New Approaches to Traditional Fluorescence Illumination

Barbara Foster, The Microscopy & Imaging Place Inc.; and Lars Dugaiczyk, Ushio America Inc.

Fluorescence microscopy presents an array of illumination challenges. Key among these is matching the spectrum of the light source to the fluorophore in the sample, to the bulb alignment and lifetime, and to the evenness of illumination.

A new lamp that optimizes many of these issues has been designed by veteran bulb designer Ushio America Inc. of Irvine, Calif. Derived from its breakthrough EmArc technology (Figure 1), the lamp integrates a very short arc, DC-enhanced metal halide light source with an integrated reflector assembly, optimizing the optics so that the lamp requires no alignment. The results are a more economical bright light source that produces even illumination, spectral capability comparable to that of other mercury arc sources and a bulb life well in excess of 1000 hours.

Figure 1. Shown is the 75-W EmArc long-focal-length lamp for fluorescence microscopy.

Challenges in existing systems

The gold standard for light sources for fluorescence microscopy today is the 100-W mercury arc lamp. This lamp is attached to the microscope via a housing fitted with a collector lens in front of the bulb for focus and, for enhanced illumination, a small concave mirror behind the arc that reflects additional intensity forward. In actual fact, this system has very poor collection efficiency, requires periodic realignment of the lamp and mirror system and, of more concern, produces a nonuniform field of illumination.

To align the system to the microscope, the microscope is set up for Koehler illumination using a high-numerical-aperture objective focused on a well-behaved slide (a thin section with good contrast). The slide is then replaced with a blank surface, such as the back of a business card, and the objective turret is rotated to an open position (no objective). The image of the arc source is focused on the blank surface using the lamp collector lens and is centered using the X and Y screws (which control actual lamp placement) on the lamp house. The process is repeated for the mirror image of the source, using the focus and centration controls for the rear reflector. Depending upon the microscope manufacturer, the direct image and the mirror image are either aligned side by side or superimposed.

If left in this state when an objective is rotated back into position for imaging, the bright spot at the cathode tip of the lamp produces a very hot spot in the center of the field of view, generating two serious problems. First, the hot spot enhances localized photobleaching. Second, the uneven illumination produces falloff at the image edges, making accurate quantitation difficult. To remedy this, the image of the arc source is typically blurred by defocusing it. However, blurring diminishes intensity and, if the full alignment is not done meticulously, still results in uneven illumination.

One solution is to increase the lamp wattage. However, arc lamps introduce unwanted heat near the delicate optics (even 100-W mercury arc lamps are known for burning holes through more delicate components such as polarizers), and, although 100-W mercury arc lamps can be convection-cooled, higher-wattage lamps require a fan that can transfer vibration to the microscope, destabilizing the image, especially at high magnification.

A second solution involves using a remote illuminator-coupled microscope via a fiber lightguide. Light sources used in this approach are typically very high pressure AC or DC arc lamps prealigned in an elliptical reflector. These sources produce a bright, uniform image and do not require alignment, but there is significant loss of efficiency both through the lightguide and the optical coupling to the microscope.

New engineering approach

The 75-W EmArc long-focal-length system incorporates a number of new engineering tactics. Its light sources are DC-enhanced metal-halide arc lamps with very short arc gaps (Figure 1). The short gap enhances overall brightness, comparing favorably to traditional 100-W mercury arc or xenon in intensity while using a lower wattage. Because mercury is used in the metal halide, the new lamp enables all the commonly used mercury arc peaks (Figure 2).

Figure 2. The 75-W EmArc spectrum contains all the commonly used mercury spectral peaks with a rated lifetime in excess of 1000 hours.

The system’s unique hybrid gas technology ensures that it is not under internal high pressure when cold, making it much safer to handle. Precise gas-filling control coupled with the electrode design produces a tightly confined, stable plasma discharge, minimizing color temperature drift over its lifetime and dramatically extending bulb lifetime. Typical 100-W mercury arcs are rated for 200 to 300 hours, while the 75-W EmArc is rated for 1000-plus hours. (Laboratory tests show that the 75-W source retains 60 percent of its intensity, even at 2000 hours.)

Proprietary EmArc DC technology requires a simpler power supply, making the lamps less expensive at the buying stage. And because they are 75- versus 100-W sources, they are more economical to operate throughout their lifetime. The same technology also provides tunability, a fairly recent development for arc lamps.

And again, because the lamp runs cooler, there is no need for a heat sink, and there is less infrared in the spectrum to contribute to unwanted background. The lower thermal load generates less strain on the illumination/microscope system, simplifying lamp house design and, again, bringing down the cost.

The reflector assembly is a key differentiating factor for this lamp. A low-numerical-aperture compound elliptical reflector with the long focal length required specifically for microscopy, it has been designed not only to be very durable but to provide a highly efficient light path, as shown by the results below.

Plug-and-play design

In the past, each time an arc source had to be replaced, the new lamp required the centering and focusing described above, a process that can be frustrating at best and, with a rated bulb lifetime typically on the order of 200 hours, time-consuming. (If a typical lamp is used eight hours per day, bulbs must be replaced approximately every five to six weeks.)

Figure 3. This diagram is of an optical test setup. All test diagrams, images and results are courtesy of Lars Dugaiczyk, Ushio America Inc.

Furthermore, as fluorescence microscopy expands out of the research lab and into secondary markets such as the clinical arena, it is being used by less sophisticated operators, presenting further challenges: Mercury arcs are high-pressure sources that require gentle, knowledgeable handling and safety precautions, including safety glasses to guard against explosion and cotton gloves to avoid fingerprints on the glass envelope. Under the thermal strain of daily use, fingerprints can weaken the envelope and, again, lead to lamp failure.

In contrast, the 75-W lamp offers a simple plug-and-play design. The lamp and reflector are integrated, so there is no alignment of direct image to mirror image. Simply rotate the objective turret to an open space and focus the lamp image at the specimen plane. With a rated lifetime in excess of 1000 hours, lamp replacement drops to one-quarter to one-fifth as often, compared with more traditional sources.

Figure 4. These results compare (a) the 200-W fiber optic illuminator with (b) the 46-W LED array.

Comparing apples to apples

To test its performance against other widely used illumination systems, an optical bench was set up (Figure 3) using the range of illuminators listed in the table. Each light source was placed so that the image of the 25-mm aperture plate could be focused on an opal glass diffuser, similar to the illumination expected at the sample plane of a fluorescence microscope. A Lumetrix camera collected the image formed on the opal glass, and all images were taken with the same pseudocolor histogram settings. Red indicates higher luminance.

Comparing the results for the 200-W fiber optic illuminator and the 46-W LED array (Figure 4) with those of the other three sources (Figures 5 and 6) reveals that, although they both produce very even illumination, they are not very efficient.

As evaluated by both the intensity maps and intensity output graph in Figure 5, when the optical setup is tuned for maximum output, the 75-W long focal length performs well in comparison with the traditional 100-W mercury arc and outperforms its standard counterpart. When the setup is tuned for optimum uniformity (Figure 6), the 75 W outperforms the traditional mercury source. Field uniformity is especially important in tissue applications and provides a consistent background, essential for even simple quantitation such as automated cell counting and sizing.

Figure 5. Adjusted for maximum output, (a) the 75-W EmArc/standard reflector, (b) the 75-W EmArc long-focal-length reflector and (c) a standard 100-W mercury arc. (d) Comparison of vertical cross section of intensity, set for maximum output, reveals that the 75-W long-focal-length lamp is a strong second to the 100-W mercury arc.

For decades, the 100-W mercury arc has been the gold standard for illumination for fluorescence microscopy in cell and tissue applications as well as for genomics and proteomics. With the rise of new lamp housings, liquid lightguides and LED sources have seen active innovation in this area over the past 10 years. As the next step in lamp evolution, the new 75-W EmArc provides a unique mix of safe, high-intensity, even illumination. Easy to use, it satisfies both the needs of today’s researchers and the future needs of more routine users in the expanding clinical fluorescence market.

Figure 6. Adjusted for maximum uniformity, (a) the 75-W EmArc/standard reflector, (b) the 75-W EmArc long-focal-length reflector, and (c) a standard mercury arc. (d) Comparison of vertical cross section of intensity, set for maximum uniformity. Both the intensity maps on the left and the graph on the right confirm that the 75-W long-focal-length lamp produces more even illumination across a wider area than does the 100-W mercury arc.

Meet the author

Barbara Foster is president and chief strategic consultant at The Microscopy & Imaging Place Inc. in McKinney, Texas; e-mail: bfoster@the-mip.com. Lars Dugaiczyk is an engineer at Ushio America Inc.; e-mail: ldugaiczyk@ushio.com. Technical questions may be addressed to Keith Cordero, director of applications engineering, Ushio America Inc.; e-mail: kcordero@ushio.com.