Techniques Series: Using Confocal Microscopy


This is the second in a series of posts on scientific techniques, and how to use them in your research.

Confocal microscopy is an essential tool in modern cell biology with a wide range of applications. It can be used to study cellular structures and subcellular components of organisms ranging from yeast to zebrafish.

The underlying principle of this imaging technique is the use of a pinhole to eliminate out-of-focus background light, providing a clearer image of the specimen at a particular focal plane. An important advantage of the instrument is its capacity to construct a 3D image of the specimen. The confocal microscope is hooked up to a computer that processes the overall image as several slices (z-stacks) of the object are taken, using the microscope and an attached digital camera.

Setting Up A Question 

Let’s say you want to study the localization of a particular protein tagged with green fluorescent protein (GFP) in an organism— for example, the localization of tubulin-tagged GFP in yeast cells with abnormal cell morphology.

Using a regular fluorescence microscope will allow you to visualize the protein, but only on a two-dimensional level. You might be able see the tubulin-GFP as a stretched line in a budding yeast cell: at best, you’d confirm whether protein is present or absent. But a confocal microscope will give you a more in-depth view of tubulin in a defined area of the cell.

Getting Started With Confocal Microscopy

Using the confocal’s automated objective lens, you can focus on the yeast cell and choose the depth range in which you want to acquire a 3D image (using a focal plane from where the tubulin-GFP is first visible till its disappearance).

A key point to decide is the number of slices you’d like to capture within the chosen depth range: if you want enhanced resolution of your protein of interest, or to capture an intricate structure in the cell, you may want to increase the number of slices the software package directs the microscope to acquire. Using other control keys, you can adjust the background light so that the specimen is clearly visible.

Once the desired settings have been programmed, a digital camera attached to the confocal microscope captures images at the specified focal planes. Imaging software can then compile all z-stacks to render the image in 3D.

Interpreting The Data

Once the image of the tubulin-GFP is generated, the localization of the protein can be studied in reference to the 3D structure of the yeast cell. Instead of just seeing the stretched line of tubulin-GFP usually captured by a fluorescence microscope, the confocal will allow you to see, for example, a triangular formation of tubulin pointing towards the bud neck region of the yeast cell (i.e, the junction between mother and daughter in a dividing cell).

This image provides additional information over that acquired by other microscopes. For example, a triangular tubulin pattern would clearly indicate an aberration. Also, since the tubulin is positioned toward the bud neck, this might mean that, although there is a defect in cell morphology and tubulin formation, the segregation of DNA by tubulin from mother to daughter is taking place as expected.

The Value of Confocal Microscopy

Confocal microscopy allows a more precise understanding of an organism’s cellular components. Construction of a 3D image permits the development of an advanced understanding of the specimen or protein of interest, so this innovative technology takes science one step further.

If you want to order confocal microscopy for your own study, check out the 25 facilities who offer the service on Science Exchange:

[about_box image=””]Mamata Thapa works at JOVE: Journal of Visualized Experiments. She previously received her PhD in Molecular and Cell Biology from the University of Maryland, Baltimore County, and researched the role of ribosomal proteins in cell cycle progression and its implication in tumorgenesis, and is also a member of the Science Exchange Advocate program.[/about_box]


Mamata Thapa


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