1. LMD of Sperm from a Cell Mixture
So far in this course you have had an introduction to of Laser microdissection, a little microscopy, and cell identification and in the lab we preparing samples for laser microdissection and been able to get your first look at the LMD instrument and its operation.
Well, why are we doing all this. What most of you are interested in is LMD of sperm cells from a mixture and how we can use LMD to separate sperm from that mixture.

2. Why is cell separation important?
DNA mixtures are frequently encountered in forensic evidence particularly in sexual assault crimes.
Identify individuals involved with a crime
Genotype mixture interpretation and statistical analysis can be challenging and time consuming
Separation of different cell types can provide cell fractions from individuals involved in a crime.
DNA mixtures are frequently encountered in forensic evidence particularly in sexual assault crimes.
Identify individuals involved with a crime
Genotype mixture interpretation and statistical analysis can be challenging and time consuming
Separation of different cell types can provide cell fractions from individuals involved in a crime.

3. Separation Methods Laser Microdissection
Preferential Lysis (differential extraction)
Flow Cytometry
Microchip separation
Membrane Filtration
Magnetic Antibodies
Y- chromosome (non-physical separation)
Laser Microdissection
There are a few different separation methods, some more successful than others,
Preferential Lysis or the differential extraction is the current gold standard for separating sperm from epithelial cells in sexual assault evidence. Tomorrow, I will talk about this in more detail and show you a comparative study with LMD.
Some other mehods
Flow Cytometry
Microchip separation
Membrane Filtration
Magnetic Antibodies
Y- chromosome (non-physical separation)
Laser Microdissection

4. LMD Technology
Microscope
Objective
Slide 
Specimen
Tube 

5. Sperm & Epithelial Cell Mixture
Technical Issues of Sperm ID under LMD using brightfield conditions
PEN film architecture
Stain is necessary
It helps to adjust focal plane
May be resolved by new EP “Director Slides”
High density of epithelial cells
May need to dilute pellet over larger area
No cover slip
Looking through 1mm slide
Set correction collar objective
If you have done sperm searches in the past one of the first things you will notice when you are looking at your slides under the LMD microscope is that they look a bit different than you may be accustomed to.
Technical Issues of Sperm ID using brightfield LMD
PEN film architecture - what do I mean by architecture. In the manufacturing of the PEN membrane there are pores that are visible in the background of the slide. (point out in slide). Originally, this was not much of a problem when the technology was developed for tissue sections but for the application of smears it can be a bit annoying. Although you would not mistake a pore for a sperm it makes screening the slide slower. What do we do….
Stain is necessary
Adjust focal plane helps - again slows screening process
BUT May be resolved by new slides in development
High density of epithelial cells
May need to dilute pellet over larger area
No cover slip
Looking through 1mm slide
Set correction collar objective (CORR - correct for spherical aberrations due to the glass
between the object and the objective)

6. Identification of sperm cells
Leica AS LMD instrument (older version of software shown)
We will be going over the software in more detail when we sit down in smaller groups but I just wanted to cover a few basic points.
This is a screen capture of a sperm/ec mixture seen under the LMD. This is the Leica AS LMD instrument (older version of software shown). All the experience that myself, kelli and pat have are on this older instrument. You fortunately will be using the new improved insturment.
You will primarily be using three cutting options for sperm dissection. Circle (reuse), live cut, maybe the line tool

7. After Laser Microdissection
Sperm cuttings in tube
After laser microdissection
You can also look into the tube to view the cutting. It is not necessary but it is a nice feature and somehow being able to see the cuttings in the tube lends a little more confinence in the final result especially if I am going to do Low copy # work.

8. Epithelial Cell Recovery
Epithelial cell in
collection tube
Epithelial cells can also be recovered. Line and circle tool used here to either collect the cell nucleus or the entire cell.
Again the cell can be visualized in the tube after cutting. (actual cutting from lower right)
If we have time in lab today you might want to collect some e cells but I warn you that the DNA extraction method that will be used in this lab does not work very well for e cells.

9. Current LMD Collection Software

10. Warning! Laser in Use