Benefits of the Pumped Phosphor Illumination System
The promise of solid state light sources has long been an enticing one for scientists performing fluorescence imaging. The benefits of solid state sources, such as LEDs, for these applications have been understood since the first papers on this topic were published in the mid-1990s. Specifically, these sources would have extremely long lifetimes, stable output and allow for high speed digital shuttering. However, the potential utility of these light sources has been limited by the fact that they did not have adequate output power in many wavelengths for all but the brightest fluorescence samples. While LEDs could be used to generate ample power for a few specific wavelengths, a much broader set of wavelengths were needed for biological experiments. One potential solution to these limitations is the use of phosphor technology, which has long been used in applications ranging from radioisotope detection to compact fluorescent lighting. The principle of these phosphors is quite simple. They absorb energy of one wavelength and emit light at another, typically longer wavelength. Phosphors have been developed with a vast number of different compositions and, therefore, can deliver a large number of different emission wavelengths.
Incorporation of phosphors, then, allows the creation of a new class of solid state light sources. These pumped phosphor illumination systems possess significant benefits which ultimately enable the full realization of the promise of solid state sources for fluorescence imaging.
How It Works
Figure 1 shows a schematic of the basic operation of a pumped phosphor illumination system. An extremely high power, solid state pump source is used to create the input light for the system. This light is collected and fed to the conversion phosphor that absorbs the input light and converts it to the desired wavelength. This emitted phosphor light is then captured and directed into the imaging system.
Advantages of the Pumped Phosphor Light Source
At present, the vast majority of fluorescence imaging systems employ either arc lamps or metal halide lamps. Though they have high intensity output, they suffer from two major downfalls. First, they typically have useful lifetimes of approximately two hundred to two thousand hours. In contrast, the pumped phosphor light sources can have useful lifetimes upwards of 50,000 hours (Figure 2). In other words, if a pumped phosphor system were turned on and allowed to run continuously at full power, it would run for over 2,000 days (5.7 years) before needing to be replaced. In contrast, a typical mercury arc lamp would run for less than 9 days and a metal halide lamp would last approximately 3 months. Along with the more frequent need for lamp replacement comes increased cost. For example, to equal the 50,000 hour lifetime of a pumped phosphor source, one would spend $37,500 on arc lamp replacements (assuming a cost of $150/lamp and 200 hrs/lamp).
The second major limitation of arc lamps and metal halide bulbs is the fact that they require several minutes to warm up and stabilize before they can be used. This means that simply turning them on and off is not an efficient means of preventing light from hitting the sample and instead, mechanical shutters must be used. This significantly limits the speed at which high-speed imaging can be done while only illuminating the sample during the exposure time of the camera. The pumped phosphor light source is based on solid-state technology. As a result, the on and off times are less than 10 microseconds, allowing for extremely high-speed digital shuttering. This means that even in the fastest time-lapse acquisitions, the excitation light can be shuttered during the times when the camera is not actively exposing, thus minimizing both photobleaching and phototoxicity during live-cell imaging experiments.
More recently, solid state light sources, such as LED systems have come on the market. LED systems solve many of the problems mentioned above. They have long lifetimes and rapid on and off times via digital shuttering. In many ways, they are the ideal solution for fluorescence imaging. The disadvantage of these systems to date is that they do not have sufficient output power across the spectrum, especially in the green and the red. As a result, their utility has been limited in high-speed, live-cell imaging applications, and they are useful with only the brightest fluorescence samples. The pumped-phosphor design effectively eliminates this problem. There are a wide range of phosphors available, from 405 nm up though the NIR region. By utilizing a high power pump source, high output powers can be generated at wavelengths where high power LEDs are not yet available. Currently, pumped phosphor illumination systems can generate wavelengths up to 670 nm with high power, and even longer wavelengths (up to 780 nm) are currently in development.
The many advantages of pumped phosphor light sources are exemplified in the Heliophor, a new high-intensity, ultrastable light engine for fluorescent imaging applications. The Heliophor features six user exchangeable wavelength modules. With advanced triggering and control capabilities, including the ability to upload macros, the Heliophor enables rapid, multidimensional imaging. This system possesses a unique combination of high output power, rapid switch times, and sub-millisecond digital shuttering to enable high-speed, live cell imaging. The Heliophor features a liquid light guide and flexible control architecture to quickly integrate into experimental set-ups. The product’s excellent stability and straightforward calibration system ensure consistent output intensity over the life of the system. Along with flexibility in control architecture, the Heliophor’s physical components, including LED modules, optical filters and dichroics, are all interchangeable by the user. The Heliophor is backed by a limited lifetime warranty, ensuring stable, high-output power for reliable quantitative fluorescence for the life of the system.