LAUSR

Fluorescence microscopy can be an excellent tool for making quantitative measurements in cells. For particularly rigorous applications, many choose to use confocal microscopes, which use pinholes to throw away the out-of-focus signal, producing an image of a thin focal plane as shown in Figure 1 (1). Confocals have many more degrees of complexity compared to flow cytometers: they share the spectral dimension, but have in addition potentially three spatial dimensions as well as time for live-cell imaging. Nevertheless, very little progress has been made toward ensuring rigor and reproducibility of confocal images. Unlike their cousins the flow cytometers, on which operators regularly flow intensity calibration beads, confocal microscopes suffer from a lack of standard intensity reference samples. Despite the beautiful images, there are therefore many pitfalls to obtaining truly quantitative data using confocal fluorescence microscopy (2,3). Many confocal microscope users know the basic steps required for comparing control and experimental conditions in a quantitative manner. First, the samples should all be prepared the same way (perhaps this is obvious to some readers), and preferably all at the same time (which is less obvious). There are many steps to preparing fixed samples (fixation, permeabilization, antibody labeling, and mounting) and live samples (fluorescent protein or vital dye labeling, incubation, and plating in coverglass-bottom chambers) and tiny changes in the protocols from 1 day to the next can dramatically affect the results. Second, the samples should all be imaged with the same settings (again, this may be obvious), and preferably all in the same imaging session (which is less obvious). A large experiment may take many hours to image, so more than one session may be required; but care must be taken to ensure that the microscope has not changed from one day to the next. For example, the intervening users might leave the objective lens dirty; or the microscope company might service and realign the confocal, resulting in a completely different laser power reaching the specimen. Furthermore, the samples themselves may change over a multiday experiment, with fixed-cell staining beginning to fade and live cells becoming more confluent. The longer the extent of your experiment, the more likely it is that the images taken along the way will have varying intensities. Consider reimaging control slides at the start of each session to help mitigate these issues. Also, ensure that you use exactly the same imaging conditions by saving and loading the settings, or even loading a previous image and reapplying the settings directly from the image.