Starting a New Series

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:

1. Have a Dendrite Analysis Spreadsheet ready and record all pertinent information as you work.
2. Make a new series & import images
3. Calibrate pixel size
4. Align EM images & propagate (if manually done in Reconstruct)
5. 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 /wiki/spaces/khlab/pages/53543137 is also available.

Step 4: Calibrate section thickness