SEM Lesson
Mixture Analysis
SEM – Mixture Analysis
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YOUR TASK  MIXTURE ANALYSIS
Phenom image of Powdered Sugar 450x
Phenom image of NaCl 355x
(Are these two crystal structures the same?)
Can we determine the relative Composition of a mixture using images from an SEM and
Optical scope? This is an Inquiry lesson that allows data collection to determine the
relative concentration of a sample mixture composed of unknowns. You'll be given up to
5g of the mixture and you'll need to determine how many substances you're given and a
relative ratio of each component.
THINGS TO CONSIDER…
1) What could you see when you look at images from an SEM?
2) Would density play a role in what you're seeing? Could crystal/granular size be an issue?
3) Could a look at relative number of crystals tell us anything?
4) How will you express your answer? (units?)
Data analysis could be based on images (observations of optical microscope and SEM
images) and mathematical analysis from your counts.
Definitions: Mixture: two or more substances which have been combined such that each
substance retains its own chemical identity. Homogeneous any combination of substances that has
uniform composition and properties; a mixture that is uniform throughout. Heterogeneous any
combination of substances that does not have uniform composition and properties; a mixture of physically
distinct substances with different properties
SEM Lesson
Mixture Analysis
Objectives:
-Use the SEM for data
collection
-Inquiry analysis of a
mixture composition
Integrates:
-Observation/Inference
-Forensics
-Chemistry
-Mathematics
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LESSON OUTLINE
At each of the following stations you should accomplish
these objectives/learning targets.
1) Scope/ Beam Time- Phenom
a. Protocols /Safety/Labels/Mapping
b. Sample prep
i. Clean/dry/stuck down
c. Image capture (tiff) / Storage/ Labeling
QuickTime™ and a
decompressor
are needed to see this picture.
2) Optical Scope
a. Protocols/Safety/Labels/Bar Scale/Mapping
b. Sample prep (thin, dry, slides, coverslip, etc..)
c. Image capture
3) Background Research
a. Important vocab for this unit, unique vocab., tool
b. Website(s), Article(s) related to the curricular topic
Standards:
-Science Inquiry
-Engineering Design
-Structure and
Function
Logistics:
2 – 90 min. class
periods
-3-6 mixture samples
-Phenom SEM
-Light Microscopes w/
cameras for image
capture
4) Image "J" tutorial
a. Beginning (opening images, saving as …
i) original, ii) enhanced, iii) scale/sized,
b. Intermediate (mapping, blowups, labels, measurements, edge detection,
(changing your data and creating an artifact…)
c. Advanced (particle analysis, thresholding, binary, counts, area, plug ins ??
5) Images to Interpret
a. Unique to the activity (pure samples, reference or standard images
b. Problem images? Charging? Challenging? Topographic? Etc…
6) How does a Phenom / SEM work
a. Creative tour of the scope (ex. write about an images' journey ….)
b. Labeled diagram with explanation of workings
c. What would make this machine or images even better? How would you do
it?
d. Visual virtual tour through the scope
(After image capture…)
7) Writeup time
a. Template/format (image plates for cut and paste that have fields to fill in)
b. Records/log/observations/labels….. Inquiry?
SEM Lesson
Mixture Analysis
System details
• Magnification range 20x – 20,000x
• Touch screen controlled
• Image options – JPEG, TIFF
•Sample load time <30 seconds
SEM – Mixture Analysis
1)Scope/ Beam Time Phenom
a) Safety/Protocols/Labels/Mapping
b) Sample prep
1. All samples MUST be dry before mounting
2. On ce a sample is dry carefully s tick it to the metal stage of a mounting
stub with carbon tape or carbon paste.
3. To assure that the samp le is stuck down and clean gentle blow a little
compressed air acro ss it.
4. Samples are then ready to be loaded onto the sample cup that can
accommod ate samp les up to25 mm (dia) x 30 mm (thick).
c) Loading Samp les
1. Place the metal stub with your dry, stuck down samp le into the center of
the sample cup and then dial down the sample so it appears to be even with
the outside rim of the cup. Once the sample is even with the outside of the
cup turn it down an additional four (4) c licks so the samp le is well below
the metal rim.
2. Place turn downed samp le into the vacuum chamber and pull the door
down until it is completely shut.
Touch Screen
Sample
Cup
Sample
Bay
Touch Screen
Settings View
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Rotary
Knob
Close-up
of Sample
Cup
d) Ima ge capture (tiff) / Storage
1. On ce the samp le is in the Phenom it will automatically create an optical
ima ge in the upper right side of the mon itor.
2. Map your samp le by pressing the mapping button on the screen once the
optical map is complete save an image by touching the camera to the rig ht
of the mapping button. Images are saved on a USB memory stick
For off-line analysis, measurements and distribution. These saved images
can be viewed on the archive screen and digitally zoomed by another 4
times. If the image appears too dark simply inc rease the brightness by
touching the brightness/contrast button. Adjust brightness by dialing the
mouse up or down.
3. Now that you have an optical image change to SEM mode by using the
toggle button.
This will take about thirty (30) seconds. Once an image is made the
sample can be easily mo ved by simpl y touching the feature of interest on
the screen. Use the touch screen to focus, change brightness, mag nification
and capture images.
4. On ce you have an image you want to capture (take a picture of) press
‘Settings’ and then ‘Labe l’ and use the keyboard on the screen to enter a
label. This label will be applied to all the images you take so remember to
change it for each samp le or when you think it is appropriate. First time
users should take some time to look over the Settings men u as it allows you
to adjust image quality. To exit settings and get back to your image just
press Imag e located on the green menu bar at the top of the screen.
5. For this activity take images in a sequence to show samp le detail. An
exam ple of an image sequence could be 500x , 1000x and 5000x. The
ima ges will be automatically saved to your thumb drive and you will use
them to create your image plates for your final report. Paste images
captured in the Image Plate temp late below.
SEM Lesson
Mixture Analysis
Mixture Analysis
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2) OPTICAL SCOPE- Light Microscope Protocols
Protocols: FOLLOW your site protocols to capture, name and properly save images.
Specimen Preparation
When observing a specimen by transmitted light (Diagram 1), light must pass through the specimen in
order to form an image. The thicker the specimen, the less light passes through. The less light that passes
through, the darker the image. Therefore, the specimens must be thin (0.1 to 0.5 mm). Many living
specimens must be cut into thin sections before observation. Specimens of rock or semiconductors are too
thick to be sectioned and observed by transmitted light, so they are observed by the light reflected from
their surfaces using a dissecting scope (Diagram 2).
Diagram (1) a typical student light microscope
Diagram (2) a typical lab dissecting scope
Some Microscope Terms
* Slide – usually a piece of glass that you use to mount your specimen (place specimen on the slide)
* Coverslip- usually glass or plastic, place this over the specimen (2-4 drops of water, 45° angle and drop,
no air bubbles?)
* Depth of field - vertical distance, from above to below the focal plane, that yields an acceptable image
* Field of view - area of the specimen that can be seen through the microscope with a given objective lens
* Magnification - product of the magnifying powers of the objective and eyepiece lenses
* Resolution - the closest two objects can be before they're no longer detected as separate objects (usually
measured in nanometers, nm)
*Wet mount = slide + speciment+ 2-4 drops of water + coverslip (no fingerprints)
A light microscope, whether a simple student microscope or a complex research microscope, has the following basic
systems:
* Illumination - shed light on the specimen
o lamp - produces the light
o condenser - lens system that aligns and focuses the light from the lamp onto the specimen
o diaphragms or pinhole apertures - alter the amount of light, (for enhancing contrast in the image)
* Lenses - form the image
o objective lens - gathers light from the specimen
o eyepiece - transmits and magnifies the image from the objective lens to your eye
o nosepiece - rotating mount that holds many objective lenses
* Focus - position the objective lens at the proper distance from the specimen
o coarse-focus knob - used to bring the object into the focal plane of the objective lens
o fine-focus knob - used to make fine adjustments to focus the image
(Source: http://science.howstuffworks.com/light-microscope5.htm)
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Mixture Analysis
SEM – Mixture Analysis
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Image Capture- - -For both the SEM and the optical scope follow your site protocols for capturing
images. REMEMBER- labels, scale bar, instrument name/model, (i.e. Nikon 155). Paste images captured
in the Image Plate template below.
Image Plate Template (YOUR TITLE HERE)
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
Magnification
and Scale Bar
Name: _____________________ Period: _____
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
included.
Magnification
and Scale Bar
included.
(paste image here and fill in info above)
(can you make them all the same size?)
(paste image here and fill in info above)
(This is still the same sample…MAGNIFIED!)
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
Magnification
and Scale Bar
included.
Magnification
and Scale Bar
included.
(paste image here and fill in info above)
(paste image here and fill in info above)
(have you used labels/arrows to highlight?)
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SEM Lesson
Mixture Analysis
3) A. Background/ Definitions (Important vocabulary/practice for this lesson). Use your classroom
resources to write definitions and answer the questions below.
1. Mixture –
2. Homeogeneous-
List at least 3 examples of Homeogeneous mixtures:
a. iced tea – brown clear liquid, consistent throughout
b.
c.
d
3.
Heterogeneous
List at least 3 examples of Heterogeneous mixtures:
a. orange juice with pulp – orange cloudy liquid, inconsistent, pulp on the surface
b.
c.
d
4. Pure substance
5. Element
6. Compound
7. Physical Property
8. List 2 examples of each of the following:
a) Solution
b) Colloid
9.
c) Suspension
List the contents of your kitchen or bathroom sink . What do you think most of the substances
would be classified as? Support your answer using your observations and background knowledge.
10. You're told that you will be receiving a heterogeneous mixture from the Gatorade company that
contains 5 ingredients. List your chemistry ideas to figure out what they are and determine a way
to separate them?
B. Related website(s) & article(s)
1)
2)
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Mixture Analysis
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4. Image J Tutorial
Use your thumb drive or your e-book folder titled, "Practice Images" and investigate the features
and abilities of 'Image J'. Start with the Beginning tutorial and then progress as required.
http://rsb.info.nih.gov/ij/docs/examples/index.html
http://rsb.info.nih.gov/ij/download.html
tutorials and examples
for downloads and information
Image 'J ' can work with variety of formats but we will usually see JPEG and Tiff. It is
helpful to establish a logical and informative pattern for saving and labeling images you
save from both the optical scope and desktop SEM - Phenom,
ex. flylegtip450xphenom.tiff
------------------------------------------------------B- Beginning
Here are the basics…
1) Launch Image J and you'll see a simple tool bar.
2) FILE Open (select your stored image) (You need to keep an original data file so after
any change or alteration make sure that you select FILE SAVE AS).
3) Improving the image? Remember you have the EDIT  Undo feature.
QuickTime™ and a
decompressor
are needed to see this picture.
B- Beginning
(opening images,
saving as tiff/JPEG…
i) original, ii)
enhanced, iii)
scale/sized,
4) Start with these options and view the results….(Remember #3 above).
a) PROCESS Enhance Contrast (equalize histogram will balance
black/white/grays) AND/OR
b) PROCESS Smooth or Sharpen
c) IMAGE  ADJUST  Size ( X 'Constrain Aspect Ratio' will keep the image
from tweaking out/distorting)
I- Intermediate
(mapping, blowups,
labels, measurements,
edge detection,
(changing your data
and creating an
artifact…)
------------------------------------------------------I- Intermediate (make sure you have completed the basics)
1) Select your tool of choice from the tool bar (straight line, rectangular selection, text
tool, etc…)
2) Measuring? For area use the rectangular/ freehand / polygon tool/ etc… and then go to
Analyze Measure.
3) Labels? Choose the text tool on the tool bar and draw a text box near where you want
your label.
4) Adding a label arrow? Use the arrow tool and draw between the text box and the image
part.
------------------------------------------------------A- Advanced
A- Advanced
(particle analysis,
thresholding, binary,
counts, area, plug ins,
(use the online
tutorial… ??
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Mixture Analysis
Mixture Analysis
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5) Images to Interpret and Critique
Image 'X'
Problem images? Charging? Challenging? Topographic? Etc…
List four constructive comments, critiques, questions related to
each image to the left. (Feel free to use labels on the diagram to
highlight your comments). (ex. Is Image 'Y' from 3 unique
samples?).
Image 'X'
Image 'Y'
Image 'Y'
Image 'Z'
Image 'Z'
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Mixture Analysis
SEM – Mixture Analysis
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6) How Does a SEM Work?
After reading the article below show your understanding of how a SEM works by
doing ONE of the activities below.
a.
b.
c.
Creative tour of the scope (write about an images' journey ….)
Label a diagram of an SEM with explanation of how it works
In a paragraph describe what would make this tool or images even better? How
would you do it?
Microscopes – Help Scientists Explore Hidden Worlds
The microscope is an invaluable tool in today's research and education. It is used in a wide range of
scientific fields, where major discoveries in biology, medicine and materials research are based on
advances in microscopy. As the need to see the world at a smaller and smaller scale has grown scientist
looked for ways to improve on the light microscope (see Fig. 1 below). In 1938 Ernst Ruska develops the
electron microscope and in 1981 Gerd Binnig and Heinrich Rohrer invent the scanning tunneling
microscope that gives three-dimensional images of objects down to the atomic level. In 1986 all three men
receive the Nobel Prize in Physics. The greater resolution and magnification of the electron microscope is
because the wavelength of an electron; its de Broglie wavelength is much smaller than that of a photon of
visible light. Conventional light microscopes use a series of glass lenses to bend light waves and create a
magnified image. The electron microscope uses electrostatic and electromagnetic lenses in forming the
image by controlling the electron beam to focus it on the surface of the specimen. The SEM shows very
detailed 3-dimensional images at much higher magnifications than is possible with a light microscope
(because electrons travel at a smaller wavelength) but the images are in black and white because aren’t
created using light (see Fig. 2 below). To prevent electrons from colliding with air molecules all SEMs
work under a vacuum. Initially before samples are stuck down on a metal stub have to be prepared
carefully to withstand the vacuum inside the microscope. No wet, magnetic or alive samples can go into an
SEM. Biological specimens are first dried in a special manner that prevents them from shriveling. Also
because the SEM illuminates with electrons, they also have to be made to conduct electricity. SEM
samples are often coated with a very thin layer of a metal (i.e. gold) by a machine called a sputter coater.
Once the specimen is prepared its ready to go into the SEM. The sample is placed inside the microscope's
vacuum column through an air-tight door. After the air is pumped out of the column, an electron gun [at the
top] emits a beam of high energy electrons. This beam travels downward through a series of magnetic
lenses designed to focus the electrons to a very fine spot. Near the bottom, a set of scanning coils moves
the focused beam back and forth across the specimen, row by row, this is called rastering. As the electron
beam hits each spot on the sample, secondary electrons are knocked loose from its surface. A detector
counts these electrons and sends the signals to an amplifier. The final image is built up from the number of
electrons emitted from each spot on the sample.
Figure 1: Resolution scale line
Figure 2: Electron Microscope
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Mixture Analysis
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6) How Does a SEM Work? (cont.)
The Scanning Electron Microscope is revealing new levels of detail and complexity in the amazing world of microorganisms and miniature structures. Follow this link to a video that shows, generally, how an SEM works.
VIDEO LINK: SEM Video Link
http://www.youtube.com/watch?v=lrXMIghANbg&eurl=http%3A%2F%2Fvideo%2Egoogle%2Ecom%2F
videosearch%3Fq%3Delectron%2Bmicroscope%26hl%3Den%26emb%3D0%26aq%3D0%26oq%3Delect
ron%2Bmic&feature=player_embedded
The good news is that the Phenom SEM that you will be using in class is a lot smaller and easier to use than
the one shown in the above video but the Phenom work basically the same way as the SEM in the video.
To see a brief preview of how to operate the Phenom go to the link below.
VIDEO LINK: PHENOM Video Link
http://www.youtube.com/watch?v=Rk7jGgMlPek&feature=related
ATTACH YOUR ACTIVITY HERE:
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TEACHER RESOURCES/REFERENCES
The Desktop SEM lesson is designed so that your students can work in groups and rotate through six
different stations. (You could have more than one group per station depending on class size). Below
is a rubric that could be used as a pre- and post- assessment tool and as a student checklist of tasks to
be accomplished.
LESSON RUBRIC
At each of the following stations you should accomplish these objectives/learning targets AND be able
to demonstrate proficiency.
1) Scope/ Beam Time- Phenom
a. Protocols /Safety/Labels/Mapping
b. Sample prep
i) Clean/dry/stuck down
ii) Coated
c. Image capture (tiff) / Storage/ Labeling
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
2) Optical Scope
a. Protocols/Safety/Labels/Bar Scale/Mapping
b. Sample prep (thin, dry, slides, coverslip, etc..)
c. Image capture
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
3) Background Research
a. Important vocab for this unit, unique vocab., tool
b. Website(s), Article(s) related to the curricular topic
1 2 3 4 5
1 2 3 4 5
4)
Image "J" tutorial
1 2 3 4 5
a. Beginning (opening images, saving as …
i) original, ii) enhanced, iii) scale/sized,
b. Intermediate (mapping, blowups, labels, measurements, edge detection, (changing your
data and creating an artifact…)
c. Advanced (particle analysis, thresholding, binary, counts, area, plug ins ??
5)
Images to Interpret
1 2 3 4 5
c. Unique to the activity (pure samples, reference or standard images
d. Problem images? Charging? Challenging? Topographic? Etc…
6)
How does a Phenom / SEM work
1 2 3 4 5
a. Creative tour of the scope (ex. write about an images' journey ….)
b. Labeled diagram with explanation of workings
c. What would make this machine or images even better? How would you do it?
d. Visual virtual tour through the scope
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TEACHER RESOURCES/REFERENCES
Optional---Warmup ACTIVITY? "CAN YOU CHEW GUM AND DO % COMPOSITION PROBLEMS TOO?"
Lab Activity: Calculate Percent Composition of Chewing Gum
We're proposing chewing gum has 2 components - gum and sugar. The sugar will dissolve when
you chew it, leaving behind only the gum. Thus we can calculate the percent of sugar in the gum.
This works best with 4-5 students in a group. Each student gets a piece of gum.
Get the mass of a paper cup. Unwrap the gum and put all sticks in the cup. Get the mass of the
gum + cup. Calculate the mass of the gum. (This is "total" gum - includes gum and sugar).
Each person chews their piece of gum for 5 minutes. Then everyone puts chewed gum in the cup.
Get mass of chewed gum + cup. Calculate mass of chewed gum. (This is just the gum, no sugar.)
Calculate the mass of sugar in the gum. (Total gum - chewed gum)
Calculate the percent sugar in the gum. (mass sugar/total gum x 100)
Conclusion Questions:
1) Would a dentist recommend chewing this gum? Why or why not?
2) Would this procedure work with a sugarfree chewing gum? Why or why not?
2) Would changing the number of pieces of gum change the results of the lab? Explain.
Optional: Homework problems related to % composition.
--------------------------------------------------------------------------------------Possible lesson using a variety of samples:
A) Relative Composition of a mixture
Inquiry lesson that allows focus on the composition of a mixture sample composed of an unknown amount of
various substances (2-5). A predictive component and hypothesis formation based on images and observations of
optical microscope and SEM images of dehydrated and crystallized substances obtained from the mixture.
Example sample prep? 250g of NaCl, 750g of Sand. Students would receive between 3.0 and 5.0 g of the sample.
We would suggest 3-5 different combos for a classroom application, (use your secret code to identify each different
sample, i.e. 6141 = 60/40 mix, 2181 = 20/80 mix, etc…) (This could be complicated by using NaCl, Sand, Sugar?)
TASKS EXPANDED/VARIATIONS*
B) Morphology and Crystal structure of various chemicals. Students would be give a solution containing 1-4
substances and be asked to dehydrate and recrystallize the solutes. Optical and SEM images would be used for
relative composition analysis. Observation / Inference lesson. (Identification possible with comparison to knowns in
the lab).
C) Forensic connection: Substance found in blood or beverage or at the crime scene…. Recrystallized and observed
and identified with a comparison to knowns. Observation /Inference lesson.
D) Standards/Quality control:
Determination of purity of a stock or purchased substance or creative sample (doctored up!)?
SEM Lesson
Mixture Analysis
SEM – Mixture Analysis
1) Operating a Scanning Electron Microscope
RULES TO LIVE
(OR DIE) BY  !!
1) KNOW BEFORE
YOU GO! (If you are
unclear about anything
ask 1st!)
2) The sample MUST
be dry and stuck
down and
nonmagnetic!
3) Make sure the
sample is 4 turns down
from the top of the
holder.
4)
5)
6)
THIS WILL BECOME… TEACHER PROTOCOLS FOR
SECURITY, TRANSPORT AND SETUP OF …A DESKTOP SEM!!
Fix fix fix fix
Before researchers can take their first SEM image of, say, a mosquito,
they have to prepare the specimen. Because SEMs, unlike optical
microscopes, operate in a vacuum and rely on electric fields to work,
sample preparation can be a complicated process. Researchers start by
making sure it is dry and then clean it of any dust or debris. Once
clean and DRY, it's ready to be mounted in the SEM if the specimen
is fairly conductive. Otherwise, it's coated in a conductive material
like gold or platinum through a process called sputter coating before
it's ready for viewing. Sputter coating allows a sample to be
grounded, preventing it from being damaged by the electron beam.
(This creates an issue called 'charging' and causes an over exposed/
bright washed out image).
This SEM Business Is All Wet
Since SEMs operate in a vacuum, researchers always assumed
samples observed using an SEM would need to be moisture-free. This
requirement kept them from observing biological specimens like
living cells. Fortunately, the newest generation of SEMs has
overcome these limitations. For instance, some SEMs now require
only a moderately strong vacuum for operation. While these
microscopes sacrifice resolution in the process, they're much more
flexible in terms of the samples they can view. Other companies have
devised a method of observing samples in solution. By separating the
samples from the vacuum chamber using a high-strength film, these
SEMs can observe objects never before subject to such high levels of
magnification.
Since specimens placed in the microscopes also are subject to a
vacuum, they sometimes undergo additional preparation to ensure that
they hold up under such extreme conditions. Biological samples, for
instance, are typically dehydrated before being placed in an SEM.
Otherwise, the low atmospheric pressure of a vacuum would cause
the water in biological samples to evaporate quickly, destroying the
sample in the process. Other specimens are frozen before they are
examined, and still others are chemically treated so that they survive
the magnification process.
Researchers, like photographers, have a variety of controls over the
images they produce. The magnification, focus, contrast and
brightness of an image are all at the fingertips of the operator of an
SEM. While some models have dedicated hardware for these settings,
the more recent integration of computerized controls has both lowered
the cost of SEMs and simplified their operation.
Finally, make sure to observe the safety precautions when operating
the instrument. In the process of scanning specimens, stay focused on
what you are doing. Your time will be limited and you'll benefit from
being organized with image prep/ notes, labels and a pattern of
sequential data collection, (have a plan!).
FOLLOW THE PROTOCOLS OF THE MACHINE!
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MARGIN
NOTES
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TEACHER RESOURCES/REFERENCES
2)Optical Scope--------------THE BASICS!--------------------A MICROSCOPE*
We have two kinds of light microscopes available in the lab. Light microscopes use light rays that are magnified and
focused by means of lenses. The binocular dissecting microscope is designed to study entire objects in three
dimensions at low magnification. The compound light microscope is used for examining small or thinly sliced sections
of objects under magnification that is higher than that of the dissecting light microscope. Illumination is from below,
and the light passes through clear sections but does not pass through opaque sections. To improve contrast, the
microscopist uses stains or dyes that bind to cellular structures and absorb light. When a specimen must be prepared
for observation, the object should always be viewed as a wet mount. A wet mount is prepared by placing a drop of
liquid on a slide or, if the material is dry, by placing it directly on the slide and adding a drop of water or stain. The
mount is then covered with a coverslip. The instructor will demonstrate how to make a wet mount.
Always observe the following rules for microscope use:
1. The lowest power objective should be in position both at the beginning and end of microscope use.
2. Use only lens paper for cleaning lenses.
3. Do not tilt the microscope when viewing a wet mount.
4. Keep the stage clean and dry to prevent rust and corrosion.
5. Do not remove parts of the microscope.
6. Keep the microscope dust-free by covering it after use.
7. Report any malfunctioning or mechanical issue.
Total Magnification
Total magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the magnification of
the objective lens. (ex. eyepiece 10x X objective lens 40x = 400X total magnification).
Focusing the Microscope
Lowest Power*
1. Turn the nosepiece so that the lowest power lens is in straight alignment over the stage.
2. Always begin focusing with the lowest power objective lens.
3. With the coarse-adjustment knob, lower the stage until it stops.
4. Place a slide of the letter e on the stage, and stabilize it with the clips (instructor will demonstrate if necessary).
5. Again, be sure that the lowest power objective is in place. Then, as you look from the side, decrease the distance
between the stage and the tip of the objective lens until the lens comes to an automatic stop or is no closer than 3
mm above the slide.
6. While looking into the eyepiece, rotate the diaphragm (or diaphragm lever) to give the maximum amount of light.
7. Slowly increase the distance between the stage and the objective lens, using the coarse-adjustment knob, until the
object – in this case the letter e - comes into view, or focus.
8. Once the object is seen, you may need to adjust the amount of light. To increase or decrease the contrast, rotate the
diaphragm slightly.
9. Use the fine-adjustment knob to sharpen the focus if necessary.
10. Practice having both eyes open when looking through the eyepieces, as it greatly reduces eyestrain.
*To locate small objects that are seen on low power, place these objects in the center of the field before rotating to high
power.
Higher Powers
Compound microscopes are parfocal; that is, once the object is in focus with lowest power, it should also be almost in
focus with the higher power.
1. Bring the object into focus under the lowest power by following the instructions in the previous section.
2. Make sure that the letter e is centered in the field of the lowest objective.
3. Move to the next higher objective (low power, 10x) by turning the nosepiece until you hear or feel it click into
place. Do not change the focus; parfocal microscope objectives will not hit normal slides when changing the
focus if the lowest objective is initially in focus.
4. If any adjustment is needed use only the fine-adjustment knob. Always use only the fine-adjustment knob with
higher powers.
5. On your drawing of the letter e that you made for exercise 1, draw a circle around the portion of the letter that you
are now seeing with the higher-power magnification. Repeat steps 2-4 with the 40x power objective lens.
6. When you have finished your observations of this slide (or any slide), rotate the nosepiece until the lowest power
objective clicks into place, and then remove the slide.
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TEACHER RESOURCES/REFERENCES
Image Capture- - -Each scope will have specifics for capturing images, follow your site protocols.
REMEMBER- labels, scale bar, instrument name/model, (i.e. Nikon 155). Paste images captured in the
Image Plate template below.
Image Plate Template (YOUR TITLE HERE)
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
Magnification
and Scale Bar
Name: ___________________Period:_____
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
included.
Magnification
and Scale Bar
included.
(paste image here and fill in info above)
(paste image here and fill in info above)
(make them all the same size? and fill the box?)
(This is still the same sample…but MAGNIFIED!)
QuickTime™ and a
decompressor
are needed to see this picture.
QuickTime™ and a
decompressor
are needed to see this picture.
(without a scale bar? ….useless? )
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
Magnification
and Scale Bar
Sample(s) :
File name: ______________________
Tool/Instrument: __________________
included.
(paste image here and fill in info above)
(have you used labels/arrows to highlight?)
Magnification
and Scale Bar
included.
(paste image here and fill in info above)
QuickTime™ and a
decompressor
are needed to see this picture.
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Image Quality
TEACHER RESOURCES/REFERENCES
When you look at a specimen using a microscope, the quality of the image you see is assessed by the following:
* Brightness - How light or dark is the image? Brightness is related to the illumination system and can be changed
by changing the voltage to the lamp (rheostat) and adjusting the condenser and diaphragm/pinhole apertures.
QuickTime™ and a
decompressor
are needed to see this picture.
Image of pollen grain under good brightness (left) and poor brightness (right)
* Focus - Is the image blurry or well-defined? Focus is related to focal length and can be controlled with the focus
knobs. The thickness of the cover glass on the specimen slide can also affect your ability to focus the image -- it can be
too thick for the objective lens. The correct cover-glass thickness is written on the side of the objective lens.
QuickTime™ and a
decompressor
are needed to see this picture.
Image of pollen grain in focus (left) and out of focus (right)
* Resolution - How close can two points in the image be before they are no longer seen as two separate points?
Resolution is related to the numerical aperture of the objective lens (the higher the numerical aperture, the better the
resolution) and the wavelength of light passing through the lens (the shorter the wavelength, the better the resolution).
QuickTime™ and a
decompressor
are needed to see this picture.
Image of pollen grain with good resolution (left) and poor resolution (right)
* Contrast - What is the difference in lighting between adjacent areas of the specimen? Contrast is related to the
illumination system and can be adjusted by changing the intensity of the light and the diaphragm/pinhole aperture.
Also, chemical stains applied to the specimen can enhance contrast.
QuickTime™ and a
decompressor
are needed to see this picture.
Image of pollen grain with good contrast (left) and poor contrast (right)
http://science.howstuffworks.com/light-microscope2.htm
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TEACHER RESOURCES/REFERENCES (need definitions filled in on this page….FIX)
3) A. Background/ Definitions (Important vocabulary for this unit) & background
1. Mixture –
2. Homogeneous-
List at least 3 examples of Homogeneous mixtures:
a. iced tea – brown clear liquid, consistent throughout
b.
c.
d
4.
Heterogeneous
List at least 3 examples of Heterogeneous mixtures:
a. orange juice with pulp – orange cloudy liquid, inconsistent, pulp on the surface
b.
c.
d
4. Pure substance
5. Element
6. Compound
7. Physical Property
8. List 2 examples of each of the following:
a) Solution
b) Colloid
c) Suspension
11. List the contents of your kitchen or bathroom sink . What do you think most of the substances
would be classified as? Support your answer using your observations and background knowledge.
12. You're told that you will be receiving a heterogeneous mixture from the Gatorade company that
contains 5 ingredients. List your chemistry ideas to figure out what they are and determine a way
to separate them?
B. Related website(s) & article(s)
1)
2)
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4. Image 'J' tutorial
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TEACHER RESOURCES/REFERENCES
http://rsb.info.nih.gov/ij/download.html for downloads and information.
http://rsb.info.nih.gov/ij/docs/examples/index.html tutorials and examples
(Use the force!)
B- Beginning (opening images, saving as
tiff/JPEG… i) original, ii) enhanced, iii)
scale/sized,
QuickTime™ and a
decompressor
are needed to see this picture.
I- Intermediate (mapping, blowups, labels,
measurements, edge detection, (changing your
data and creating an artifact…)
A- Advanced (particle analysis, thresholding,
binary, counts, area, creating plug ins, (Use the
videos and online assistants… ?)
Image 'J ', IJ, can work with variety of formats but we will usually see JPEG and Tiff. It is helpful to
establish a logical and informative pattern for saving and labeling images you save from both the optical
scope and desktop SEM like the Phenom, ex. flylegtip450xphenom.tiff
B- Beginning
Here are the basics…
1) Launch Image J and you'll see a simple tool bar.
2) FILE Open (select your stored image) (You need to keep an original data file so after any change or
alteration make sure that you select FILE SAVE AS  'tiff' or 'jpeg' and name logically and detailed).
3) Improving the image? Remember you have the EDIT  Undo feature.
4) Start with these options and view the results….(Remember #3 above).
a) PROCESS Enhance Contrast (equalize histogram will balance black/white/grays) AND/OR
b) PROCESS Smooth or Sharpen
c) IMAGE  ADJUST  Size ( X 'Constrain Aspect Ratio' will keep the image from tweaking out/distorting)
I- Intermediate (make sure you have completed the basics)
1) Select your tool of choice from the tool bar (straight line, rectangular selection, text tool, etc…)
(double-click to change the properties of the tool).
2) Measuring? For area use the rectangular/ freehand / polygon tool/ etc… and then go to Analyze
Measure; For a length use the line tool and select the two points and then go to Analyze Measure.
3) Labels? Choose the text tool on the tool bar and draw a text box near where you want your label.
4) Adding a label arrow? Use the arrow tool and draw between the text box and the image part
A- Advanced
Use the online assistants and web tutorials.
SEM Lesson
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SEM – Mixture Analysis
TEACHER RESOURCES/REFERENCES
5) Images to Interpret and Critique
A. Unique to the activity (pure samples, reference or standard images
Problem images? Charging? Challenging? Topographic? Etc…
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7) How Does a SEM Work?
After reading the article below show your understanding of how a SEM works
by doing ONE of the activities below.
d.
e.
f.
Creative tour of the scope (write about an images' journey ….)
Labeled diagram of an SEM with explanation of workings
In a paragraph describe what would make this machine or images even better?
How would you do it?
Microscopes – Help Scientists Explore Hidden Worlds
The microscope is an invaluable tool in today's research and education. It is used in a wide range of
scientific fields, where major discoveries in biology, medicine and materials research are based on
advances in microscopy. As the need to see the world at a smaller and smaller scale has grown scientist
looked for ways to improve on the light microscope (see Fig. 1 below). In 1938 Ernst Ruska develops the
electron microscope and in 1981 Gerd Binnig and Heinrich Rohrer invent the scanning tunneling
microscope that gives three-dimensional images of objects down to the atomic level. In 1986 all three men
receive the Nobel Prize in Physics. The greater resolution and magnification of the electron microscope is
because the wavelength of an electron; its de Broglie wavelength is much smaller than that of a photon of
visible light. Conventional light microscopes use a series of glass lenses to bend light waves and create a
magnified image. The electron microscope uses electrostatic and electromagnetic lenses in forming the
image by controlling the electron beam to focus it on the surface of the specimen. The SEM shows very
detailed 3-dimensional images at much higher magnifications than is possible with a light microscope
(because electrons travel at a smaller wavelength) but the images are in black and white because aren’t
created using light (see Fig. 2 below). To prevent electrons from colliding with air molecules all SEMs
work under a vacuum. Initially before samples are stuck down on a metal stub have to be prepared
carefully to withstand the vacuum inside the microscope. No wet, magnetic or alive samples can go into an
SEM. Biological specimens are first dried in a special manner that prevents them from shriveling. Also
because the SEM illuminates with electrons, they also have to be made to conduct electricity. SEM
samples are often coated with a very thin layer of a metal (i.e. gold) by a machine called a sputter coater.
Once the specimen is prepared its ready to go into the SEM. The sample is placed inside the microscope's
vacuum column through an air-tight door. After the air is pumped out of the column, an electron gun [at the
top] emits a beam of high energy electrons. This beam travels downward through a series of magnetic
lenses designed to focus the electrons to a very fine spot. Near the bottom, a set of scanning coils moves
the focused beam back and forth across the specimen, row by row, this is called rastering. As the electron
beam hits each spot on the sample, secondary electrons are knocked loose from its surface. A detector
counts these electrons and sends the signals to an amplifier. The final image is built up from the number of
electrons emitted from each spot on the sample.
Figure 1: Resolution scale line
Figure 2: Electron Microscope
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7) How Does a SEM Work? (cont.)
The Scanning Electron Microscope is revealing new levels of detail and complexity in the amazing world of microorganisms and miniature structures. Follow this link to a video that shows, generally, how an SEM works.
VIDEO LINK: SEM Video Link
http://www.youtube.com/watch?v=lrXMIghANbg&eurl=http%3A%2F%2Fvideo%2Egoogle%2Ecom%2F
videosearch%3Fq%3Delectron%2Bmicroscope%26hl%3Den%26emb%3D0%26aq%3D0%26oq%3Delect
ron%2Bmic&feature=player_embedded
The good news is that the Phenom SEM that you will be using in class is a lot smaller and easier to use than
the one shown in the above video but the Phenom work basically the same way as the SEM in the video.
To see a brief preview of how to operate the Phenom go to the link below.
VIDEO LINK: PHENOM Video Link
http://www.youtube.com/watch?v=Rk7jGgMlPek&feature=related
QuickTime™ and a
decompressor
are needed to see this picture.
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