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THE FUNDAMENTALS OF... Becky Hohman

LED Source: Major Advance in

Becky Hohman Downloaded from http://meridian.allenpress.com/bit/article-pdf/41/6/461/1484418/0899-8205(2007)41[461_llsmai]2_0_co_2.pdf by guest on 26 September 2021

luorescence microscopy re- gation. In other words, an ideal light mechanical switching devices like quires an intense light source source would start with a baseline fi lterwheels or shutters required by Fat the specifi c that contributes zero peripheral light traditional illumination systems. that will excite fl uorescent and and would allow the user to precisely LEDs of different can be . The traditional method control the addition of only those used in combination, giving users employs a light, typically from selective of light that the option of seeing multiple fl uo- a or . Al- match the particular excitation wave- rochromes simultaneously or rapidly though such broad lamps lengths of the fl uorochromes. The capturing sequential images of each can generate ample light at desired user should also be able to control fl uorochrome. wavelengths, only a small percentage the intensity and duration of the light of the projected light is useful in any to achieve the highest possible image The Principle of particular application. The other quality while protecting the sample Fluorescence wavelengths need to be suppressed against bleaching and phototoxicity. To understand how LEDs compare to avoid background noise that re- Recent advances in high perfor- to other illumination technologies, it duces image contrast and obscures mance Light Emitting Diode (LED) is useful to examine the illumination the fl uorescent light emissions. technology have enabled the practi- requirements of various fl uorescence This process of subtraction is com- cal implementation of this theoretical microscopy applications. plex, expensive, and only partially ef- model. High-intensity monochro- Fluorescence illumination is fective. Even after decades of refi ne- matic LEDs are now available in a defi ned as the emission of ments, the best fi lters are not 100% variety of colors that match the exci- by molecules whose are successful at blocking the bleed- tation bandwidth of many commonly transiently stimulated to a higher through of non-specifi c photons. used fl uorescent dyes and proteins. excitation state by radiation from Some mitigation techniques end up This new LED technology uses an outside source. In other words, not only suppressing peripheral light, specifi c wavelength windows with when a specifi c wavelength of light but also signifi cantly diminishing the much less need to suppress unwanted (the excitation wavelength) excites intensity of the desired wavelengths. peripheral wavelengths from a white fl uorescent dyes or proteins, the To address the root cause of the light arc lamp. One such system has fl uorescent material will appear to problem—the presence of non-spe- up to four LEDs, each individually glow as it gives off light of a specifi c cifi c photons—a radically different and instantly approach is coming to light. controlled by Due to the speed of changing excitation wavelengths and In theory, a design that only intro- electrical cur- attenuating intensity, LEDs are ideally suited to image duces specifi c photons is preferable rent without living cells. Shown here are time lapse images of Hela Cells to one that requires subsequent miti- any of the labeled with DS Red and expressing mutant YFP.

Biomedical Instrumentation & Technology 461 THE FUNDAMENTALS OF... LED Light Source: Major Advance in Fluorescence Microscopy

wavelength (the emission wavelength). The process is age—that is, an image with a high signal-to-noise ratio. practically instantaneous: the emission occurs less than Wavelengths that match the fl uorochrome strengthen 10-6 seconds after the end of excitation. the signal, but any peripheral wavelengths produce back- Because energy is used in this process, the emission ground noise that can overshadow the signal emitted by

wavelength will always have less energy than the excita- the object of interest. Downloaded from http://meridian.allenpress.com/bit/article-pdf/41/6/461/1484418/0899-8205(2007)41[461_llsmai]2_0_co_2.pdf by guest on 26 September 2021 tion wavelength, and by defi nition, lower energy light always has a higher wavelength. The difference in wave- lengths between the excitation and emitted fl uorescent Contrast = photons is called the “.” Depending on the (Brightness of Specimen – Brightness of Background) fl uorochrome, the shift can range from just a few nano- (Brightness of Specimen + Brightness of Background) meters to several hundred nanometers.

Expanding Use of This formula, while a good starting point, is a bit sim- Fluorescence Microscopy ple. Other factors like detector noise, internal straylight, Fluorescence microscopy utilizes optical fi lters to sepa- etc., contribute to the contrast as well. rate excitation light from the emitted fl uorescence, which A second and related requirement is illumination in- is observed visually or detected by a camera equipped tensity, i.e., the number of photons specifi c to the excita- with a charge-coupled device (CCD) or other detectors. tion wavelength that reach the specimen. Obviously, the Fluorescence microscopy is an increasingly widespread intensity must be suffi cient to illuminate the objects of medical and biological laboratory technique that pro- interest, but the optimal level of intensity will vary by vides highly sensitive detection for medical diagnostics application. The human eye is less sensitive than most and allows for the detection of cellular components and automated detection systems, and therefore applications inter- and intra-cellular communication. Fluorescence involving visual observation typically require higher lev- microscopy is capable of detecting single molecules els of illumination intensity. However, lower intensity and sub-microscopic structures, which are too small to may be preferable in live cell imaging applications to be resolved by other conventional tech- protect against photobleaching and phototoxicity. niques. Intensity of illumination and signal intensity do not The availability of () fl uorescent proteins (GFPs) just follow a linear correlation. Saturation effects and derived from jellyfi sh, corals, etc., and their -shifted dark states become more and more important with in- genetic derivatives has greatly expanded the use of fl uo- creasing illumination intensity. rescence microscopy during the past 10 years. Fluores- An additional consideration is protection of the speci- cent proteins such as GFP are less phototoxic than the men, especially in live cell imaging applications. We have small fl uorescent molecules in most chemical dyes such already mentioned the dangers of overexposure to light: as fl uorescein isothiocyanate (FITC), which can harm the phototoxicity of the specimen, and photobleaching of the specimen when illuminated during live cell fl uorescence fl uorescent or . Overexposure can be avoided microscopy. This attribute of fl uorescent proteins has in- by attenuating the light intensity and by limiting the du- spired the development of highly automated time-lapse ration of the illumination to exactly the exposure time live cell imaging systems. of the sensor. The cells also need to be protected from Even more important is the fact that fl uorescent pro- the heat generated by light source lamps and from the teins can be expressed by the cells while other fl uorescent vibrations caused by mechanical fi ltering and switching dyes usually cannot penetrate the membranes of living devices. cells. This limits their use mainly to fi xed cells or needs Another factor to consider when evaluating light microinjection, electroporation, etc. sources is the lifetime and stability of the lamp. Some light sources exhibit short-term intensity fl uctuations Light Source Requirements and a substantial deterioration of over time. The most basic requirement of a fl uorescence microscopy Limited lifespan results in more frequent bulb replace- light source is closely matching the excitation wavelength ments, and poor stability diminishes the reproducibility of the fl uorochrome to achieve a high-contrast im- of illumination conditions.

462 November/December 2007 THE FUNDAMENTALS OF... Becky Hohman

Traditional White Light Lamps two different wavelengths, such as calcium experiments. Xenon lamps also have the advantage of more stable Mercury Lamps emissions over time. The lower fl uctuations facilitate Historically, mercury-vapor high-pressure arc lamps more reliable measurements in ratio imaging and other

(commonly referred to as HBO lamps) have been the quantitative applications. The long-time stability relates Downloaded from http://meridian.allenpress.com/bit/article-pdf/41/6/461/1484418/0899-8205(2007)41[461_llsmai]2_0_co_2.pdf by guest on 26 September 2021 most prevalent light source for fl uorescence microscopy. to the relatively long life span of XBO (400 to 2,000 An ecosystem grew around this illumination method hours, depending on the bulb). during the past few decades, as chemical fl uorescent dyes The disadvantages of XBO include the inability to at- were developed and selected based upon conformance to tenuate intensity without the need for proper alignment. the arbitrary emission peaks of the HBO spectrum, and XBO also generates considerable heat that could damage many suitable fi lter sets became available. the specimen, and the high-pressure bulbs are danger- Mercury light is characterized by very high emission ously explosive. peaks in the (UV) and green regions, while between the peaks the excitation energy of the light Fiber-Coupled White Light Systems source is very low. The high excitation energy produces a bright signal from fl uorochromes, making HBO a good Metal Halide for applications with weak (if the fl uo- For many years, the decision about which light source rochrome is stable enough to resist the effects of photo- to choose was limited: mercury or xenon. In the last fi ve bleaching). As mentioned above, a weak staining will not years, new types of light sources have been emerging in result in a bright signal by just increasing the excitation fl uorescence microscopy. One of the fi rst newcomers was intensity above a certain level. The high-illumination the metal halide (HXP) white light source. HXP has a intensity of mercury lamps is diffi cult to attenuate, which similar to mercury, but in contrast to increases the risk of phototoxicity of living cells. mercury light sources, it is fi ber-coupled to the micro- The short life span of the HBO lamps (approximately scope. The lamp and its power supply are housed in an 300 hours) necessitates relatively frequent bulb changes, external box, which is joined to the microscope, typically which can be an expensive and cumbersome process. with a liquid light guide. The fi ber-coupling reduces heat Newer systems simplify the adjustments required to align transfer from the light source to the microscope. The a new bulb or even automate this process. HXP light sources provide high intensity comparable to HBO at mercury peaks. Xenon Lamps The liquid light guide acts as a scrambler to homog- A second high-pressure white light source, xenon-vapor enize the arc output, eliminating the need to center the arc lamps (XBO), produces relatively even emission levels light and creating an even fi eld of illumination without across the visible spectrum. Without the so-called mer- any need for alignment. HXP bulbs have a long lifetime, cury peaks, the nonspecifi c photons emitted by XBO are approaching 2,000 hours, but the liquid light guide has a somewhat easier to suppress with fi ltration, and therefore recommended lifetime of 18 to 24 months. XBO is often thought to provide better image contrast and to better protect living cells and tissue from pho- High-Speed Xenon Systems totoxicity. While the fl at output spectrum of the xenon For live-cell applications involving high-speed image bulb means that it is signifi cantly lower in intensity at the acquisition, several manufacturers offer external il- wavelengths typically associated with the mercury peaks, lumination systems utilizing a xenon lamp with a fi ber the mercury output can be up to 20 times brighter than coupling to the microscope. These systems employ a the xenon light source. However, outside of those peak combination of mechanical and optical techniques to areas, the intensity is comparable. One area of particular achieve extremely high-speed switching (1 to 3 milli- interest is the typical 480- to 500-nanometer excitation seconds) to change intensity and wavelength. The fi ber range of GFP, where Xenon has an intensity similar to coupling has the advantage of reducing heat transfer that of mercury HBO. from the light source to the microscope. The maximum The more even emission levels make XBO better illumination intensity is low and can be insuffi cient for suited to applications that compare the intensity between visual observation.

Biomedical Instrumentation & Technology 463 THE FUNDAMENTALS OF... LED Light Source: Major Advance in Fluorescence Microscopy

Light-Emitting Diodes LEDs have been met with widespread acceptance in A light-emitting diode is a compact de- the fi eld of . High-brightness LEDs are widely vice that emits incoherent narrow-spectrum light when used for endoscopes and other invasive optical equipment. electrical current is applied. The color of the emitted The highly specifi c light emissions make LEDs effective

light depends on the composition and condition of the at diagnostic techniques like blood glucose measurement Downloaded from http://meridian.allenpress.com/bit/article-pdf/41/6/461/1484418/0899-8205(2007)41[461_llsmai]2_0_co_2.pdf by guest on 26 September 2021 semiconducting material used, and can be near-ultravio- or pulse oxymetry, as well as for therapeutic applications let, visible, or . such as acne therapy (blue or blue/red), neonatal jaundice Unlike arc lamps, which are inherently very bright, therapy (blue), and when com- LED technology has dramatically evolved from humble bined with specifi c drug treatment. beginnings. When the fi rst commercial LEDs were in- troduced in 1968, they were capable of providing only Advantages of LED 0.001 lumens of red light, a level of brightness suitable Now that high-performance LEDs provide suffi cient only for use as indicators. During the past four decades, intensity at the specifi c wavelengths required for many LED technology has advanced at a rapid pace, compa- applications, fl uorescence microscopy is able to take rable to the rate of advancement of microprocessors. advantage of the benefi ts of LEDs, including compact It is common knowledge that Gordon E. Moore pre- size, low power consumption, minimal heat output, fast dicted that the number of transistors on a chip would switching and adjusting properties, high-emission stabil- double about every two ity, and extremely long lifespan years. Less well known, An advantage of LEDs is that they instantly illuminate except among LED en- at full intensity as soon as electrical current is applied. gineers, is that a scientist Unlike arc lamps that are turned on continuously, LEDs at Agilent Technologies, can be switched on or off when needed, with no deleteri- Roland Haitz, predicted ous effects to their lifespan. Additionally, with no moving that the amount of light parts, the all-electronic system is vibration free. generated by an LED The intensity of every LED module can be adjusted would increase by a fac- in percentage steps, enabling equidistant multichannel tor of 20 every 10 years. images to be easily realized in time-lapse series. Instead “Haitz’ Law” has proven of adapting the integration times of the camera to the to be a reliable forecast, illumination intensity, LED technology makes it possible as LEDs have histori- to simply set the illumination intensity for the required cally doubled in bright- integration time. The LED illumination intensity is also 10 different LED modules, ness every two years, and highly stable over time, making quantitative analyses which can be easily ex- performance is predicted easier and more reliable. changed, are currently avail- to continue to increase by While the intensity of the LEDs has evolved signifi - able, from UV to dark red. 20% annually. cantly over the past few years, it is still not as high as con- As LEDs have evolved ventional arc lamps. However, in most live cell imaging in both brightness and in the range of available colors, environments, intensity from conventional light sources they have been put to use in a wide variety of new ap- are typically reduced to minimize phototoxic effects to plications. The most prevalent application is illumina- cells and tissues. White light sources have been in use for tion as an energy-effi cient and durable replacement for decades, and much expertise has been developed in the incandescent light bulbs. In fact, the U.S. Department ways of reducing the problems associated with periph- of Energy has been investing heavily in this technol- eral light. Many labs have invested a great deal in fi lters ogy—$91.8 million in 2007—as a core component of the used to suppress non-specifi c light. In the past, it may federal government’s energy conservation plans. have been diffi cult to imagine that any other light source High-performance LEDs are employed in many in- method would ever be viable. „ dustrial and medical applications. For example, the fi ber optic gyroscope (FOG) is used in navigation, robotics, and Becky Hohman is the product manager for light microscopy at Carl Zeiss MicroImaging, Inc. (www.zeiss.com/micro). machine-vision systems for automated manufacturing.

464 November/December 2007