For a conventional laser scanning confocal microscope, around 5 different laser sources can be needed to cover the excitation wavelengths of commonly used fluorophores. For example, a commonly used laser is the argon-ion laser which can produce a range of excitation wavelengths which are selected by filters. The argon-ion laser covers the green wavelengths of the excitation spectrum and these are used to excite fluorophores like FITC (fluorescein isothiocyanate). The yellow to red wavelengths of the excitation spectrum are covered by a helium-neon laser in which the range extends from approximately 543 to 632 nm. This spectrum is used to excite fluorophores like Texas Red and rhodamine. However, now there are confocal microscopes using white light lasers with a wavelength range from 440 nm to 790 nm [5,6].
Before light-emitting diodes (LED) were introduced as fluorescence light sources for widefield microscopy, the main sources of excitation light were gas arc lamps and these are still widely used today. The 2 arc lamps which are commonly found in widefield microscopes are the mercury- (also referred to as a “mercury burner” or “mercury vapor lamp”) and the xenon-arc lamp. The mercury-arc lamp provides excitation wavelengths across much of the visible spectrum (refer to figure 2), however, this illumination is not uniform and the main peaks are within the near-ultraviolet (UV) wavelengths (313, 334, 365, 405, and 436 nm) with 2 other peaks in the green/yellow part of the spectrum at 546 and 579 nm.
Compared to mercury-arc lamps, xenon-arc lamps provide excitation wavelengths across most of the visible spectrum, but the peaks within this range do not reach the intensity of mercury ones. Although xenon-arc lamps do not extend as far into the UV part of the spectrum compared to mercury ones, their excitation range is shifted further into the infrared wavelengths.
Although these lamps are extremely intense light sources for fluorescence microscopy , they are not without inherent problems. The lifetime of these bulbs is limited with a mercury-arc lamp lasting typically 200 to 300 hours and a xenon-arc lamp lasting between 400 and 600 hours. Because they have restricted lifetimes, a careful note of the hours used should be kept with the microscope (although some systems have a built-in recorder of hours used). If gas-arc lamps are used beyond their recommended lifetime range, there is a danger that the tubes can explode. Furthermore, if the lamps are regularly switched on and off, it can significantly reduce the lifetime of the bulbs, so this needs to be taken into consideration. Used arc lamps need to be disposed of carefully and should be done so according to laboratory or institute regulations. Replacement and alignment of such lamps are covered in these 2 referenced articles [8,9].
Despite some of the drawbacks highlighted above, the mercury-arc lamp is still considered to be a fundamental light source for widefield fluorescence microscopy due to the intensity of light produced.
The new generation of LED light sources for microscopy provide not only a full spectrum of excitation wavelengths (from around 365 to 770 nm), but also an intensity comparable to arc lamps. A major advantage of them over the arc lamps is the LED lifetime which can be up to 50,000 hours with no warm-up or cool-down periods required. This also saves time as an LED unit needs only to be aligned when initially installed. Finally, waste heat is a problem with arc lamps and, as a consequence, they are housed in special units next to the microscope. As most of the electrical input with an LED is converted to light, they produce little waste heat. An example of a Leica LED light source for fluorescence microscopy is shown below (refer to figure 3).
As highlighted above, the confocal scan head contains an array of photomultiplier tubes (PMTs)  for the collection of photons from the sample. Typically, a confocal scan head will contain at least three PMTs which are responsible for collecting red, green and blue light, but additional PMTs are commonly used for the collection of transmitted or reflected light. The PMTs are not the same as digital cameras, but are comprised of vacuum tubes which have a photon entry window at one end and an electron multiplying component in the body of the tube (refer to figure 4). The amount of photons collected is converted to an electrical signal before the image is finally assembled and displayed. Many confocal systems are also equipped with cameras similar to those used for widefield microscopy.
Image capture in widefield microscopy is facilitated by a digital microscope camera (refer to figure 4) [10,11]. These digital cameras contain semiconductor-based detectors and the most common sensors are charge coupled devices (CCDs), complementary metal oxide semiconductors (CMOSs), and sCMOS (scientific