Stability in Fluorescence Light Sources
Fluorescence imaging has become a common technique in biomedical research applications. The ability to visualize and track molecules, both big and small within living cells has enabled researchers to ask questions previously difficult or impossible to answer. As fluorescence imaging-based applications have grown and developed, there has been an ever increasing trend away from more qualitative studies to more quantitative types of research. Researchers are no longer sufficient asking “Where is my molecule with in the cell.” They now want to know where it is and how much is there and has that amount changed over some defined period of time. In addition, a host of ratiometric fluorescence probes are available where the fluorescence intensity of sequentially taken images is compared on a pixel by pixel basis, resulting in quantitative data on the concentration of the target molecule within the cell.
In order to perform quantitative fluorescence imaging, it is essential that every component of your imaging system is stable. Most of the system related errors associated with quantitative fluorescence imaging result from the inherent instabilities of the fluorescence light source. For any quantitative fluorescence imaging technique, the stability of the fluorescence light source is critical. If there are changes in the intensity of the fluorescence lamp during an experiment or between experiments, the ability to accurately quantitate the resulting data is significantly reduced. Therefore, the stability of the fluorescence light source is of paramount importance to quantitative imaging applications.
In addition to having adverse effects on fluorescence microscopy, other fluorescence imaging applications can also be effected by the lamp instability. Foremost of these is in vivo or whole animal imaging applications. These types of experiments can often last days, weeks or months where the same animal or group of animals are imaged over and over again and changes in the fluorescence signal are tracked over time. It is quite easy to see where a 20-30% change in the intensity of the fluorescence lamp could have significant effects on the quantitation and results of these studies.
What is Stability?
When discussing stability in fluorescence light sources, people often define “stability” differently. For this article, we will use the terms short term and long term stability. Short term stability is defined as the stability of the light source during a given experiment. This can refer to the stability of the lamp between subsequent acquisitions or over the course of the experiment. Reduction in short term stability can have detrimental effects on ratiometric imaging applications since changes in lamp intensity between sequential images can artificially alter the subsequent ratios. In addition, if monitoring the amount of a fluorescence signal within a region of the cell, changes in the intensity of the light source during the experiment will result in changes in intensity within that the defined region that have no biological relevance. Long term stability is defined as the stability of the light source between subsequent experiments. For example, if an experiment needs to be repeated (say in response to a reviewer’s comments) and there is a difference in lamp intensity, the resulting data set can be quite different. The change in lamp intensity will change the photon flux impinging upon the sample, and it is well established that the photon flux at the cell can have significant effects on the biology of the cell. Changes in the photon flux at the cell can therefore have significant effects on the biological activity of your sample and thus change the resulting data and conclusions.
Types of Lamps
Historically, arc lamps have been the preferred light source for fluorescence imaging applications. While very bright, these types of lamps suffer from both short term and long term instability. In the short term, these types of lamps can suffer “arc instability” that can manifest itself as arc wander, arc flutter or arc flare. In arc flare, there is a temporary change in the lamp’s intensity as the arc changes position on the cathode. Arc wander occurs as the arc slowly moves around the tip of the cathode resulting in a several seconds of instability of the lamp output. Finally, arc flutter results from the convection currents that occur from the differences in temperature between the arc and the area surrounding the arc. Together, these three factors can cause significant changes in output power and significantly reduce the short term stability of these types of lamps.
Long term stability is also an issue with arc lamps. As arc lamps age (over the course of ~200 hours), the distance between the electrodes of the lamp increases due to the extreme heat and pressure of the arc slowly breaking down the conical tips of the anode and cathode. As the distance between the anode and cathode increases, the amount of current that passes between them is reduced, resulting in a decrease in lamp intensity. This drop in intensity can be significant, on the order of 20-30% over the ~200 hour life of the lamp.
Metal Halide Lamps
Metal halide lamps have become a popular alternative to mercury and xenon arc lamps. Metal halide lamps are a type of high power arc lamp, mounted in a reflector that collects the light and, typically, launches the light into a liquid light guide. Light sources that use metal halide lamps can be divided into two main categories, those powered by an AC power supply and those powered from a DC power supply. The AC-powered lamps are typically less stable then their DC counterpart, such as the PhotoFluor® II light source from 89 North (Figures 1 and 2). AC powered lamps have significant fluctuations in their intensity due to the alternating nature of the AC power supply (Figure 1, gray line). DC powered systems such as the PhotoFluor II have much greater output stability. In addition, AC powered metal halide lamps have significant arc instabilities as compared to their DC counterparts.
Figure 2 shows a data set comparing an AC powered and a DC powered light source. The sample is an auto fluorescence test slide from Chroma Technology Corp that was continually imaged over the course of 14 hours. The slow, gradual decrease in fluorescence is simply photobleaching of the sample but could easily represent the loss of fluorescence in a specific region of a biological sample. Comparing the blue and gray lines, you can see that the intensity of the gray line (AC powered lamp) has significant fluctuations as compared to the blue line (DC powered lamp). These fluctuations are the manifestations of the arc instability discussed above. From this graph, it is clear that if this was an actual experiment, it would be very difficult to analyze this data and come to the conclusion that the fluorescence intensity is dropping over time. Comparing the intensity at hour 1 and hour 10, the hour 10 intensity is actually brighter though this does not reflect the reality of the sample. It is purely related to arc instability.
Metal halide lamps also suffer from long term stability issues though they are much less pronounced than standard arc lamps. Where arc lamps have lifetimes in the range of 200 hours, metal halide lamps have lifetimes up to 2000 hours. Though the absolute amount of intensity loss over the life of the lamps are similar, with up to a 2000-hour lifetime metal halide lamps will offer greater long term stability over the life of the lamps.
LED Light Sources
LED-based light sources are starting to become more popular in fluorescence imaging applications. LEDs are solid-state, digitally controlled semiconductor materials that emit light in a fairly narrow band (as compared to arc lamps) in response to an applied electrical current. Because they are not arc-based, they do not suffer from any of the arc related instabilities that many of the previous light sources described above do. As such, these types of light sources are much more stable in both the short and long term. However, LEDs do have several attributes related to their design that can contribute to both short term and long term instability issues. These are especially true for many of the higher power LEDs. Higher power LEDs, by nature of how they work, require more current to drive them. This in turn, creates more heat, and heat is the enemy of LEDs. Many of the high power LEDs will shift their peak wavelength as they warm up. In terms of total output power, the peak wavelength shift may not have a significant effect.
However within a defined wavelength band (as you would have in a microscope system), the shift in peak wavelength will manifest itself as a change in overall output intensity, thus decreasing the short term stability of the system. In addition to the wavelength shift, nearly all LEDs will suffer from a drop in total output as they warm up. This drop in output can take several seconds to several minutes to stabilize, depending on the specific LED. An example of this is seen in the gray trace in figure 3. Here, the LED light source takes around 1 minute of constant “on time” to stabilize its output intensity. The drop in intensity can be as much as 10% of the output power. Again, this can have dramatic effects on the short term stability of these systems.
In addition to these short term instability issues, LEDs also suffer from long term instability issues though these are far less significant than both arc lamps and metal halide. With expected life times of 20,000 hours or more, even though LEDs can lose up to 10-30% of their power over this lifetime, the rate of loss is extremely small as compared to other light sources.
As fluorescence light sources have progressed from less stable (arc lamps) to more stable (LEDs), the types of experiments researchers want to perform has also changed, moving more and more towards a more quantitative approach. As a result, the demands and expectations of fluorescence light sources have increased significantly. Recently, an LED-based light source has been designed that achieves the highest level of stability possible. The Heliophor, a pumped-phosphor light source from 89 North, has implemented a closed-loop feedback system into its LED-based light engine. The feedback loop samples the light intensity in real time and makes constant adjustments to the current being applied to the system. These real time adjustments ensure that the output stability of the Heliophor is extremely high. Two examples of this stability are shown. The blue line in figure 3 shows single pulse of about 50 seconds. As can be seen, the system comes to power (in <10 microseconds) and stays stable over the duration of the pulse. Figure 4 demonstrates the stability over 10 hours. Again, the stability of the system is extremely high.
As can be seen, with closed loop feedback, the output stability of the Heliophor is extremely high. Over both 60 seconds and 10 hours, the stability is better than 0.2% RMS. In fact, over the life of the system, the output intensity will vary by 1% or less. This level of stability is essential to achieve truly quantitative imaging.