In WF microscopy, the whole specimen on the microscope stage will be exposed to a light source (s. Figure 1). The most basic form of widefield microscopy is ‘brightfield microscopy’ in which the entire specimen is illuminated by white light either from above (in an inverted configuration), or below (in a standard upright microscope).
In a confocal laser scanning microscope, the light source for excitation of fluorescent dyes and proteins comes from laser units which are an integral part of the whole confocal system. The main advantages of confocal microscopy are that user-defined regions of interest can be selected negating the need for the entire specimen to be exposed to a fluorescent light source. In addition, the confocal microscope can be used to obtain optical sections through a specimen which can have the advantage of excluding much of the out-of-focus or background fluorescence.
Standard WF microscopes are less complex than confocal microscopes usually comprising of a white and fluorescence light source, microscope and camera (with or without an attached computer). In a confocal system, the microscope itself is only one part of a configuration including laser units, a confocal ‘scan head’ (containing pinholes for excluding out of focus light and photomultiplier tubes to collect photons from the specimen) and computers for controlling multiple parameters in the system as well as image processing.
In a conventional laser scanning confocal microscope, around five 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 lasers cover the green wavelengths of the excitation spectrum and are used to excite fluorophores such as 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 such as Texas Red and rhodamine.
Before light emitting diodes (LED) were introduced as fluorescence light sources for WF microscopy, the main sources of excitation light were gas arc-lamps and these are still widely used today. The two arc-lamps which are commonly found in WF microscopes are the mercury arc-lamp (also referred to as a ‘mercury burner’ or ‘mercury vapour lamp’) and the xenon arc-lamp. The mercury arc-lamp provides excitation wavelengths across much of the visible spectrum (s. Figure 2), however, this illumination is not uniform and the main peaks are within the near-ultraviolet (UV) wavelengths (313, 334, 365, 405, 436 nm) with two 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 burners. Although xenon arc-lamps do not extend as far into the UV part of the spectrum compared to mercury burners, their excitation range is shifted further into the infra-red wavelengths.
Although these lamps provide extremely intense light sources for fluorescence microscopy, they are not without inherent problems. The life time of these bulbs is limited with a mercury burner 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 out with their recommended lifetime range, there is a danger of explosion of the tubes. Furthermore, if the lamps are regularly switched on/off, this 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 respectively institute regulations. Replacing and alignment of such lamps is covered in these two tutorials in the Leica Science Lab (s. Figure 3):
Despite some of the drawbacks highlighted above, the mercury arc-lamp is still considered to be a fundamental light source for WF 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 provide an intensity comparable to arc-lamps. A major advantage over the arc-lamps is the lifetime of LED sources 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 one alignment adjustment when initially fitted. Finally, waste heat is a problem with arc-lamps and they are consequently fitted in special housing units next to a microscope. As most of the electrical input with an LED is converted to light, they produce practically no waste heat.
As highlighted above, the confocal scan head contains an array of photomultiplier tubes (PMT’s) for the collection of photons from the sample. Typically, a confocal scan head will contain at least three PMT’s which are responsible for collecting red, green and blue light, but additional PMT’s are common for the collection of transmitted or reflected light. The PMT’s are not cameras as such, 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 (s. 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 WF microscopy.
Image capture in WF microscopy is facilitated by a more conventional camera based on photodiodes (s. Figure 4). Digital microscopy cameras contain semi-conductor detectors and the most common sensors are the Charge Coupled Devices (