Light Sources for Optical Microscopy
The overall performance of the various illumination sources available for optical microscopy depends on the emission characteristics and geometry of the source, as well as the focal length, magnification and numerical aperture of the collector lens system. These, in turn, are affected by the shape and position of lenses and mirrors within the system. In gauging the suitability of a particular light source, the important parameters are structure (the spatial distribution of light, source geometry, coherence, and alignment), the wavelength distribution, spatial and temporal stability, brightness, and to what degree these various parameters can be controlled.
Fundamentals of Illumination Sources for Optical Microscopy - This discussion addresses brightness, stability, coherence, wavelength distribution, and uniformity in the most common light sources currently employed for investigations in transmitted and fluorescence microscopy. Included are comparisons of the radiant energy output of the various sources, a review of the origins of instability in arc lamps, an explanation of the halogen regenerative cycle, as well as the fundamentals of Köhler and critical illumination.
Tungsten-Halogen Lamps - Incandescent light sources, including older versions with tungsten and carbon filaments, as well as the newer, more advanced tungsten-halogen lamps, have been successfully employed as a highly reliable light source in optical microscopy for many decades and continue to be the one of the illumination mechanisms of choice for a variety of imaging modalities. Tungsten-halogen lamps, the most advanced design in this class, generate a continuous distribution of light across the visible spectrum, although most of the energy emitted by these lamps is dissipated as heat in the infrared wavelengths.
Mercury Arc Lamps - High pressure mercury vapor arc-discharge lamps range between 10 and 100 times brighter than incandescent lamps (such as the tungsten-halogen) and can provide intense illumination over selected wavelength bands throughout the visible spectral region when combined with the appropriate filters. These illumination sources are highly reliable, produce very high flux densities, and have historically been widely used in fluorescence microscopy. Unfortunately, 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.
Xenon Arc Lamps - In contrast to mercury and metal halide illumination sources, the xenon arc lamp is distinct in that it produces a largely continuous and uniform spectrum across the entire visible spectral region. Because the xenon lamp emission profile features a color temperature of approximately 6000 K (very close to that of sunlight) and lacks prominent emission lines, this illumination source is more advantageous than mercury arc lamps for many applications in quantitative fluorescence microscopy.
Metal Halide Arc Lamps - Metal halide illumination sources are rapidly emerging as a serious challenger to the application of mercury and xenon arc lamps for investigations in fluorescence microscopy. These light sources feature a high-performance arc discharge lamp housed in an elliptical reflector that focuses the output into a liquid light guide for delivery to the microscope optical train. Advanced versions also contain internal filter wheels for wavelength selection, shutters, and neutral density filters to control intensity. The metal halide lamps that are most useful for microscopy have an emission output featuring pressure-broadened versions of the prominent mercury arc spectral lines in addition to higher radiation levels in the continuous regions between lines.
Light-Emitting Diodes - Among the most promising of emerging technologies for illumination in optical microscopy is the light-emitting diode (LED). These versatile semiconductor devices possess all of the desirable features that incandescent (tungsten halogen) and arc lamps lack, and are now efficient enough to be powered by low-voltage batteries or relatively inexpensive switchable power supplies. The diverse spectral output afforded by LEDs makes it possible to select an individual diode light source to supply the optimum excitation wavelength band for fluorophores spanning the ultraviolet, visible, and near-infrared regions. Furthermore, newer high-power LEDs generate sufficient intensity to provide a useful illumination source for a wide spectrum of applications in fluorescence microscopy.
Light Source Power Levels - Choosing the appropriate light source for investigations in optical microscopy is highly dependent upon the illumination strategy (transmitted or episcopic), specimen parameters, microscope configuration, and the detector sensitivity. In fluorescence microscopy, the class of fluorophore (synthetics, fluorescent proteins, quantum dots, etc.), filter set characteristics (bandwidth and profile), and detector acquisition speed are variables that must be also considered. As a general rule, synthetic fluorophores and quantum dots can be used with fixed cells with high illumination power, whereas in live-cell imaging, fluorescent proteins and other probes should be excited at much lower power levels. The tables presented in this section are compilations of the comparative output powers (measured at the objective focal plane) for the most popular non-coherent light sources used in epi-fluorescence optical microscopy.
Interactive Flash Tutorials
Arc Lamp Instability - Illumination sources based on plasma discharge (arc lamps) require a considerable period after ignition to reach thermal equilibrium, a factor that can affect temporal, spatial, and spectral stability. Generally, arc discharge lamps are the most unstable illumination sources currently used in optical microscopy. Besides the fact that the arc exhibits a significant degree of chaotic, flickering discharge that worsens with age, the light output can also be affected by ambient electromagnetic fields or an unstable power supply.
The Halogen Regenerative Cycle - The halogen compounds found in tungsten-halogen lamps serve to initiate a reversible chemical reaction with tungsten evaporated from the filament to yield gaseous tungsten oxyhalide molecules in the vapor phase. Thermal gradients formed as a result of the temperature differential between the hot filament and the cooler envelope contribute to interception and recycling of tungsten to the lamp filament through a phenomenon known as the halogen regenerative cycle.
Coherence of Light - One of the important parameters of illumination sources is their coherence, which is somewhat related to brightness due to the fact that extremely bright light sources are more likely to be highly coherent. Light sources that are relatively incoherent limit their interference to the microscope focal plane while highly coherent sources generate reflections from virtually every dust particle and imperfection in the optical system, and thus are less desirable.
Elliptical Reflectors - Light sources suitable for use in high-performance fluorescence microscopy couple advanced metal halide arc lamps with elliptical collection mirrors and high-speed filter wheels for rapidly shifting the output wavelength. These sources also provide fiber optics or liquid light guides for coupling the output to the microscope optical train. This interactive tutorial explores how careful positioning of the arc with respect to elliptical reflector focal points is critical to the formation of a focused beam at the input of a liquid light guide.
Mercury Lamphouses - 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. This interactive tutorial examines advanced mercury arc lamphouses that are capable of automatic bulb alignment and intensity control.
Light-Emitting Diode Operation - The fundamental key to manipulating the properties of LEDs is the electronic nature of the p-n junction between two different semiconductor materials. When dissimilar doped semiconductors are fused, the flow of current into the junction and the wavelength characteristics of the emitted light are determined by the electronic character of each material. This interactive tutorial explores how two dissimilar doped semiconductors can produce light when a voltage is applied to the junction region between the materials.
LED Illumination for Microscopy - High-power LEDs generate sufficient intensity to provide a useful illumination source for a wide spectrum of applications in fluorescence microscopy, including the examination of fixed cells and tissues, as well as live-cell imaging coupled to Förster resonance energy transfer (FRET) and lifetime measurement (FLIM) techniques. The interactive tutorial featured in this section explores the ZEISS Colibri LED illumination system for widefield fluorescence microscopy.
Andreas Nolte - ZEISS AG, Goettingen, Germany.
Lutz Höring - ZEISS AG, Oberkochen, Germany.
Christopher S. Murphy, Kristin L. Hazelwood, Adam Rainey, Tony Gines, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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