procedure
13. Step 12) Remove the sample from the chamber
When the analysis is completed, click “Stop”
Take the detector out by tunning the knob in the “OUT” direction until it stops.
In Vacuum, select Air.
Click Finish 
Pull the gate of the chamber to open the equipment.
Take the sample holder out.
Close the chamber.
Click “Evac” (the equipment must remain in High vacuum when not used).
Clean the working station
SECTION: Notes
14. "dark"areas have lower average atomic number
Detectors:
o  BSE is used to find differences in composition. High energy elements.
o  Secondary electrons (SE) has lower energy. It is used to study the sample surface and texture (this detector didn’t work well because of the substrate for LIG samples)
A higher pressure from a low vacuum setting will scatter the electrons, and the image will be distorted 
For EDS, 5 kV is adequate for Carbon but not for copper or platinum
For elements such copper, platinum and gold an adequate potential ranges 15 – 20 kV
For LIG samples, it is adequate to work in non-conductive samples and low vacuum
For high resolution images, it is recommended to use the autofocus and auto contrast
from the program and slow 3 
SECTION: Overview
1. 
Protocol SEM/EDS 1.1
Scanning Electron Microscopy (SEM)
and Energy-Dispersive X-ray Spectroscopy (EDS) analysis
Saylen Sofia Paz, David
Bahamon-Pinzon, Diana Vanegas Gamboa
Clemson University

This protocol describes the process of performing the Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDS) analysis of laser-inscribed graphene (LIG) electrodes (e.g. Protocol L-Cu1.1) using a Scanning Electron Microscope SU5000. Fig 1 depicts the process for the analysis. The time must be change according with the number of the samples that you have. 

 

MATERIALS
       LIG sensors (Protocol L-Cu1.1)
       Tweezers
       Tape. NEM. Nisshin em.co.ltd
       Sampleholder
       USB 
 
HARDWARE
       Scanning Electron Microscope SU5000.