Papers you should read before you start:
- Fiala JC (2005) Reconstruct: A free editor for serial section microscopy. J Microscopy 218:52-61. (PDF)
- Fiala JC, Harris KM (2001) Cylindrical diameters method for calibrating section thickness in serial electron microscopy. J Microscopy 202(3):468-472. (PDF)
- Harris KM, Spacek J, Bell ME, Parker PH, Lindsey LF, Baden AD, Vogelstein JT, Burns R (2015) A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development. Sci Data Sep 1;2:150046. PMCID: PMC4555877. (PDF; Tables)
- Harris KM (1994) Serial electron microscopy as an alternative or complement to confocal microscopy for the study of synapses and dendritic spines in the central nervous system. In: Three-dimensional confocal microscopy: volume investigation of biological specimens (Stevens JK, Mills LR, Trogadis JE eds), pp 421-445. New York: Academic Press, Inc. (PDF)
Steps for starting a new series from scratch:
- Have a Dendrite Analysis Spreadsheet ready and record all pertinent information as you work.
- Make a new series & import images
- Calibrate pixel size
- Align EM images & propagate (if manually done in Reconstruct)
- Calibrate section thickness
Step 1: Make a new series & import images
Please see the Starting a New Series PDF for complete details.
Step 2: Calibrate pixel size
Please see the Calibration Protocol PDF for complete details.
Here is the Calibration Protocol - using a scale bar PDF if you need to calibrate using a scale bar.
WARNING: CALIBRATION MUST BE DONE BEFORE YOU START TRACING.
Step 3: Manual alignment of serial EM images in Reconstruct
Please see the Reconstruct Alignment Protocol. (PDF, Word)
Protocol for serial EM image alignment using AlignEM-SWiFT or TrakEM2 is also available.
