In confocal microscopy, the scan line speed refers to how many lines of your sample are being scanned per second. This determines the time resolution of the images; the higher the frequency, the higher the time resolution i.e. the longer the acquisition takes.

As the scan line speed increases, the pixel sampling time reduces, resulting in a decreased signal to noise ratio and reduced image resolution. The loss in image intensity can be countered by increasing the gain, but image quality cannot be improved this way. Other techniques such as increased laser power or increased pinhole size have to be used to increase the photon input; these techniques have their disadvantages and also take more time, which defeats the point of using a higher scan line speed.

When the scan line speed is too fast, the field of view is also reduced. This is due to the scanning mirrors becoming out of sync. A high scan line speed is used when imaging live specimens because time is more important than the image quality.

At slower speeds, pixels can be scanned for longer and more signal is detected, leading to higher quality images. However, if the scan line speed is too low, the sample has to tolerate laser scanning for longer and the sample may become photobleached. This is when fluorophores in the sample are photochemically altered such that they can no longer fluoresce. It is, therefore, important to compromise between quality of image and damage to the sample to find the optimum scan line speed.