Sensitivity and Dynamic Range
Before taking a closer look at the quality of an image, it is important to ensure that the system is sensitive enough to capture the fluorescence signals, which can be very weak, depending on the individual application. Sensitivity should be understood as the minimum amount of light that is required to generate a signal that can be distinguished from noise. An important value is the quantum efficiency (QE) describing the ratio between the incident photons of the light source and the generated electrons of the pixel. It depends on the wavelength, and to get the best result, the spectrum of a given sensor should fit with the emission spectra of the fluorophores in the application. The higher the QE, the better the yield of photons, enabling shorter exposure times, reducing photo bleaching of fluorophores, and potentially improving overall imaging speed.
Often it is also beneficial to have a wide range of light intensities that can be resolved with one exposure. Here the full well capacity is relevant. It describes the maximum number of electrons that can be generated by one pixel per exposure. The higher the full well capacity, the more light can be captured before a pixel is saturated, reducing the requirement of additional exposures due to saturation.
Combining the maximum number of electrons with the lowest number of electrons required to produce a true signal (see “read noise” in the next section), the dynamic range characterizes a camera’s overall ability to measure and distinguish different levels of light.
Finally, there is the absolute sensitivity threshold, which is the number of photons required by one pixel to generate a signal-to-noise ratio (SNR) of 1 — meaning the signal is equivalent to the noise. The smaller this value, the less light is required to produce a true signal. Because it does not take into account the pixel size, it cannot be directly used to compare two cameras when their pixel sizes are different.