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These illumination sources are highly reliable, produce very high flux densities, and have historically been widely used in fluorescence microscopy. Classically referred to by the registered trademark as HBO lamps (H for Hg or mercury; B is the symbol for luminance; O for unforced cooling), a large number of fluorescent probes have been developed for this ubiquitous light source. First introduced as a commercial product in the 1930s, many thousands of microscopes equipped with mercury arc lamp illuminators have been sold by the manufacturers over the past few decades. However, compared to traditional incandescent lamps, the significant increase in brightness afforded by mercury arc lamps is accompanied by the inconvenience of critical mechanical alignment, shorter lifetime, decreased temporal and spatial homogeneity, specialized lamphouse and power supply requirements, potential explosion hazards, and higher cost. Regardless of the pitfalls, the mercury arc lamp remains a workhorse in fluorescence microscopy and is still considered one of the best illumination sources, especially for low abundance (in effect, those that have sparse targets) or weak fluorophores whose excitation maxima coincide with the spectral lines emitted by the hot mercury plasma. The most popular mercury lamp for optical microscopy is the HBO 100 (a 100-watt high-pressure mercury plasma arc-discharge lamp), which has the highest radiance and mean luminance, due to its very small source size, of the commonly used lamps of any wattage. For the microscopist, the unique spectral content of the mercury arc light output (in effect, the spectral irradiance) is an important consideration when comparing various illumination sources. Only about a third of the output lies in the visible portion of the spectrum, the remainder being confined to the ultraviolet and infrared regions. Ultraviolet emission accounts for about half of the output of the mercury arc lamp, so great care must be taken to protect the eyes as well as living cells that are being illuminated with this source. The remainder of the mercury lamp output is dissipated as heat in the form of infrared radiation. Mercury arc discharge lamps produce among the highest luminance and radiance output levels of any continuously operating light source for optical microscopy and very closely approach the ideal model for a point source of light. However, mercury lamps exhibit significantly greater fluctuation in intensity than do incandescent lamps, light-emitting diodes (LEDs), or laser sources, primarily because the gas plasma is inherently unstable and affected both by magnetic fields and erosion of the electrodes. Short term lamp stability is affected by three artifacts of the arc plasma created between the tungsten electrodes. Arc wander occurs when the attachment point of the arc on the conical cathode tip surface traverses the electrode in a circular pattern, usually requiring several seconds to rotate in a full circle. Flare refers to the momentary change in brightness when the arc relocates to a new region of the cathode with a higher emissive quality than the previous attachment point. Finally, convection currents in the mercury vapor arising from a temperature differential between the plasma and envelope generate arc flutter, which is manifested by rapid lateral displacement of the arc column. These combined artifacts limit the utility of mercury arc lamps in quantitative fluorescence measurements. Aside from the numerous artifacts associated with mercury arc lamps, they also suffer from limited life spans of approximately 200 hours and significant variations in spatial and temporal stability. Because the arc image is focused onto the rear aperture of the objective (in Köhler illumination), the most important aspect of mercury lamps is the intensity of the arc image. Surprisingly, even though arcs with higher power ratings produce more light, the actual arc size is larger and its corresponding image must be reduced below the actual size to accommodate the objective rear aperture. Minimizing the arc size leads to a reduction in image intensity and, for this reason, lamps having smaller arcs actually produce more intense light. Illumination in the microscope viewfield is the most evenly distributed when a sharp image of the arc is centered in the objective rear aperture. Although the highly defined and focused arc image leads to regions of the aperture having minute fluctuations in light intensity, the net effect is the potential restriction of some illumination angles from reaching the specimen. Due to the fact that fluorescence excitation is insensitive to illumination angle, however, this inhomogeneity (unless severe) does not usually compromise image quality. In contrast, when the arc image is not properly focused on the objective aperture, intensity fluctuations are often observed in various regions of the specimen. Optical Power of Mercury (HBO) Arc Lamps Filter Set Excitation Filter Bandwidth (nm) Dichromatic Mirror Cutoff (nm) Power mW/Cm2 DAPI (49)1 365/10 395 LP 23.0 CFP (47)1 436/25 455 LP 79.8 GFP/FITC (38)1 470/40 495 LP 32.8 YFP (S-2427A)2 500/24 520 LP 20.0 TRITC (20)1 546/12 560 LP 43.1 TRITC (S-A-OMF)2 543/22 562 LP 76.0 Texas Red (4040B)2 562/40 595 LP 153.7 mCherry (64HE)1 587/25 605 LP 80.9 Cy5 (50)1 640/30 660 LP 9.1 1ZEISS Filters 2Semrock Filters Table 1 Presented in Table 1 are the optical output power values of a typical 100-watt HBO light source after passing through the microscope optical train and selected fluorescence filter sets. Power (in milliwatts/cm2) was measured at the focal plane of the microscope objective (40x fluorite dry, numerical aperture = 0.85) using a photodiode-based radiometer. Either a mirror with greater than 95% reflectivity from 350 to 800 nanometers or a standard fluorescence filter set was used to project light through the objective and into the radiometer sensor. The light throughput loss in a microscope illumination system can vary between approximately 50 and 99 percent of the input power, depending upon light source coupling mechanism and the number of filters, mirrors, prisms, and lenses in the optical train. As an example, for a typical research-grade inverted microscope coupled to an HBO lamphouse at the entrance port of the epi-illuminator, less than 50 percent of the light exiting the collector lens system is available for excitation of fluorophores positioned at the objective focal plane. The rated lifetime of mercury arc lamps depends upon how they are used, and the usual 200-hour limit can be compromised by an excessive number of starts (ignitions) or by repeated ignition of warm or hot lamps. Normal operation requires burn periods for a minimum of 30 minutes and a total number of ignitions not to exceed one-half the total number of rated hours (around 100 maximum). Therefore, a typical HBO 100 lamp should be ignited no more than 100 times and burned for an average of two hours per ignition. This is not a hard and fast rule because some burn cycles are much longer (lasting, for example, an 8-hour day). As mercury arc lamps age, they blacken and become increasingly more difficult to ignite due to degeneration of the cathode and anode. Furthermore, during use the lamp alignment is subject to drift so that the arc image can slowly become de-centered in the objective rear aperture, necessitating repeated adjustment of the alignment mechanism. Generally, the end of a mercury arc lamp is the point at which the ultraviolet light output has decreased by approximately 25 percent and the arc instability has increased beyond 10 percent, or if the lamp will no longer ignite. Once the lamp has reached or moderately exceeded its usable life span, it should be replaced. The emission profile of mercury arc lamps is distinct from incandescent lamps in that several prominent emission lines are present in the ultraviolet, blue, green, and yellow spectral regions, which are significantly brighter (up to 100 times) than the continuous background (see Figure 1). Approximately 45 percent of the radiant output from a standard 100 watt HBO mercury lamp falls between the useful fluorescence microscopy wavelengths of 350 to 700 nanometers. In addition, much of the ultraviolet and visible light energy is not distributed equally across the spectrum, but rather is concentrated in spectral lines at 365 nanometers (near ultraviolet; 10.7 percent), 405 nanometers (violet; 4 percent), 436 nanometers (deep blue; 12.6 percent), 546 nanometers (green-yellow; 7.1 percent), and 579 (yellow doublet band; 7.9 percent). Mercury arc lamps also have a significant number of spectral lines in the ultraviolet region between 250 and 350 nanometers and several lesser lines in the infrared wavelengths exceeding 1000 nanometers. In contrast, the mercury lamp spectral emission region between 600 and 1000 nanometers is relatively continuous and no brighter in output than xenon arc lamps, which span a broad spectral range with only a few spectral lines in the blue and infrared regions. The 546-nanometer green-yellow line of the mercury arc lamp has become a universal reference for calibrating wavelengths in a wide variety of optical devices and is a favorite among scientists in the biology community for examining living cells. Selected Fluorophores for Mercury Arc Excitation Fluorophore Excitation (nm) Emission (nm) Mercury Line DAPI 358 461 365 Marina Blue 365 460 365 Nuclear Yellow 365 495 365 Alexa Fluor 405 401 421 405 Cascade Yellow 400 550 405 Alexa Fluor 430 433 541 436 Cerulean FP 433 475 436 Lucifer Yellow 430 535 436 Alexa Fluor 546 556 573 546 Cy3 552 570 546 Tetramethylrhodamine 549 574 546 tdTomato FP 554 581 546 Kusabira Orange FP 548 559 546 MitoTracker Red 579 599 579 Alexa Fluor 568 578 603 579 LysoTracker Red 579 590 579 Table 2 A considerable effort has been expended on developing specialized fluorophores that have absorption maxima located near the prominent mercury spectral lines (see Table 2). The classical fluorescent probes DAPI (4',6-diamidino-2-phenylindole) and rhodamine efficiently absorb the 365 and 546 nanometer mercury lines, respectively, however the absorption maximum of fluorescein (perhaps one of the universally most widely used fluorophores) lies in the region between 450 and 500 nanometers, which is devoid of a prominent mercury line (Figure 1). Newer synthetic fluorophores, including the MitoTrackers, Cyanine (Cy) series, and Alexa Fluor dyes have been specially tailored to match mercury spectral lines. For example, the MitoTracker Red absorption maximum of 579 nanometers almost exactly matches the corresponding mercury line, whereas Cy3 (maximum at 548 nanometers) efficiently absorbs the 546 mercury line. Several of the Alexa Fluor dyes are named in reference to their equivalent mercury absorption profiles: Alexa Fluor 350 (mercury-365), Alexa Fluor 405 (mercury-405), Alexa Fluor 430 (mercury-436), and Alexa Fluor 546 (mercury-546). In general, when exciting fluorophores with a mercury arc illumination source, it is wise to choose among the widely available fluorophores that closely match the spectral lines. It should be noted that mercury arc lamps are not a suitable light source for several ratiometric dyes, such as Fura-2 and Indo-1, where comparison of the signals at two excitation wavelengths is compromised by the fact that one of the wavelengths overlaps with a mercury peak to a much greater degree than does the other. Also, the relatively weak emission by mercury lamps in the 450 to 540 nanometer region renders these illumination sources less useful for many of the popular dyes that absorb strongly in the blue-green region, including fluorescein, Alexa Fluor 488, Cy2, and the many varieties of green fluorescent protein. back to top ^Mercury Arc Lamp Construction The extremely high flux density (brightness) generated by mercury arc lamps is achieved by producing the arc in a limited region between two closely spaced electrodes in a high-pressure gas medium. The gas and electrodes are contained within an optically transparent, elliptically shaped envelope (or bulb) composed of fused silica (see Figure 2). The electrodes are fabricated from tungsten alloys that have a melting point exceeding 3400° C, one of the few materials capable of withstanding the high arc plasma temperature. Additionally, tungsten has the lowest vapor pressure of all metals, another positive feature when considering the high temperatures required during operation. Mercury arc lamps are filled with an inert (rare) gas such as argon or xenon under low pressure and a carefully measured aliquot of metallic mercury. The mercury dosage is calculated so that the lamps generate an internal pressure of up to 75 atmospheres (1,087 pounds per square inch) during operation. Arc lamp electrode manufacturing variables are critical in determining the starting characteristics, lifetime, and performance variables of mercury lamps. Cathodes designed for mercury arc lamps are cone-shaped rods (see Figure 2) fabricated from thoriated (thorium oxide) tungsten to improve starting and emission characteristics as well as to lower the open-circuit voltage. Because a majority of the heat produced by the arc discharge is generally retained within the electrode area, the cathode is able to quickly attain the optimum electron emission temperature with insignificant levels of tungsten evaporation that lead to premature lamp blackening. The cathode tip is also radiused to stabilize the discharge. The anode in mercury lamps is fabricated from pure swaged (forged) tungsten and is noticeably more massive than the cathode. The larger size of the anode enables it to withstand intense electron bombardment from the plasma and to more efficiently dissipate heat. Mercury arc lamps usually feature starting coils on one or both of the electrodes to assist in arc formation during ignition and have an anode-cathode gap ranging from 0.25 to several millimeters, depending upon the lamp power rating. The mercury arc lamp envelope is produced with pure fused silica or quartz glasses, which are impermeable to most gasses at high temperature and pressure, and are thus ideal for containing the hot plasma. Furthermore, the low expansion coefficient and high mechanical durability of these glasses render them dimensionally stable and capable of operating under the extreme lamp operating conditions. Envelopes are manufactured from high-quality tubing to prevent lamp failure from localized stress points that arise from air pockets and impurities. Quartz transmits light with high efficiency from approximately 180 nanometers to 4 micrometers, but lamps designed for optical microscopy are fabricated with doped quartz to absorb shorter ultraviolet wavelengths and minimize the generation of ozone. Most of the glass alloys used for mercury arc lamp construction feature very little hydroxyl (OH) content, thus eliminating infrared absorption at 2.7 micrometers and reducing the thermal load on the envelope. One of the most critical features of arc lamp construction is the hermetic metal-to-quartz seal that is necessary to isolate the electrodes from the surrounding atmosphere and for mechanical support of the lamp. These seals must be impermeable to gasses while simultaneously being able to withstand currents of hundreds of amperes, temperatures ranging between 200° and 300° C, and pressures of 30 atmospheres or higher. The most popular technique for sealing electrodes involves wrapping thin ribbons of molybdenum foil in a concentric, parallel configuration sandwiched between a quartz rod and the coaxial envelope tube, which is then covered by a temperature-resistant adhesive cement. The exceedingly thin width and tapered edges of the foil enable an efficient seal to the quartz tube despite the difference in thermal expansion coefficients. Additionally, the hermetic nature of the seal permits the application of high current loads without significant oxidation. The lamp seals are capped by ferrules or bases that serve as both a firm electrical connection and a precise mechanical mechanism for locating the point source within the microscope optical system. Ferrule design varies, but most contain a threaded or smooth locating pin and some feature a cable that links the lamp to a terminal in the lamphouse. The ferrules are designed to aid in lamp cooling and are usually fabricated from nickel-plated brass. back to top ^Mercury Lamphouses and Power Supplies In a typical optical microscope configuration, the mercury lamp is positioned inside a specialized illuminator consisting of a lamp housing containing the lamp, a concave reflector mirror, an adjustable collector lens system to focus the lamp output, an electrical socket for securing and alignment of the bulb, and the external power supply (Figure 3). Depending upon the design, mercury arc lamphouses may also contain filters to block ultraviolet wavelengths and hot mirrors to block heat from entering the microscope optical train. Many lamphouses also contain external heat sinks to dissipate heat and vents that allow the dissipation of hotter air, while others also feature a large cooling fin attachment to the lamp itself (see Figure 3). In addition, the lamphouse must contain an adjustment knob for the collector lens position and provisions (either knobs or screws) for alignment of the lamp and the reflector. Of primary concern is that the lamphouse itself must not leak harmful ultraviolet wavelengths and should incorporate a switch to automatically shut the lamp down should the housing be compromised or opened during use. As discussed above, mercury arc lamps contain a precisely measured amount of metallic mercury within the envelope, and they are filled with argon or xenon, which acts as a starter gas as the mercury vaporizes. When the lamps are cold, small droplets of mercury can often be observed on the inside walls and the gas pressure inside the envelope is lower than the ambient pressure of one atmosphere. Once the lamp is ignited, the mercury vaporizes over the course of a 5 to 10 minute transition phase. During this period, the lamp is operated at higher than normal current, requiring the anode to be positioned at the bottom of the lamp to ensure proper vaporization of the mercury. For this reason, the ferrule sockets in a mercury lamphouse have different diameters (one smaller than the other) to enable correct positioning of the lamp, which itself has a larger ferrule on the anode end of the tube. Thus, mercury arc lamps are positioned vertically within the lamphouse with the anode pointing toward the bottom and the cathode pointing upward. Operating a mercury lamp at an angle exceeding 30° from the vertical position deflects the arc toward the quartz envelope resulting in uneven heating and premature darkening of the bulb. Several mercury lamp designs incorporate a reflective coating on part of the envelope to speed the vaporization transition phase and to improve thermal distribution. Because the envelope temperature influences the internal mercury pressure to a significant degree, mercury arc lamps are sensitive to airflow over the bulb and this aspect must be carefully controlled by the lamphouse. Mercury arc lamps require a direct current (DC) power supply that is specifically designed to meet the ignition and operational requirements for each lamp design. A typical power supply must provide up to a 50 kilovolt starting pulse to ionize the gas in the arc gap, as well as an open circuit voltage three to five times the rated lamp operating voltage in order to heat the cathode to thermionic emission temperatures. Additional requirements include a maximum level of inrush current to prevent excessive thermal shock during ignition. Inrush current can be several orders of magnitude greater than the lamp circuit steady-state value and is often a contributor to ignition failure. The lamp power supply must also limit current ripple to less than 10 percent (peak to peak) to ensure long lamp life and light stability. Finally, the power supply must be able to adjust the applied current over a wide range as the voltage can significantly increase during the lamp warm-up period. Power supplies for the HBO 100 mercury arc lamps used in optical microscopy are usually equipped with several features that enable the operator to monitor the operating conditions and lifetime. Included are an indicator light for lamp ignition, a light that signifies when the transformer has reached an internal temperature within the permissible range, a safety light to alert the operator that the safety circuit of the lamp housing is closed, and a voltage light that is enabled when the transformer is performing within the permissible voltage range. All commercial mercury lamp DC power supplies also feature a re-settable display of the total time (in hours) that the lamp has been in operation. Lamphouses for arc lamps require continuous inspection and maintenance. The lamp socket assembly and power cord should be examined periodically for oxidized metal surfaces (socket electrodes) and the integrity of the cord. The socket electrodes are prone to oxidation and should be lightly brushed with an emery cloth (or extra-fine sandpaper) each time the lamp is changed to assure good electrical contact. The bulb, rear mirror reflector, and front collector lens should be inspected and cleaned if necessary to remove dirt, lint, and fingerprint oils. Each time the lamp is replaced, the collector lens assembly and reflector positioning mechanisms should be inspected for proper operation. The illuminator adjustment knobs or screws should be adjusted while examining the resulting motion of the collector and reflector to ensure they travel in the expected manner. The high-current power line connecting the power supply and lamphouse should not be crimped (as might occur when the line is shoved between a table and a wall) as this maneuver can stretch or loosen internal wires and lead to malfunction. Contributing Author Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310. Back to Microscope Light Sources
