Tips and Tricks

Advance Imaging Facility 

Image analysis or data manipulation?

Alternatives for fluorescent proteins in live cell imaging

Wound healing assay; protocol for imaging and analysis of data.

Antibody page; Information about antibodies and a comprehensive list of antibody producers and suppliers.

Rabbit monoclonal antibodies; antibodies with a higher affinity than mouse monoclonal antibodies.

List of suppliers for petri dishes/plates with cover glass (like) bottom.

Organelle markers.

 

Imaging

The images you take are never better than the sum of the components used to take the images. This means that clean objectives, clean and correct cover glasses, proper microscope setup and image settings are all important to get the optimal image!

Image analysis or data manipulation?

Coverslip thickness

Most objectives are designed for a coverslip thickness of 0.17 mm. Using the wrong coverslip thickness will introduce spherical aberration resulting in loss of contrast and sharpness in the image. The list below gives the standards for the most common coverslips. From this it is clear that #1.5 fits best to a coverslip thickness of 0.17 mm.

Coverslip number
Coverslip thickness (mm)
#0
0.08 - 0.12
#1
0.13 - 0.17
#1.5
0.16 - 0.19
#2
0.17 - 0.25

Keep in mind however that most coverslips in a batch of #1.5 are still not optimal (= 0.17 mm) and often a few percent of coverslips in a box are outside the range of 0.16-0.19 (dependent on the manufacturer). Only when you have a thick sample or there is a relative thick layer of mounting medium between your coverslip and sample you might try using #1 or #0 coverslips.

Slides

There are also different quality slides on the market sold by different companies. This linked page gives a good overview of what is available.

Data storage

You can not store the data on the microscope computer or analysis computer for extended periods. Usergroups can store their data on the Research File Store(R-drive). Request an account via Dr Kees Straatman.

Fluorescence microscopy in the spot light

Special issues:

Microscopy and Research Technique Vol 77 (2014) Fluorescence microscopy in the spotlight.
Methods in Cell Biology Vol 114, (2013) Digital Microscopy
Methods in Enzymology Vol 505, (2012) Imaging and Spectroscopic Analysis of Living Cells — Live Cell Imaging of Cellular Elements and Functions.

Articles:

Designing a rigorous microscopy experiment: Validating methods and avoiding bias (2019) Jost and Waters, J Cell Biol. DOI: 10.1083/jcb.201812109.
Imagining the future of bioimage analysis (2016) Meijering et al. Nat. Biotech 34: 1250-1255. 
Quantitative assessment of fluorescent proteins (2016) Cranfill et al. Nat Meth 13: 557-562.
Cellular growth defects triggered by an overload of protein localization processes (2016) Kintaka et al. Sci Rep. 6: 31774.
Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM (2012) Ishikawa-Ankerhold et al. Molecules 17: 4047-4132.
Immunolabeling artifacts and the need for live-cell imaging (2012) Schnell et al. Nat Meth 9: 152-158.
Antibody validation (2010) Bordeaux et al. Biotechniques. 48:197-209.
A guide to super-resolution fluorescence microscopy (2010) Schermelleh et al. J. Cell Biol. 190: 165-175.
Accuracy and precision in quantitative fluorescence microscopy (2009) Waters. J Cell Biol. 185: 1135-1148.
Live-cell microscopy – tips and tools (2009) Frigault et al. J Cell Sci. 122: 753-767.
New ways to see a smaller world (2008) Nathan Blow. Nature 456: 825-828.
Spatial quantitative analysis of fluorescently labeled nuclear structures: Problems, methods, pitfalls (2008) O. Ronneberger1, D. Baddeley, F. Scheipl, P. J. Verveer, H. Burkhardt, C. Cremer, L. Fahrmeir, T. Cremer5 and B. Joffe. Chrom. Res. 16: 523-562.
Fluorescence microscopy - Avoiding the pitfalls (2007) Claire M. Brown. J. Cell Sci. 120(10): 1703-1705.
The good, the bad and the ugly (2007) Helen Pearson. Nature 447: 138-140.
Seeing is believing? A beginners' guide to practical pitfalls in image acquisition (2006) Alison North. JCB 172: 9-18.
Counting Cytokinesis Proteins Globally and Locally in Fission Yeast (2005)
Jian-Qiu Wu1 and Thomas Pollard. Science 310: 310-314. This approach can be used to measure the global and local concentrations of any fusion protein.
Fluorescence microscopy (2005) Jeff Lichtman and Jose-Angel Conchello. Nature Methods 2: 910-919.

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