Many labs use one standardized protocol for labeling with all antibodies, irrespective of whether the targets are membrane- or cytoskeleton-associated, nuclear or cytosolic. Thus, incomplete penetration of antibodies can lead to false data interpretation. The GFP and DAPI labeling extend throughout the whole tissue slice, but the antibody labeling (red) is restricted to a few sections at the top and bottom, although all of the dendrites should contain MAP2 label. A and B show merged xy images taken from the top and middle of the stack, respectively C shows a merged xz reconstruction of the stack (i.e., a “slice” through the stack, perpendicular to the image plane). The tissue was labeled with three colors: blue (DAPI), marking the nuclei green (GFP), marking the dendrites and red (C圓), marking the microtubule-associated protein MAP2 by indirect antibody labeling. A z-stack of optical sections, 18.2 μm in total thickness, was captured from a mouse brain tissue slice using a confocal microscope (LSM 510 Carl Zeiss MicroImaging, Inc.) with a C-Apochromat 40×/1.2 NA objective (1-μm optical slice thickness, 50 z-sections collected at 0.33-μm intervals). Because one short article cannot be an exhaustive “how to” guide, I have also assembled a bibliography of a few highly recommended textbooks and microscopy web sites, which readers should consult for more extensive treatments of the critical issues introduced here. Throughout, we will be on the look-out for problems that can produce misleading results, using colocalization as the most common example. Here, I take the reader through the imaging process, from sample preparation to selection of the imaging and image-processing methods. How can this be? Many factors contribute. But on the confocal microscope, there is no yellow in the merged images. Upstairs in the lab, a researcher collects a predominantly yellow merged image on a basic microscope, naturally interpreted as colocalization of green and red signals. As an example of a common problem, take colocalization.

Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!ĭeliberate scientific fraud exists, but in modern microscopy a far greater number of errors are introduced in complete innocence.

Instead, this is for beginners who gulp with alarm when they hear the word “confocal pinhole” or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. You see something you report what you see. Imaging can be thought of as the most direct of experiments.

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