Institute for Molecular Bioscience

Confocal Microscopes

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Confocal microscopy is an advanced light microscopy method which utilises a ‘pinhole’ to eliminate out of focus light and is suitable for both live and fixed cells and tissues. The advantage of a confocal microscope over conventional wide-field microscopes is that discrete optical sections can be collected while eliminating the out of focus light above and below the current plane of focus. Because of this, high powered lasers are used to illuminate the sample in order to collect enough light only from the desired focal plane.

Focused beams of laser light are scanned across the sample (Laser Scanning Microscopy, LSM) and light only from the desired focal plane is allowed to enter the detector. Depending on the fluorophores in your sample, and the number of detectors available, multiple fluorophores can be excited and detected simultaneously. Traditional detectors are photo-multiplier tubes, however more recent Gallium Arsenide Phosphide (GaAsP) detectors have been developed to increase sensitivity (Confocal 2 and 4, Airyscan; Confocal 5, BiG detectors). Leica have also developed their own higher sensitivity detector, known as a HyD - a hybrid detector combining the best properties of traditional PMTs and the highly sensitive avalanch photo-diode detectors (Confocal 1, HyD). 

These microscopes provide excellent axial resolution, and very good signal to noise sampling, however this often comes with a sacrifice in temporal resolution due to the slow nature of scanning pixel-by-pixel across the image during capture. Some systems overcome this speed issue with either faster scanners (Leica SP8  - Confocal 1 uses an 8 kHz resonant scanner) or with multiple pinholes (Andor Dragonfly Spinning Disc Confocal and Yokogawa CSU-X1) solving this speed limitation in unique ways.