In this hands-on course  you will learn standard TEM sample preparation procedures for biological and soft matter samples (negative staining; bench-top and microwave-assisted fixation, dehydration, epoxy embedding; ultramicrotomy and staining of sections for TEM imaging), for a fraction of cost normally charged for such training.  Theory presentations on Day 1 are followed by demonstration and lab sessions over the course of 2 weeks.

• Day 1: Theory of TEM sample preparation; Typical preparation workflow
• Demonstration and hands-on lab sessions: negative staining; fixation, dehydration, epoxy embedding, ultramicrotomy, staining of sections for TEM
• Manual bench-top and microwave-assisted processing; using the automated EM tissue processor; making and using glass knives for ultramicrotomy;
• Demonstration and use of Leica UC 7 and the UC Enuity next-generation ultramicrotome   

Program

WEEK 1:

Day 1 – Tuesday, October 14

9am-noon       presentations/lectures (ILSB 1143) – sample preparation principles, overview, workflow. Presentation by MIC staff and a remote presentation by Mercedeh Yeganeh, Ph. D. (Leica)

1pm-5pm: Demonstration by MIC staff (ILSB 1121, 1127) :

• Using EM TP tissue processor for automated sample preparation
• Making glass knives for ultramicrotomy
• Trimming epoxy-embedded sample blocks, cutting semi-thin sections for LM, ultramicrotomy

Hands-on follow up: Days – 2, 3, 4:  Students split into two groups.

Group A: Wednesday October 15 all day  and ~1 hour the following morning, 9am-10am

Group B: Thursday October  16, starting at 10am, whole day and 1 hour the following morning

• Dissection of samples, microwave fixation, wash, osmium tetroxide post-fix; loading samples into the EM TP processor. Samples provided by MIC: plant tissue (leaf or root tips), animal tissue, such as liver or muscle; yeast or bacterial suspension culture (?).  At the end of the run of EM TP ne next morning, samples will be transferred to embedding molds or embedding capsules and put in the oven to polymerize for 24-36 hrs.     
• Glow discharge treatment of grids with carbon support film, negative staining of bacterial and phage or macromolecular samples.  

WEEK 2:

Tuesday October 21-Thursday October 24 (= Day 5-8) – half-day sessions for groups of 3-4 (two ultramicrotomes , Leica + MIC staff Tuesday to Thursday; one ultramicrotome and MIC Staff on Friday)

• Trimming of epoxy embedded samples
• Using the ultramicrotomes for semi-thin and ultrathin sectioning with glass knives  (1-2 h per person);

Monday October 27- Thursday October 30 (Day 9 – 12): half-day sessions for groups of 4:

• Staining of sections with Uranyl Acetate and Lead Citrate
• TEM viewing of selected negatively stained samples and UA+LC-stained grids, on JEOL 1200 TEM

Friday October 31 (Day 13):  9am-10am: Quiz, Short Course Certificates are issued.