Scanning Electron Microscopy Lab

MSE 2021
Magnification Calibration and Depth of Field

Objectives:
To perform a magnification calibration and measure the depth of field of the LEO 1550
SEM from micrographs of a calibration standard at a variety of magnifications.

Introduction:
In many applications, the knowledge of the precise magnification of the image on the
Cathode Ray Tube (CRT) screen or printout micrograph is essential. For example, one must
know the exact magnification when measuring size distributions. In general, there are two ways
of obtaining accurate magnification information: 1) work under preset conditions for which the
magnification is calibrated; and 2) calibrate the magnification for the conditions of use.

Three major instrumental factors control magnification. First, the deflection current
applied to the scan coils within the objective lens determines the magnitude of beam deflection
in the scan raster. The scan raster is made up of a fast horizontal deflection, the sweep, and a
slower vertical deflection, the scan. The second factor determining magnification is the CRT
size. The ratio of the magnitudes of the sweep and scan deflections on the sample surface relative
to the same sweep and scan on the CRT determine the magnification.

Length on printout or CRT LP

MAG = --------------------------------------- = ------ (1)
Length scanned on sample LS

The third factor is the working distance, or the distance of the sample below the objective
lens pole piece. Within the scan coils is the scanning pivot point, which is some fixed distance
above the objective lens pole piece. The magnitude of the sweep, and therefore, the area
scanned, is determined by the distance of the sample surface beneath the pole piece.

Figure 1. Actual sample picture elements and screen picture element.

Depth of field is the ability of a lens to focus on a three dimensional object. It is defined
to be the thickness of the specimen that can be seen with acceptable sharpness in a magnified
image. It is dependent on the divergence angle and the magnification. An understanding of depth
of field requires the concept of picture element (pixel). Picture element is the size of the area on
the specimen from which information is transferred to the CRT image or computer memory
(usually is considered as a circle or square). For a given choice of magnification, images are
considered to being sharpest focus if the signal measured when the beam is addressed to a given
picture element on screen comes only from that picture element on sample. Overlap of
information from adjacent picture elements on sample occurs as the magnification is increased
because of the size of the interaction volume. This overlap of pixel information is manifested as
blurring in the image. To determine the limiting magnification for a given beam size, energy, and
specimen composition, we must calculate the signal-producing area and compare it to the pixel
size. How can the signal “leak out” to overlap adjacent picture elements? The most important
source of signal leakage occurs as a result of electron scattering – volume from which the signal
emanates.
To calculate the depth of focus field we must know at what distance above and below the
plane of optimum focus the beam has broadened to a noticeable size. At this point the beam
reaches a condition of overlapping adjacent pixels.

Figure 2. Schematic illustration of the depth of focus (field) in a SEM image .

If the saved image has 1024x1024 pixels, the picture element diameter is:

dpicture element = lactual (2)

1024
where lactual is the length of the actual scanned area (you can calculate it form the micron bar in
the micrograph).
The image becomes out of focus when the beam diameter is larger than the diameter of
the picture element addressed on the sample. The point at which an observer will notice the out
of focus effect depends on his/her visual acuity, but generally it occurs when the beam overlaps
at least two picture elements. Thus,

2 dpicture element 2 lactual

Depth of Field = ------------------ = -------------- (3)

 l pr int ed
l actual =
Since lactual is a function of magnification ( M ), then depth of field is also a function
of magnification. Decreasing the divergence angle (or the magnification (M) yields an
increase in the depth of field; for small features, it is not practical to decrease the magnification,
therefore the divergence angle must be adjusted.
R
 = ------- (4)
Dw

where R = radius of objective aperture and Dw = working distance. So, to decrease the
divergence angle, one must decrease the aperture size or increase the working distance.

Procedure:
Magnification Calibration:
1. Log in into the log book record.
2. Follow the LEO 1550 operation procedure to put sample inside the SEM and get decent
image on the screen.
3. Center the sample and rotate if necessary to align the grid sample to the screen
4. Set the image acquisition to 1024 resolution and 4 points average. Take pictures at
different magnifications according to your own lab session.

Depth of Field:
1. At lowest magnification, tilt stage to 45 and adjust the stage both in X and Y directions
to center the sample (rotate the stage to align the grid if necessary).
2. Adjust focus and stigmatism up to 30kx to 50kx magnification
3. Take pictures at magnification in your lab session (Note that Z=Z1 and Apt=A1).
4. Change the aperture to 1 step by step and notice the brightness on the screen (Increase or
decrease ?)
5. Adjust brightness and contrast. Focus and adjust stigmatism at 30kx - 50kx magnification
6. Take pictures at magnification in your lab session (Note that Z=Z2 and Apt=A1).
7. Decrease magnification to 40x and Set Z=9
8. Adjust brightness and contrast. Focus and adjust stigmatism at 30kx - 50kx
magnification.
9. Take pictures at magnification in your lab session (Note that Z=Z1 and Apt=A2).
10. Change the aperture back to 3 step by step and notice the brightness on the screen
(Increase or decrease ?)
11. Adjust brightness and contrast. Focus and adjust stigmatism at 30kx - 50kx magnification
(rotate stage if necessary to align grid to screen).
12. Take pictures at magnification in your lab session (Note that Z=Z2 and Apt=A2).
13. Turn off the beam;
14. Follow the SEM operating procedure to take out sample and end the session.
Tasks:
For the magnification calibration, the student will print the images from 0 tilt sample
and measure the distance across as many lines as possible, in both the horizontal and vertical
directions and divide that number by the actual distance given by the grid manufacturer (see the
grid information in the next page).
Distance measured from SEM Image (cm)
Nominal Magnification = -------------------------------------------------- (5)
“Real” distance shown SEM (μm)

Distance measured from SEM Image (cm)

Actual Magnification = -------------------------------------------------- (6)
Real distance from manufacturer (μm)
Error= (1-Nominal Mag./Actual Mag.) (7)

Next calculate the magnification based on the micron marker. Measure the length of the
micron marker and divide by the specified length of the marker. Comparison of the Calibrated
Magnification with the given Marker Magnification will be made to determine the percent error.

For the depth of field, on the bottom and top of the 500x-magnification photo, the image
will be out of focus or blur where the electron beam diameter is covering at least two picture
elements. Measure the length, l, of the image feature that is in focus, and find the depth of field
from the following equation:

D = lmeasured tan  

where  is the tilt angle. Now, calculate the divergence angle and the actual diameter of the
aperture from equations 3 and 4.
From the images taken at 20000x, compare the sharpness of the image. Assuming the
microscope conditions (focus, stigmatism, etc) are properly adjusted, the sharpness of an image
will indicate how good the resolution is. You may need to rank the sharpness in a scale from 1
to 4 with 1 is the sharpest.
Put the calculated numbers and observations in a table (see in the report template)

Reference
Joseph I. Goldstein et al., Scanning Electron Microscopy and X-Ray Microanalysis, 2nd ed., (New York: Plenum
Press, 1992).
Grid information: http://www.tedpella.com/grids_html/Pelco-TEM-Grids.htm
Mesh Specifications
Mesh (lines/inch) 200
Pitch µm 127
Bar Width µm (31)
Hole Width µm (54)
Laboratory Report Template: The laboratory report
should contain the following elements and components
within each section.
Title

SEM Lab I: Magnification Calibration and Depth of Field

Lab Participation Lab Report

Possible Points Possible Points

Points Awarded Points Awarded
Attendance 10 Name and 20
Introduction
Participation 5 Procedure 10
Operation 10 Results 20
Safety 5 Discussions 10
Conclusion 10
SUBTOTAL 30 SUBTOTAL 70

Lab Location: _ ____________________

Student Name: _ _______________________

Section ___________________________________

Lab Session Date(s): ___ _______________________________

Lab Report Date: _______ _______________________________

(2 pts)

Introduction
Briefly describe
1. The general working mechanism of SEM using necessary words (4
pts) and one figure (4 pts), list the references (2 pts);
2. State at least two applications in material research and reference (8
pts).

Procedure
Write a summary procedure stating what was done. List all relevant equations used for
deriving the results in the next section. Specify the meaning of each parameter in the equations.
For instance, a detailed description of the depth of field measurement which includes a drawing
showing tilt angle and lmeas.should be used to justify equation (6) above.
1. Magnification Calibration (5 pts);
2. Depth of Field (5 pts).

Results
1. For Magnification Calibration, provide the following chart, ONE graph of actual vs. marker
magnification for both the horizontal and vertical directions, and each image taken for this
part. Label each figure and table appropriately (graphs count as figures) (0.5 pts each)

Figure #
Ave. Distance measured from SEM
Image (cm)
“Real” distance shown SEM (μm)
Real distance from sample
manufacturer (μm)
Nominal Magnification
Actual Magnification
Error(%)

2. For depth of field, provide the following chart and each of the images taken to determine
depth of field with the lines used to calculate depth of field on them. Use a few sentences to
explain how you calculated depth of field. (0.5 pts each)

WD = __ WD = __ WD = __ WD = __
Apt = __ Apt = __ Apt = __ Apt = __
Depth of field at 100x (m)
Sharpness/resolution at (what
magnification you have in your data)
(Ranked from 1 to 4 of sharpest to
blurriest)

Discussion

A larger aperture results in a brighter/darker image.

A larger working distance results in a closer/further view.
Smaller/larger aperture size and shorter/longer working distance results in a longer depth of field.
We adjust ( ), ( ), and ( ) to focus the beam of the microscope.

Conclusion

Like your previous lab reports, provide a summary of the results you obtained including numbers
where appropriate. In addition, please address the following questions in the body of your
response.
1. Why do we need to do magnification calibration?
2. Why do we need to get the depth of field? How does the value compare to that of optical
microscope?