Microscopy in general

• A quick guide to light microscopy in cell biology – Kurt Thorn MBoC 2016 (27) p.219
• Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition– Allison North JCB 2006 (172) 9
• Confocal Principles– Zeiss poster explaining confocal microscopy
• Multiphoton microscopy explained

Resources on microscopy:

• MicroList.org a searchable microscopy resources dbase of the HMS (Jennifer Waters / Talley Lambert and Anne Jost)
• iBiology Microscopy Courses (UCSF/HHMI)
  A collection of online microscopy lectures explaining different aspects of microscopy.
• Molecular Expressions
• Nikon Microscopy U
• Zeiss MicroImaging Campus
• Olympus Microscopy Resource Center

Fluorescent spectra tools:

• FPbase (great interactive fluorescent protein database by HMS Talley Lambert)
• Fluorescence SpectraViewer (Life Technologies), FluoScout (Leica), spectral compatibility for many fluorophores.
• Comparative plotting of fluorescent proteins characteristics (and of photoconvertable ones)

Considerations before performing Microscopy data acquisition

• Choose the right technique for your experiment (JCS 2007 (122) 753)
• Have a plan before acquiring images.
• Choose proper materials:

coverslip thickness (#1.5), live cell chambers, fixation buffers, dyes, affinity labeling tools, mounting media.

• Test labelling strategies thoroughly before performing large scale sample sets or intravital microscopy.

nuclear counterstain, cell mask, specific stain, multi dye stain, live cell timing, in vivo tile/stack size, fiducial marker etc.

• Consider that data analyses often require labeling of defined structures to which you can relate your stain of interest.
• Rethink your experiment to implement a workflow from doing the experiment, imaging it and analyzing the data. All three aspects will take time and effort to complete.
• Estimate the resolution (spatially, temporally, spectrally) you need to draw a conclusion
• Estimate the optimal image data volume you need to draw a conclusion (consider population variation of a parameter and group size to reliably measure changes).