When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope’s ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision.
Figure 1. Quantitative confocal microscopy example. a and b, Widefield image (a) and a single confocal image slice (b) of an organoid expressing a fluorescently labelled nuclear protein (green: H2B-Venus) and with a fluorescent membrane probe (red: DiI, Thermo Fisher) (c), Confocal 3D volume rendering of the organoid. (d), 3D quantification of the mean nuclear intensities with color coding from 9,000 counts (violet) to 30,000 counts (red). Scale bars = 10 μm. Cells courtesy of Hui Wang (Princess Margaret Cancer Centre, Toronto, ON, Canada). Images in a and b used with permission from the Journal of Biomolecular Techniques (JBT), ©Association of Biomolecular Resource Facilities, http://www.abrf.org
However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, and choosing the most suitable microscope for a given application and configuring the microscope parameters. Every microscope comes with trade-offs between parameters such as cell viability, speed of image acquisition required, resolution, contrast and depth penetration.
Figure 2. Comparing inter-related key instrument performance parameters of different microscopy techniques and configurations. When choosing between different microscopy techniques, it is important to consider how their relative performance attributes, their strengths and weaknesses, make them more or less suitable for a particular experiment. The tutorial has limited analysis to five key parameters (contrast, depth of penetration, speed, resolution and sample viability), although your experimental needs may require you to consider others too (e.g., noise). (a) The relative performance of typical widefield, spinning-disk confocal microscopes and confocal laser-scanning microscopes (CLSM) are compared with the outer position indicating the best performance for that attribute. It is important to remember that microscopes can be configured differently to optimize for a particular parameter (or combination of parameters), either through the hardware choices or the software settings. (b) This panel illustrates that the same CLSM instrument can be optimized for either contrast or live-cell imaging, but this comes at the expense of other performance parameters—it is always a trade-off.
Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are also addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. These include non-uniform illumination, focus drift, laser instability and power variation and looking into various causes of jitter and stripes in the confocal images.
Finally, guidelines for analysing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. While the primary responsibility for rigor and reproducibility in confocal microscopy rests with the experimenter, core facility staff and microscope manufacturers also play critical roles. Confocal microscope users, developers and manufacturers must partner to identify and develop methods and standards that will make reproducibility more inherent to all systems.
The full article can be accessed here, and a visual summary of this tutorial is available as a poster here.
1Advanced Optical Microscopy Facility (AOMF), University Health Network, Toronto, Ontario, Canada. 2Advanced BioImaging Facility (ABIF), McGill University, Montreal, Quebec, Canada. 3A*STAR Microscopy Platform (AMP), Skin Research Institute of Singapore, A*STAR, Singapore, Singapore. 4Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London, UK. 5Bio-Imaging Resource Center, The Rockefeller University, New York, NY, USA.
