Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut

Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal conditions. However, optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. While the resolution of optical microscopes does not reach that of electron microscopes, they are the most valuable tools in the imaging of biological macrostructures and are a staple in any biological lab.

In conventional light microscopes, the signal produced from the imaged object is from the full thickness of the specimen, which does not allow most of it to be in focus to the observer. This causes the image to have "out of focus blur". The confocal microscope, on the other hand, illuminates the sample through a pin-hole, and is therefore able to filter out the out-of-focus light from above and below the point of focus in the object.

This demonstration provides an introduction to image acquisition using optical and confocal microscopy methods. Here, a sectioned piece of mouse brain will be studied.  Image acquisition and analysis, including the tools to generate topographical maps and composite images, will be covered. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to provide more information on optical and confocal microscopes to determine if these microscopy modules are the best fit for a type of biological sample.

1. Confocal Imaging

1. Load the sample onto the stage. Center it beneath the lens. It should not exceed the weight limitation of the stage, which in this case is 5 kg. The sample should not be more than 100 mm thick.
2. Open the imaging software and select "Create Job."
3. Under the Topographies Column, choose the assistant button.
4. Create an overview image at the lowest magnification, 2.5X. Before switching the magnifications, ensure that the sample is in focus by changin

The following images give an overview of results that can be obtained of a mouse brain using a confocal microscope. They show how varying levels of information can be obtained and how a topographical map of the results reveals the height of the sample.

Figure 5: Confocal images at

In this demonstration, the depth of focus, field of view and maximum resolution and magnification of optical and confocal microscopes were optimized to view biological samples. This demonstration was designed to help the participant decide which microscopy module is the best for a certain application. Both modes of microscopy have advantages in analyzing biological samples for their ease of preparation and high resolution composite images.