Confocal microscopy is a technique that is commonly used in biological research to take high-resolution 2D images deep inside a sample. Two pinholes in the optical path remove out-of-focus light, providing excellent contrast and resolution. In addition, blocking the out-of-focus light means only a thin region, or slice, of the sample near the focal plane will be imaged. This allows us to take many slices at different depths and combine them to form a 3D image of the sample, also known as an image stack or z-stack.
Our microscope (Leica TCS SPE) is a laser scanning confocal microscope (LSCM) meaning that it illuminates and observes a single spot at a time by using mirrors to scan a focused laser spot across the sample. Detectors then collect light that is transmitted through, reflected back, or fluorescently emitted from the sample. We have three different colored lasers to use and can choose which color of light we want to detect. This allows us to stain a single sample with multiple fluorescent dyes or labels and then image them all in sequence.