An innovative new fluorescence filter block design by Nikon helps to eliminate the possibility of residual stray light that occurs in the microscope fluorescence optical pathway, vastly improving the emission signal-to-noise ratio. Termed the Noise Terminator, this technology directs deviated stray light away from the objective light collection path, resulting in a dramatic improvement in image contrast. This interactive tutorial demonstrates how the Noise Terminator technology functions.
The tutorial initializes a traditional fluorescence filter optical block (from an inverted microscope) containing an excitation and barrier filter, as well as a dichromatic mirror, appearing in the window. Photons (yellow spheres) from the light source enter the filter block from the right-hand side of the window and pass through the excitation filter, which is a bandpass filter allowing primarily wavelengths between 510 and 560 nanometers (green light) to pass through. The light leaving the excitation filter (green spheres) is deflected at a 90-degree angle by the dichromatic mirror and up to the objective and specimen. Some of the shorter and longer wavelengths that are able to pass through the excitation filter (represented in the tutorial as red and blue spheres) are not deflected by the dichromatic mirror and pass through. A portion of these photons, which are a major source of noise in fluorescence microscopy, reflect from the interior of the optical block and are able to traverse the barrier filter at oblique angles to ultimately reach the detector.
In order to operate the tutorial, use the Illumination Intensity slider to regulate the number of photons entering the filter optical block. The Activate Noise Terminator checkbox (disabled by default) can be enabled to demonstrate the Nikon deflector technology, which effectively traps photons that would otherwise generate noise. When the Noise Terminator checkbox is activated, photons flow through the rear of the optical block into the terminator tube and are absorbed by the neutral density material, thus escaping the microscope optical path. Any light that does not get absorbed by the neutral density material is reflected into the end of the terminator tube where it is finally dispersed. The Microscope Configuration radio buttons can be employed to choose between the optical block and noise terminator orientation for an Inverted (default) tissue culture or traditional Upright instrument. Use the Tutorial Speed slider to control the rate of photon flow through the optical block.
The essential feature of any fluorescence microscope is to provide a mechanism for excitation of the specimen with selectively filtered illumination followed by isolation of the much weaker fluorescence emission signal using a second filter to enable image formation on a dark background with maximum sensitivity. Localized probe concentration in biological specimens is so low in many experiments that only a small fraction of the excitation light is absorbed by the fluorescent species. Furthermore, of those fluorophores that are able to absorb a quantity of illumination, the percentage that will emit secondary fluorescence is even lower. The resulting fluorescence emission brightness level will range between three and six orders of magnitude less than that of the illumination. Thus, the fundamental problem in fluorescence microscopy is to produce high-efficiency illumination of the specimen, while simultaneously capturing weak fluorescence emission that is effectively separated from the much more intense illumination band. These conditions are satisfied in modern fluorescence instruments by a combination of filters that coordinate excitation and emission requirements based on the action and properties of the dichromatic beamsplitter.
The principles behind dichromatic beamsplitter (mirror) function in reflected light fluorescence microscopy are outlined in the tutorial for excitation illumination in the green region (550 nanometers) and fluorescence in the red (620 to 660 nanometers) wavelengths of the visible light spectrum. A high-intensity light source outputs a wide spectrum of excitation wavelengths at high flux density (usually covering most of the ultraviolet and the entire visible spectrum), which travels through the illuminator and first encounters a filter that selects the proper wavelength band for excitation (labeled the excitation filter in the tutorial). In this case, the filter passes light having wavelengths between 510 and 560 nanometers with high efficiency, but also allows other wavelengths to pass at a much lesser extent. The excitation light next reaches the dichromatic mirror and is reflected into the objective rear aperture to form a cone of illumination that bathes the specimen. The dichromatic mirror is positioned in the light path at a 45-degree angle and designed to selectively reflect wavelengths between 490 and 565 nanometers (essentially the blue-green and green wavelengths), while simultaneously transmitting both shorter and longer wavelengths.
Because only a narrow bandwidth of light is reflected by the dichromatic mirror, illumination wavelengths shorter than 490 nanometers and longer than 565 nanometers that manage to pass through the excitation filter are also transmitted through the dichromatic mirror. Note that the reflection of excitation light is not 100-percent efficient, and thus, a small amount of green light passes through the dichromatic mirror without being reflected. In addition, not all of the light having wavelengths above 565 or below 490 nanometers is transmitted through the mirror. A small percentage of this light is reflected by the mirror through the objective and onto the specimen. Light transmitted from the excitation filter through the dichromatic mirror is partially absorbed by the flat black coating on the interior of the filter block, but some reflects from the surface and passes through the barrier filter at an oblique angle, contributing to the fluorescence background noise.
Joseph LoBiondo and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.
Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.