Introduction and History
Wide field fluorescence microscopy is used in research and health care because it offers extreme sensitivity in the detection of pathogens, cellular organelles and molecular complexes. A wide field fluorescent microscope can be either upright or inverted. These microscopes use intense light sources, excitation filters, barrier filters and are designed to detect substances that emit fluorescent light when excited. Subjects are usually viewed against a black background. Fluorescent microscopy can even detect single fluorescent molecules under optimum conditions (H. Goi et. al. 2007, S. Shashkova and M. C. Leake 2017). Wide field fluorescent microscopes are more common than those designed for super resolution such as confocal microscopy. Scanning confocal microscopes have features in common with a wide field fluorescent microscope, but confocal scopes use lasers for illumination, methods to reduce out of focus light (deconvolution) and are more costly. For more information about confocal microscopy see J. B. Pawley’s comprehensive book (2006).
If you are considering purchasing a wide field fluorescence microscope I describe some of the feature you need to consider. Before purchasing any microscope I recommend testing it with samples you intend to examine. There are several factors to consider that affect the performance of a wide field fluorescent microscope including: light source, filters, objectives, the fluorophore and its properties, background fluorescence, fading of the fluorescent probe, and the camera’s sensitivity.
Epifluorescence microscopes direct light through the objective where the objective also works as a condenser. The excitation light after passing through a filter allows fluorescent light to return through the objective where it passes a barrier filter which eliminates the excitation light. The fluorescent light from the specimen is directed to the viewer or camera.
Some specimens and organelles are autofluorescent (e.g. algae, wood, mitochondria, extracellular matrix) while other specimens require staining with a fluorescent dye like Acridine orange. The excitation light wavelengths or spectrum of arc lamps are broader than those shown in the diagram above. With phosphorescence the light emitted occurs in a similar manner, but the excited state lifetime lasts longer.
The first fluorescent microscopes were developed around 1911-1913 by physicist Otto Hemstaedt and Henrich Lehmann (Zeiss Campus). Fluorescence was encountered in optical microscopy by August Köhler, Karl Reichart and others who reported it was a nuisance in ultra violet microscopy. These first fluorescent microscopes were used to observe autofluorescence in bacteria, plant and animals cells. Stenislav von Provazek is thought to be one of the first to use fluorescent dye binding in fixed tissues and Adolf von Bayer synthesized the first fluorescent dye, fluorescein in 1871 (A. Heinrichs, 2009).
Albert Coons first attached fluorescent molecules to antibodies in the 1940’s and showed they could be used to localize proteins in the liver (Y. Hamashima et al., 1964). Antibodies were made to specific proteins then covalently attached to Fluorescein isothyocynate and the fluorescent probe localized in sections. Fluorescent probes can also be micro-injected into cells e.g. carboxyfluorescein, and Lucifer yellow (W.W. Stewart, 1978, R.C. Berdan, 1987) to study cell-cell communication and trace neuronal pathways. Other fluorescent techniques can be used to examine the production and distribution of RNA or DNA within a cell. One technique is fluorescence in situ hybridization (FISH) a powerful tool to visualize target DNA sequences or messenger RNA (mRNA) transcripts in cultured cells, tissue sections or whole-mount preparations (see review by A. P. Young et.al., 2020). Today binding of fluorophores to various molecules has produced powerful probes for studying the function and location of various molecules in cells.
In 1992, (D.C. Prasher et al.) cloned the gene for a green fluorescent protein (GFP) from a chemiluminescent jellyfish. The gene was fused with other genes and transfected into a host and the resulting protein produced in the living cell was fluorescent. Roger Tsien won a Nobel prize in Chemistry in 2008 for his work of novel fluorescent and photo labile molecules including the GFP. Fluorescence microscopy and fluorescent probes can also measure diffusion coefficients, pH, viscosity, refractive index, ionic concentration, membrane potential and solvent polarity in living cells.
Wide field fluorescent microscopes and light sources
Upright microscopes are ideal for examining tissue sections or small samples on microscope slides. An inverted microscope is good for studying living cells in tissue culture and embryos. An inverted microscope can also be modified for microinjection of fluorescent probes into live cells and is designed for studying cells grown in culture.
One of the major decisions when purchasing a wide fluorescent microscope is the choice of an appropriate light source. The excitation light source and its spectrum need to be matched to the fluorophore. The microscope might include: bright-field, phase contrast, fluorescence or DIC optics. Fluorescent images may be combined with other types of imaging in post-production. The challenge in wide field microscopy is to obtain high energy illumination in order to capture the weak fluorescence emission. Fluorescence is often 3-6 orders of magnitude lower in brightness than the excitation light. Therefore the microscope must be equipped with an appropriate illumination source, excitation and barrier filters specific for the fluorophore (fluorescent molecule). Most fluorescent molecules absorb light of a limited wavelength with a span of about 20-100 nm.
The main light sources for fluorescence microscopy are a mercury arc lamp, xenon arc lamp, metal halide, (LED) light emitting diodes or laser light. Each source has a different spectral profile. Proper alignment of arc lamps is critical to provide uniform illumination. Lamp houses should not leak harmful ultra violet and should be sturdy enough to withstand a lamp explosion. LED lights are emerging as a stable alternative to arc lamps and have numerous advantages over arc lamps. Laser light is used mainly with confocal microscopes. Mercury and Xenon lamps provide an intense light source, but the bulbs have a short lifetime of 200-600 hours and their intensity fades with time. Mercury lamps may explode if used over 200 hours and the bulbs need to be carefully centered. Tungsten lamps are often of insufficient brightness for fluorescence microscopy.
Newer fluorescent microscopes use specific LED lights that emit a narrow wavelength band. The LED bulbs are rated for about 10,000 hours, and they are not as hot as Mercury or Xenon bulbs and can be turned on and off at will. Mercury lamps once turned on require a warm up period and should be left on for a period of time before turning off. LED light sources are currently expensive, but their price is falling. Arc lamps decay in intensity with time and do not provide even illumination across the visible spectrum; their main peaks are in the UV with two peaks in the green yellow part of the spectrum. LED lights should be tested with the fluorophore molecule you are using. The lowest wavelength of light emitted by LED is about 200nm (LED professional web site) and some UV dyes cannot be excited by LEDs. LED disadvantages are that they emit lower intensity light at wavelengths in the UV part of the spectra. Arc discharge lamps are about 10-100X brighter than 12 V quartz halogen bulbs, but their high intensity peaks need to be matched to the fluorophore absorption spectrum. For a review of light sources for fluorescence microscopy see K. Aswani et al., (2012).
The wavelength range of commercially available LEDs with single-element output power of at least 5 mW ranges from 275 to 950 nm. Each wavelength range is made from a specific semiconductor material family. Below the diagram shows that LED lights have narrow excitation bands.
J. Zi and H. Bi (2023) introduced a new LED fluorescence light source called the LED-integrated excitation cube (LEC). The design can be used in any fluorescent microscope. According to the developers LEC is compact, low in cost and easy to install. Their work provides a new solution by integrating LED, filters, Fresnel lenses, and dichroic mirrors into a filter cube.
Because fluorescent light is often weak the best objectives are those with a high numerical aperture. A small increase in numerical aperture will result in a large increase in brightness. Numerical aperture is engraved on the objective and characterizes the range of angles over which the objective can accept or emit light. Choosing the best objective is important and those objectives that use oil immersion fluid will be even brighter because oil increases the effective numerical aperture (NA). Objectives designed for fluorescence microscopy like Fluorite and Apochromats have higher numerical apertures are brighter and more expensive. The brightest images will be gathered by an objective with high numerical aperture and low magnification such as a NA 0.75/20x objective. Any microscope objective can potentially work but those specifically designed for fluorescence microscopy work better. There are phase contrast objectives that can be combined with fluorescence illumination. You can also use DIC objectives for fluorescence and combine the two different images in post processing.
Objectives specifically designed for use with fluorescence illumination feature high light transmission values in both the ultraviolet and visible portions of the spectrum and are themselves not autofluorescent. While oil immersion fluid boosts brightness, check that the oil and any fluid the specimen might be bathed in is itself not fluorescent. Also block the excitation light when the specimen is not being viewed or photographed to avoid photobleaching (fading). Some fluorophores can lose much of their fluorescence after only a few seconds exposure to the excitation light source. Orange plastic filters attached to the outside of a fluorescent microscope function to block any reflected UV light and damaging the operator’s eyes.
There are a number of chemical agents that can reduce photobleaching (e.g. p-Phenylenediamine (PPD), n-Propyl gallate (NPG), and 1,4-Diazabicyclo-octane (A. Longin et al., 1993). Citifluor is a mixture of phenylamine in glycerol that works well with fluorescein, but not rhodamine. Most of antifade agents are used to examine fluorescence in fixed cells and tissues, however some companies sell antifade agents for use with live cells made from the plasma membranes of E.coli (e.g. Thermofisher). Most antifade agents are reactive oxygen scavengers.
With film or a digital camera select a high ISO (International Organization for Standardization) speed or gain setting on the camera. Increasing gain will introduce more digital noise so use the lowest setting required to capture detail. Digital cameras with a larger sensor size and those that use a pixel binning option are more sensitive to the fluorescent light. Some cameras offer intensification and can detect fluorescent light even when the specimen appears completely invisible to the viewer. I have used film, digital single lens reflex cameras, C mount cameras and intensified cameras with success but it is important to test the cameras. Check out recent reviews about cameras for low light imaging, astrophotography, and fluorescence microscopy e.g. Scientifica website. What camera you chose will also depend on the resolution and size of the images required and whether you need black and white or color images.
The Fluorescent Probe or Fluorophore
There are thousands of fluorescent molecules and ligands commercially available, including antibodies, lectins and agents sensitive to pH, and ionic concentration. A description of the full range of probes is beyond the scope of this article, but I recommend downloading “The Molecular Probes Handbook ”Guide to Fluorescent Probes and Labelling Technologies” available for free online – see reference section.
The concentration of the fluorophore also must be carefully optimized because too high a concentration can result in quenching of the signal and reduction in overall intensity. Injecting live cells with a fluorophore will also sensitize the cells to damage by light so you should minimize live cells exposure. Fluorescent probes tend to be expensive and some are difficult to obtain, you might want check out Vector labs website that promotes economical costing fluorophores.
Some specimens like algae, and wood are autofluorescent and need no staining. Fluorescein is a cheap and easy to buy which emits green light and is membrane permeable. Carboxyfluorescein is similar but not membrane permeable. Rhodamine is a fluorescein derivative that appears bright red in color. Other dyes stain mitochondria (Rhodamine 123), cytoskeleton (Phalloidin-TRITC), and nuclei of live cells (membrane-permeant Hoechst 33342 dye). Acridine orange is a cell-permeable probe which allows the dye to interact with DNA by intercalation, or RNA via electrostatic interactions. An important consideration is the fluorophores quantum yield which is defined by the ratio of photons absorbed to photons emitted through fluorescence. Quantum yields of 0.10 are considered quite fluorescent. Fluorescein and green fluorescent protein has a very high quantum yield of 0.79. Other factors including concentration, pH, and temperature can also affect fluorescence emission (T. Wenzel).
The resolution of a wide fluorescence microscope is about 200 nm (Leica website, 2017), confocal microscopes and other image processing techniques can resolve objects down to about 120 nm. Fluorescence can be detected at low levels of light, but is difficult to quantify and identify small differences in intensity. The main disadvantage of fluorescence microscopy is that not all compounds are fluorescent.
A wide field fluorescence microscope tends to be suitable for viewing specimens less than 30 microns thick including paraffin and frozen sections. A confocal laser scanning microscope (CLSM) is better suited for thicker sections, and embryos because it can eliminate out of focus light by taking a stack of images, combining them and then removing the out of focus light by digital processes called deconvolution. I describe deconvolution methods using Adobe Photoshop to remove out of focus light from wide field fluorescence images (R. Berdan, 2020). Some fluorescence microscopes come with only two excitation filters blue and green. The excitation and barrier filters need to be carefully chosen to work with specific fluorophores.
CAUTION never look directly at the high energy light without filters in place and avoid touching the arc bulbs, and install bulbs wearing gloves and protective glasses. Follow all the instructions if using an arc lamp, its disposal and track the time the bulb is used so as not to exceed its lifetime.
Before purchasing a wide field fluorescent microscope test it with the fluorescent probes you intend to use to be sure it offers sufficient brightness and excitation light. If pictures are required for publication or documentation test the camera and associated software. I have used digital single lens reflex cameras for over a decade with good results. Wide field fluorescent microscopes with arc lamps are still in common use. LED microscope lighting is becoming the method of choice because these systems are more stable, longer lasting, energy efficient, safer, and more environmentally friendly. Wide field microscopes have a low maintenance cost compared to confocal microscopes, and are relatively easy to purchase fluorescent probes that have high sensitivity, and permit tracking dynamic processes in real-time and in vivo. Processes in live cells, such as neuronal signaling and cell-cell communication can be observed in real time.
By Robert Berdan Ph.D.
fluorescence in situ hybridization PeerJ. 8: e8806. doi: 10.7717/peerj.8806
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