High and Partial Vacuum SEM on SeaMonkeys®
OPT 307 Spring 2010
Title Image
By: Jonathan Brand
The Institute of Optics
University of Rochester



Introduction
Materials and Methods
Results
Discussion and Analysis
Conclusion
Works Cited
Comment




 

Introduction

SEM is a powerful tool to evaluate biological specimens.  Working with unknown specimens however, can prove difficult when their reaction to certain drying processes may or may not alter a sample.  Working with SeaMonkeys, or brine shrimp, HMDS and air-drying preparation were compared when collecting micrographs in the SEM.  An anaglyph of one of the micrographs was constructed and colorization of micrographs was attempted.  Identifying different microscopic organisms is important for monitoring the health and changes in marine and estuarine environments.

Materials and Methods

Specimen

SeaMonkeys are a form of brine shrimp, from the Artemia genus, usually grown by young children for early science experiments.  Beyond entertainment, brine shrimp are an important component of fish food as well as specimens for various research.  Males and females are differentiated by the presence of graspers on the male's heads.  In the wild, brine shrimp feed on phytoplankton, in domesticated environments the specimens are fed an algal powder three times a week.  The specimens took two weeks to grow to a size appropriate for collection and SEM preparation.

HMDS Drying

Brine Shrimp were placed in a water/5% Glutaraldehyde mixture to polymerize the specimens.  Using the miscibility of water and alcohol, water is removed from the samples by gradually increasing the percentage of alcohol in a water/alcohol solution.  After this, hexamethyldisilazane (HMDS) replaced alcohol using the same miscibility property.  Leaving the HMDS prepared brine shrimp under a chemical hood, the specimens dried and were placed onto SEM stubs.

Partial Vacuum SEM Operation

Running the SEM in PV mode allowed air dried samples to interact with the electron beam.  The specimen chamber pumps down to high vacuum and it is slowly increases to allow the recording of micrographs.  The electron beam operated at 20 kV as well.  A working distance range increased to 10 to 15mm

Air Drying

To compare the effects of the HMDS drying process and damage done to specimens, some were air dried and placed onto slides.  These specimens were observed used the SEM Partial Vacuum mode. 

Light Microscopy

To optimize the quality of micrographs, a light microscope was used as an aid to place specimens onto SEM stubs.  A desktop microscope was used to observe a live specimen for light microscopy imaging.

Sputter Coating

A 12nm layer of gold was sputtered onto HMDS dried samples with the Denton Vacuum Sputter Coater in the sample prep lab.  At a vacuum of about 50 mT a 15 mA current was run across an Argon gas allowing layer of gold to form on the sample.  The thick gold layer was used because charging in SEM Micrographs were observed in specimens with thinner coatings.

High Vacuum SEM Operation

The Supra 40VP SEM at the University of Rochester SEM /TEM Lab acquired all the micrographs seen in this report.    The electron beam operated a 20 kV and a working distance between 10 and 12mm was kept while acquiring micrographs.  The larger working distance maximized the depth of focus for the relatively large object viewed in the SEM.

Results

Light Microscopy

A light microscope without a recording device was used to transfer dried samples onto SEM stubs.  To replicate this, A desktop microscope captured images of live brine shrimp.

fig 1

Figure 1.

Fig 2

Figure 2.

Legs of the specimen are the darkened region next to the body

Figures 1 and 2 show the entire brine shrimp, however there are few details that are recognizable from the image.  The black dots at one end are the eyes.  The microscope could not resolve the legs that propel the brine shrimp, only a dark spot along the body represents

Partial Vacuum SEM Operation

Running the SEM in PV mode allowed air dried samples to interact with the electron beam.  The specimen chamber pumps down to high vacuum and it is slowly increases to allow the recording of micrographs.  The electron beam operated at 20 kV as well.  A working distance range increased to 10 to 15mm.  

Fig 3

Figure 3

This is potentially a female in light of no graspers present on the head, a key feature on male brine shrimp.

Fig 4

Figure 4

The more complete specimen between the air dried samples.  The sex of the sample is less evident.  In both cases, PV SEM and air drying performed better than a light microscope, but could not compare to HV SEM micrographs.

High Vacuum SEM Operation

High Vacuum SEM is the standard mode the SEM is operated at and HMDS dried samples were observed in this environment

Fig 5

Figure 5

A male brine shrimp, identified by its graspers. 
These appendages are used to hold onto females while mating. 

Fig 6

Figure 6

A close up of one graspers used to hold onto females while mating

Fig 7

Figure 7:

The top portion of a brine shrimp.  The food track is cracked
in the middle of the torso.  Some of the 11 pairs of legs that
propel the shrimp and move food to the mouth are seen..

Fig 8

Figure 8

The feeding appendages deliver algae to the mouth of brine shrimp. 
Food is either caught by these or it is delivered from the legs that catch algae.

Fig 9

Figure 9

The main protection of the body is seen here.  Along with the
top of the food track, there are also plats the hinge off the
side of the exoskeleton

Fig 10

Figure 10

Swimming legs of a brine shrimp, at the ends are grabbers that
can take algae and phytoplankton out of the water and feed the brine shrimp

Discussion and Analysis

Colorization and 

Using Adobe Photoshop Fig 5 and 6 were colorized, an anaglyph was created with Fig 7.

Fig 11

Figure 11: Closeup of an eye and antenna

Fig 12

Figure 12: Protective plates and legs with feelers that grab onto food.

Anaglyph Simulation

Fig 13

Figure 13:  

Upper portion of brine shrimp showing eyes, antenna and graspers.
Use a pair of red/cyan glasses to see the three dimensional effect.

Conclusion

Environmental SEM has can collect micrographs of biological samples, however the sample will determine how much detail in the data becomes available.  In PV SEM the outer layer was present, however the greater details of anatomy only became apparent through HMDS drying.  Using a combination of both techniques may be the best procedure for observing smaller organisms and PV SEM should not be disregarded.  HMDS did destroy the outer part of specimens through the drying process.

Works Cited

"BRINE SHRIMP." Clinton High School Science. Web. 25 Apr. 2010. <http://www.drwhitey.com/Ecology/Artemia/BrineShrimp.html>.

"USGS Utah Water Science Center: Great Salt Lake." USGS Utah Water Science Center - Home Page. Web. 25 Apr. 2010. <http://ut.water.usgs.gov/greatsaltlake/shrimp/>.

 









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