This webpage is merely a guide. You are strongly recommended to read the papers, and browse the URLs given in the references below.
I regard reference (1) “Seeing is believing? A beginners´ guide to practical pitfalls in image acquisition” by Alison North, and the accompanying two papers listed below as key references as mandatory reading before you embark on any science project, or series of experiments, involving the use of core facility light microscopes.
Cited from the references below: “Journals… [should] require more details about microscopy techniques in methods sections or supplementary information”. Do seek advice before your project and also when writing up.
A) Check that the microscope is set up properly
If you are using a widefield (non-confocal) microscope, do ensure that it is set up properly. Read the pages on Köhler illumination on this website. Check the cleanliness of the objective before you start work. Use the correct immersion oil.
Microscopes – e.g. the DeltaVision deconvolution microscope – do not default to a standard resting state between sessions. Do double-check to ensure that the settings that the last user employed do not disrupt your work.
B) Avoiding Spherical aberration
Spherical aberration manifests itself as blurring in the image, and elongation and asymmetry in the axial z-direction. It reduces the intensity, resolution and clarity of the image. To reduce or, hopefully, avoid the effect of spherical aberration, pay particular attention to the thickness of the coverslip, the refractive index of the mounting medium and immersion medium (where used). If using an objective with a correction collar, ensure you take the time and trouble to set this correctly.
C) Select the right fluorophores to match the lasers and filter sets available
Do this at the planning stage before you buy/apply your fluorescent stains. This will ensure that you get the best signal, avoid or reduce undue noise in the image, and manage bleed-through or cross-talk. Colocalisation can only be claimed in the definite absence of bleed-through between selected fluorophores.
C) Be aware about bleed-through and co-localisation
This is important when imaging more than one fluorophore. You must ensure that you have the properly-stained controls to allow for bleed-through, so that this does not disrupt any co-localisation studies that you undertake. Click on the hyperlinks to read more about these topics. Also, be aware that chromatic aberration can lead to an apparent lack of colocalisation in an image z-stack of fluorophores that are, in fact, properly colocalised in the sample. Check this with labelled bead samples beforehand.
D) Do not over-saturate your image
This is important, particularly in confocal microscopy and when collecting z-stack for 3-D studies. Read the PDF on how to set the confocal PMT controls elsewhere on this website. More advice can be found in “Seeing is believing? A beginners´ guide to practical pitfalls in image acquisition” (reference 1, below).
E) Ensure adequate Nyquist sampling in all three axes
Your spatial sampling intervals must be more than two times smaller than the smallest resolvable feature in your specimen. If this sampling requirement is not met, you will have gaps in your data and you can also introduce spurious features into your image which are not present in the sample. Read the webpage on Nyqist sampling elsewhere on this website.
ALWAYS add a properly-calibrated scale bar – no exceptions. See the relevant page on this website.
G) Correcting uneven illumination
Have a look at the following URL for advice: http://jcp.bmj.com/cgi/reprint/56/8/619
From: Leong, FJ; Brady, M & McGee, J O´D (2003) Correction of uneven illumination (vignetting) in digital microscopy images. Jour. Clin. Pathology 56: 619-621.
H) Use of Adobe Photoshop
Be careful how you post-process images in Adobe Photoshop. Have a look at these URLs for further help and advice:
Douglas W. Cromey, SWEHSC Cellular Imaging Core, has a good collection of links to Photoshop tutorials on his web site. He has also some tips for highlighting text and overlaying confocal images in Photoshop. Especially you should check his article “Potentially the most dangerous dialog box in Adobe Photoshop“, which deals with image size and resolution. Also have a look at: http://www.chainstyle.com/tutorials/res1.html and http://www.rawlight.com/resolution.pdf. If you want to merge red/green images in Photoshop, check those from the AIF http://www.aecom.yu.edu/aif/instructions/merging01/merging01.htm and Birgit Ehmer´s instructions http://microscopy.uc.edu/ia/PS_tutorial2.php, or Paul Rigby’s instructions.
Also look at the Other Useful Links given at the bottom of the front page of this website.
1. North, AJ (2007) Seeing is believing? A beginners´ guide to practical pitfalls in image acquisition. Journal of Cell Biology 172(1): 9-18
Accompanying paper and URL: Nature overview article on image quality.
Helen Pearson (2007) The good, the bad and the ugly Nature 447: 138-140 10 May issue.
Accompanying paper and URL: short half-page Nature leader comment on microscope complexity.
Anonymous (2007) Under the microscope: use of `black box´ techniques carries risks
Nature 447: page 116 10 May issue
2. Brown, CM (2007) Fluorescence microscopy – avoiding the pitfalls. Journal of Cell Science 120: 1703-1705.<
3. Rossner, M & Yamada, KM (2004) What´s in a picture? The temptation of image manipulation.
Journal of Cell Biology 166 (1): 11-15.
4. Mackenzie, JM; Burke, MG; Carvalho, T & Eades, A (2006) Ethics and Digital Imaging. Microscopy Today, January 2006, pages 40-41. MSA sub-committee report on the Ethics of digital imaging.
5. Centonze, V & Pawley, JB (2006) Tutorial on practical confocal microscopy and use of the confocal test specimen. Chapter 35, pages 627-649 in: Handbook of Biological Confocal Microscopy, 3rd Edition. (Ed.) JB Pawley. Springer. ISBN 0-387-35921-X