Microscopy School Lesson 6 - Principles in Confocal Microscopy

The advent of fluorescence microscopy allowed the revelation of a whole new world previously hidden inside cells and tissues. Epifluorescence microscopes are extremely useful to visualise thin samples or tissue sections. Still, when samples are a bit thicker, the out of focus light will decrease significatively the Signal to Noise Ratio of the image. Because of reduced signal to noise, the structures of interest will be surrounded by “haze” becoming more difficult or nearly impossible to visualise. It became necessary to develop another type of imaging that could deal with the out-focus light, and that would allow optical sectioning of the sample. The confocal microscope was developed to address these issues.   

Confocal microscopes discard the out of focus light using pinholes. Optical sectioning of the sample is achieved using the pinholes in which the in-focus light reaches the detector, while the out of focus light is discarded.

There are two types of confocal microscopes:

• single pinhole confocal, or point scanners, where a single pinhole will discard the out of focus light.
• multiple point confocal, or spinning disks, where multiple pinholes will discard the out of focus light.

In this lesson, Dr Claudia Florindo, Microscopy Specialist, Andor, will explain what a confocal microscope is and discuss the differences between single-point confocal and multipoint confocal. By the end of this talk, we hope that you will have a clear understanding of confocal microscopy and it´s applications in life sciences.

Learning Objectives:

• Understand what a confocal microscope is.
• Recognize the differences between point scanners and multipoint confocal then review the advantages and disadvantages of each technology.
• List life sciences application for which a confocal is an ideal system for imaging acquisition. 

Questions Answered:

• What is a pinhole?
• Why pinholes have different sizes?
• When should a confocal system be used?