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).