Confocal microscopy

Instrument: Leica TCS SP1 Confocal Microscope

Instrument specifications

Detector: Photomultiplier

Magnifications: Objectives up to 63x, giving a total maximum magnification of 630x

Image Output: 8-bit TIFF, up to 1024 x 1024 pixels

Key instrument features

• Offers transmitted and reflected light imaging
• Enhanced contrast is provided by phase contrast and differential interference contrast (DIC), a technique similar to Nomarsky phase interference
• UV fluorescence from mercury vapour lamp (for imaging only)
• For confocal imaging, a three laser system offers:
  • emission lines at 476 and 488nm using an argon laser for FITC excitation and similar
  • a krypton laser at 568nm for Rhodamine, Texas red or TRITC-like dyes
  • a helium-neon laser at 633nm for CY5 and comparable dyes
• The software allows for simultaneous or sequential scanning of up to three fluorescence channels and one transmitted light channel in any combination.

Sample preparation

Users are reminded to only choose dyes which are within the excitation range of the available lasers.

How laser-scanning confocal microscopy works

In this technique an object is scanned using a laser beam to build up the image a line at a time. 

The laser is either reflected off the object or excites fluorescence from within it. The returning light rays are fed through a pinhole which excludes out of focus information. Thus only the focused image plane is viewed.

Sequential image planes can be scanned and built up into a stack of images. These can be manipulated to provide extended focus images and 3D images, or the image stack can be rotated to provide views from tilted viewpoints or at right angles to the plane of section.

Confocal techniques work exceptionally well for:

• epifluorescence - by reducing glare and showing single image planes
• reflected light applications