JUNE 1998
`
`HANDBOOK OF
`OPTICAL FILTERS
`FOR FLUORESCENCE
`MICROSCOPY
`
`by JAY REICHMAN
`
`CHROMA TECHNOLOGY CORP. An Employee-Owned Company
`72 Cotton Mill Hill, Unit A9 Brattleboro VT 05301 USA Phone 802/257-1800 Fax 802/257-9400
`
`Edmund Optics(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)
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`COPYRIGHT © 1994,1998 CHROMA TECHNOLOGY CORP. All or parts of this handbook may
`be freely copied. Chroma Technology Corp. requests appropriate attribution.
`
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`Fluorescence microscopy requires optical filters that
`
`have demanding spectral and physical characteris-
`tics. These performance requirements can vary greatly
`depending on the specific type of microscope and the
`specific application. Although they are relatively minor
`components of a complete microscope system, the ben-
`efits of having optimally designed filters can be quite
`dramatic, so it is useful to have a working knowledge of
`the principles of optical filtering as applied to fluores-
`cence microscopy.
`
`This guide is a compilation of the principles and know-
`how that the engineers at Chroma Technology Corp.
`use to design filters for a variety of fluorescence micro-
`scopes and applications, including wide-field micro-
`scopes, confocal microscopes, and applications involving
`simultaneous detection of multiple fluorescent probes.
`Also included is information on the terms used to de-
`scribe and specify optical filters, and practical informa-
`tion on how filters can affect the optical alignment of a
`microscope.
`
`Finally, the handbook ends with a glossary of terms that
`are italicized or in boldface in the text.
`
`For more in-depth information about the physics and
`chemistry of fluorescence, applications for specific fluo-
`rescent probes, sample-preparation techniques, and mi-
`croscope optics, please refer to the various texts devoted
`to these subjects. One useful and readily available re-
`source is the literature on fluorescence microscopy and
`microscope alignment published by the microscope
`manufacturers.
`ABOUT CHROMA TECHNOLOGY CORP.
`Employee-owned Chroma Technology Corp. specializes
`in the design and manufacture of optical filters and coat-
`ings for applications that require extreme precision in
`color separation, optical quality, and signal purity:
`
`low-light-level fluorescence microscopy and cytometry
`
`spectrographic imaging in optical astronomy
`
`laser-based instrumentation
`
`Raman spectroscopy
`
`Our coating lab and optics shop are integrated into a
`single facility operated by a staff with decades of experi-
`ence in both coating design and optical fabrication. We
`are dedicated to providing the optimum cost-effective
`solution to your filtering requirements. In most cases
`our staff will offer, at no extra charge, expert technical
`assistance in the design of your optical system and selec-
`tion of suitable filtering components.
`
`AN INTRODUCTION TO
`FLUORESCENCE MICROSCOPY
`Excitation and emission spectra
`Brightness of the fluorescence signal
`The fluorescence microscope
`Types of filters used in fluorescence microscopy
`The evolution of the fluorescence microscope
`
`A GENERAL DISCUSSION OF OPTICAL
`FILTERS
`Terminology
`Available products
`Colored filter glass
`Thin-film coatings
`Acousto-optical filters
`
`DESIGNING FILTERS FOR
`FLUORESCENCE MICROSCOPY
`Image Contrast
`Fluorescence spectra
`Light sources
`Detectors
`Beamsplitters
`Optical quality
`Optical quality parameters
`Optical quality requirements for wide-field
`microscopes with K(cid:246)hler illumination
`FILTERS FOR CONFOCAL MICROSCOPY
`Optical quality requirements
`Nipkow-disk scanning
`Laser scanning
`Spectral requirements
`Nipkow-disk scanning
`Laser scanning
`
`FILTERS FOR MULTIPLE PROBE
`APPLICATIONS
`
`REFERENCES
`
`GLOSSARY
`
`Page
`3
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`11
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`17
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`25
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`29
`30
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`G-1
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`© 1997, 1994 Chroma Technology Corp. An Employee-Owned Company 72 Cotton Mill Hill, Unit A-9, Brattleboro, Vermont 05301 USA
`BRATTLEBORO VERMONT 05301 USA
`Telephone: 800 / 8-CHROMA or 802 / 257-1800 Fax: 802 / 257-9400 E-mail: sales@chroma.com
`
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`
`AN INTRODUCTION
`TO FLUORESCENCE
`MICROSCOPY
`
`Fluorescence is a molecular phenomenon in which a substance absorbs
`
`light of some color and almost instantaneously1 radiates light of another
`color, one of lower energy and thus longer wavelength. This process is known
`as excitation and emission. Many substances, both organic and non-or-
`ganic, exhibit some fluorescence. In the early days of fluorescence micros-
`copy (at the turn of the century) microscopists looked at this primary
`fluorescence, or autofluorescence, but now many dyes have been developed
`that have very bright fluorescence and are used to selectively stain parts of a
`specimen. This method is called secondary or indirect fluorescence. These
`dyes are called fluorochromes, and when conjugated to other organically
`active substances, such as antibodies and nucleic acids, they are called fluo-
`rescent probes or fluorophores. (These various terms are often used inter-
`changeably.) There are now fluorochromes that have characteristic peak
`emissions in the near-infrared as well as the blue, green, orange, and red
`colors of the spectrum. When indirect fluorescence via fluorochromes is
`used, the autofluorescence of a sample is generally considered undesirable: it
`is often the major source of unwanted light in a microscope image.
`
`EXCITATION AND EMISSION SPECTRA
`Figure 1 shows a typical spectrum of the excitation and emission of a fluoro-
`chrome. These spectra are generated by an instrument called a
`spectrofluorimeter, which is comprised of two spectrometers: an illuminating
`spectrometer and an analyzing spectrometer. First the dye sample is strongly
`illuminated by a color of light that is found to cause some fluorescence. A
`spectrum of the fluorescent emission is obtained by scanning with the ana-
`lyzing spectrometer using this fixed illumination color. The analyzer is then
`fixed at the brightest emission color, and a spectrum of the excitation is
`obtained by scanning with the illuminating spectrometer and measuring the
`variation in emission intensity at this fixed wavelength. For the purpose of
`designing filters, these spectra are normalized to a scale of relative intensity.
`
`Emission
`
`Excitation
`
`100
`
`%
`
`0
`
`300
`
`400
`
`500
`
`600
`Wavelength (nm)
`
`700
`
`800
`
`900
`
`FIGURE 1
`Generic excitation and emission
`spectra for a fluorescent dye.
`
`1 The time it takes for a molecule to fluoresce is on the order of nanoseconds (10 -9
`seconds). Phosphorescence is another photoluminescence phenomenon, with a lifetime
`on the order of milliseconds to minutes.
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`These color spectra are described quantitatively by wavelength of light. The
`most common wavelength unit for describing fluorescence spectra is the
`nanometer (nm). The colors of the visible spectrum can be broken up into
`the approximate wavelength values (Figure 2):
`
`violet and indigo
`
`400 to 450 nm
`
`blue and aqua
`
`green
`
`450 to 500 nm
`
`500 to 570 nm
`
`yellow and orange
`
`570 to 610 nm
`
`red
`
`610 to approximately 750 nm
`
`BRIGHT
`RED
`
`DARK
`RED
`
`NEAR IR
`
`YELLOW~
`
`~ORANGE
`
`BLUE GREEN
`
`VIOLET
`
`NEAR UV
`
`FIGURE 2
`Color regions of the spectrum.
`
`320
`
`400
`
`450
`
`500
`
`570
`
`610
`
`670
`
`750
`
`Wavelength (nm)
`
`On the short-wavelength end of the visible spectrum is the near ultraviolet
`(near-UV) band from 320 to 400 nm, and on the long-wavelength end is the
`near infrared (near-IR) band from 750 to approximately 2500 nm. The
`broad band of light from 320 to 2500 nm marks the limits of transparency of
`crown glass and window glass, and this is the band most often used in
`fluorescence microscopy. Some applications, especially in organic chemis-
`try, utilize excitation light in mid-ultraviolet band (190 to 320 nm), but spe-
`cial UV-transparent illumination optics must be used.
`
`There are several general characteristics of fluorescence spectra that pertain
`to fluorescence microscopy and filter design. First, although some substances
`have very broad spectra of excitation and emission, most fluorochromes
`have well-defined bands of excitation and emission. The spectra of Figure 1
`are a typical example. The difference in wavelength between the peaks of
`these bands is referred to as the Stokes shift. Second, although the overall
`intensity of emission varies with excitation wavelength, the spectral distribu-
`tion of emitted light is largely independent of the excitation wavelength.2
`Third, the excitation and emission of a fluorochrome can shift with changes
`in cellular environment, such as pH level, dye concentration, and when
`conjugated to other substances. Several dyes (FURA-2 and Indo-1, for
`example) are useful expressly because they have large shifts in their excita-
`tion or emission spectra with changes in concentration of ions such as
`
`2 The emission spectrum might change "shape" to some extent, but this is an
`insignificant effect for most applications. See Lakowicz (1983) for an in-depth
`description of the mechanism of fluorescence.
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`H+ (pH level), Ca2+, and Na+. Lastly, there are photochemical reactions that
`cause the fluorescence efficiency of a dye to decrease with time, an effect
`called photobleaching or fading.
`
`BRIGHTNESS OF THE FLUORESCENCE SIGNAL
`Several factors influence the amount of fluorescence emitted by a stained
`specimen with a given amount of excitation intensity. These include 1) the
`dye concentration within stained sections of the specimen, and the thickness
`of the specimen; 2) the extinction coefficient of the dye; 3) the quantum
`efficiency of the dye; and, of course, 4) the amount of stained material
`actually present within the field of view of the microscope.
`
`The extinction coefficient tells us how much of the incident light will be
`absorbed by a given dye concentration and specimen thickness, and reflects
`the wavelength-dependent absorption characteristics indicated by the excita-
`tion spectrum of the fluorochrome. Although many of the fluorochromes
`have high extinction coefficients at peak excitation wavelengths, practical
`sample preparation techniques often limit the maximum concentration al-
`lowed in the sample, thus reducing the overall amount of light actually ab-
`sorbed by the stained specimen.
`
`The quantum efficiency, which is the ratio of light energy absorbed to fluo-
`rescence emitted, determines how much of this absorbed light energy will be
`converted to fluorescence. The most efficient common fluorochromes have
`a quantum efficiency of approximately 0.3, but the actual value can be
`reduced by processes known as quenching, one of which is photobleaching.
`
`The combination of these factors, in addition to the fact that many speci-
`mens have very small amounts of stained material in the observed field of
`view, gives a ratio of emitted fluorescence intensity to excitation light inten-
`sity in a typical application of between 10-4 (for very highly fluorescent
`samples) and 10-6. Current techniques (e.g. fluorescence in situ hybridiza-
`tion), which utilize minute amounts of fluorescent material, might have ra-
`tios as low as 10-9 or 10-10.
`
`Thus, in order to see the fluorescent image with adequate contrast, the
`fluorescence microscope must be able to attenuate the excitation light by as
`much as 1011 (for very weak fluorescence) without diminishing the fluores-
`cence signal. How does the fluorescence microscope correct for this
`imbalance? Optical filters are indeed essential components, but the inherent
`configuration of the fluorescence microscope also contributes greatly to the
`filtering process.
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`THE FLUORESCENCE MICROSCOPE
`Figure 3 is a schematic diagram of a typical epifluorescence microscope,
`which uses incident-light (i.e., episcopic) illumination. This is the most com-
`mon type of fluorescence microscope. Its most important feature is that by
`illuminating with incident light, it need only filter out excitation light scatter-
`ing back from the specimen or reflecting from glass surfaces. The use of
`high-quality oil-immersion objectives (made with materials that have mini-
`mal autofluorescence, and using low-fluorescence oil) eliminates surface re-
`flections, which can reduce the level of back-scattered light to as little as 1/
`100 of the incident light. In addition, the dichroic beamsplitter, which re-
`flects the excitation light into the objective, filters out the back-scattered
`excitation light by another factor of 10 to 500. (The design of these
`beamsplitters is described below.)
`
`FIGURE 3
`Schematic of a wide-field
`epifluorescence microscope,
`showing the separate optical paths
`for illuminataing the specimen and
`imaging the specimen:
`illumination path
`imaging path
`
`Eyepiece
`
`Filter slider
`
`Fluorescence
`filter cube
`
`Heat filter
`
`Field
`diaphragm
`
`Aperture
`diaphragm
`
`Filter
`slider
`
`Collector
`lenses
`
`Arc lamp
`
`Objective
`
`Specimen
`
`An epifluorescence microscope using oil immersion, but without any filters
`other than a good dichroic beamsplitter, can reduce the amount of observ-
`able excitation light relative to observed fluorescence to levels ranging from
`1 (for very bright fluorescence) to 105 or 106 (for very weak fluorescence.)
`If one wants to achieve a background of, say, one-tenth of the fluorescence
`image, then additional filters in the system are needed to reduce the ob-
`served excitation light by as much as 106 or 107 (for weakly fluorescing
`specimens), and still transmit almost all of the available fluorescence signal.
`Fortunately, there are filter technologies available (described in the section
`beginning on page 13) that are able to meet these stringent requirements.
`
`TYPES OF FILTERS USED IN FLUORESCENCE MICROSCOPY
`The primary filtering element in the epifluorescence microscope is the set of
`three filters housed in the fluorescence filter cube, also called the filter
`block : the excitation filter, the emission filter, and the dichroic beamsplitter.
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`Figure 4
`Schematic of a fluorescence
`filter cube
`
`A typical filter cube is illustrated schematically in Figure 4.
`
`1) The excitation filter (also called the exciter) transmits only those wave-
`lengths of the illumination light that efficiently excite a specific dye. Common
`filter blocks are named after the type of excitation filter:
`
`UV or U
`
`Ultraviolet excitation for dyes such as DAPI and
`Hoechst 33342
`
`B
`
`G
`
`Blue excitation for FITC and relateted dyes
`
`Green excitation for TRITC, Texas Red®, etc.
`
`Although shortpass filter designs were used in the past, bandpass filter de-
`signs are now used almost exclusively.
`
`2) The emission filter (also called the barrier filter or emitter) attenuates
`all of the light transmitted by the excitation filter and very efficiently trans-
`mits any fluorescence emitted by the specimen. This light is always of longer
`wavelength (more to the red) than the excitation color. These can be either
`bandpass filters or longpass filters. Common barrier filter colors are blue or
`pale yellow in the U-block; green or deep yellow in the B-block; and orange
`or red in the G-block.
`3) The dichroic beamsplitter (also called the dichroic mirror or dichro-
`matic beamsplitter 3 ) is a thin piece of coated glass set at a 45 degree angle
`to the optical path of the microscope. This coating has the unique ability to
`reflect one color, the excitation light, but transmit another color, the emitted
`fluorescence. Current dichroic beamsplitters achieve this with great efficiency,
`i.e., with greater than 90% reflectivity of the excitation along with approxi-
`mately 90% transmission of the emission. This is a great improvement over
`the traditional gray half-silvered mirror, which reflects only 50% and trans-
`mits only 50%, giving only about 25% efficiency. The glass (called the sub-
`strate) is usually composed of a material with low autofluorescence such as
`UV-grade fused silica.
`
`Most microscopes have a slider or turret that can hold from two to four
`individual filter cubes. It must be noted that the filters in each cube are a
`matched set, and one should avoid mixing filters and beamsplitters
`unless the complete spectral characteristics of each filter component
`are known.
`
`Other optical filters can also be found in fluorescence microscopes:
`
`1) A heat filter, also called a hot mirror, is incorporated into the illuminator
`collector optics of most but not all microscopes. It attenuates infrared light
`(typically wavelengths longer than 800 nm) but transmits most of the visible
`light.
`
`2) Neutral-density filters, usually housed in a filter slider or filter wheel
`between the collector and the aperture diaphragm, are used to control the
`intensity of illumination.
`
`3 The term "dichroic" is also used to describe a type of crystal or other material that
`selectively absorbs light depending on the polarization state of the light. (Polaroid®
`plastic film polarizer is the most common example.) To avoid confusion, the term
`"dichromatic" is sometimes used.
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`3) Filters used for techniques other than fluorescence, such as color filters
`for transmitted-light microscopy and linear polarizing filters for polarized
`light microscopy, are sometimes installed.
`
`THE EVOLUTION OF THE FLUORESCENCE MICROSCOPE4
`The basic configuration of the modern fluorescence microscope described
`above is the result of almost one hundred years of development and innova-
`tion. By looking at its development over the years, one can gain a better
`understanding of the function of these various components.
`
`The first fluorescence microscopes achieved adequate separation of excita-
`tion and emission by exciting the specimen with invisible ultraviolet light.
`This minimized the need for barrier filters.5 One of these turn-of-the-
`century microscopes used for its light source a bulky and hazardous 2000 W
`iron arc lamp filtered by a combination Wood’s solution (nitrosodimethylaniline
`dye) in gelatin, a chamber of liquid copper sulfate, and blue-violet colored
`filter glass. This first excitation filter produced a wide band of near-UV light
`with relatively little visible light, enabling the microscopist to observe the
`inherent primary fluorescence of specimens. Microscopists were aided by
`the fact that most substances will fluoresce readily when excited by UV
`light. In 1914, fluorochromes were first used with this type of microscope
`to selectively stain different parts of cells, the first utilization of secondary
`fluorescence.6
`These first fluorescence microscopes, illustrated schematically in Figure 5,
`used diascopic (i.e., transmitted light) illumination. Both brightfield and
`darkfield oil-immersion condensers were used, but each had certain impor-
`tant disadvantages. With the brightfield condenser, the maximum intensity
`of illumination was severely limited by the capabilities of the optical filters
`that were available at the time. The darkfield condenser, which directed the
`
`excitation light into a cone of light at oblique angles, prevented most of the
`excitation light from entering the objective lens, thus reducing the demands
`on the optical filters. However, the efficiency of the illumination was greatly
`reduced, and the objective lens required a smaller numerical aperture, which
`resulted in a further reduction in brightness as well as lower resolution.7
`
`4 Most of this information is taken from the following excellent reference: Kasten
`(1989).
`5 The first barrier filter to be used was a pale yellow coverslip, which protected the eye
`from hazardous radiation, but some of the early fluorescence microscopes might have
`lacked even this.
`6 Several fluorescent dyes were synthesized in the eighteenth century for other
`purposes, including use as chemotherapeutic drugs that stained parasitic organisms
`and sensitized them to damaging rays.
`7 Abramowitz (1993).
`
`FIGURE 5
`Schematic of an early transmitted-
`light fluorescence microscope.
`(After Kasten, 1989.)
`
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`The most important advance in fluorescence microscopy was the develop-
`ment of episcopic illumination for fluorescence microscopes in 1929. Episcopic
`illumination was first utilized to observe the fluorescence of bulk and opaque
`specimens. These first epifluorescence microscopes probably used half-sil-
`vered mirrors for the beamsplitter, with a maximum overall efficiency of
`25%, but important advantages were 1) the ability to use the high numerical
`aperture objective as the condenser, thus achieving greater brightness; 2) the
`fact that the intensity of excitation light that reflects back into an oil-immer-
`sion objective is, as discussed above, roughly 1% of the incident light;8 and
`3) ease of alignment. The introduction of dichroic beamsplitters by Brumberg
`in 1948, and their subsequent commercialization by Ploem in 1967, im-
`proved the efficiency of the beamsplitter to nearly 100% and further im-
`proved the filtering effect of the microscope.
`
`The following important technical advances, together with the advent of the
`epifluorescence microscope, helped revolutionize fluorescence microscopy
`during this historical period: 1) the development of compact mercury-vapor
`and xenon arc lamps (1935); 2) advances in the manufacture of colored filter
`glasses, which enabled the use of fluorochromes that were efficiently excited
`by visible light (thus allowing, for example, the use of simple tungsten fila-
`ment light sources); 3) advances in microscope objective design; and 4) the
`introduction of anti-reflection coatings for microscope optics (c. 1940).
`
`These developments were, of course, driven by great advances in the bio-
`logical and biomedical sciences over the years, and revolutionary develop-
`ments have continued with the advent of ultrasensitive cameras,9 laser
`illumination, confocal microscopy, digital image processing, the continuing
`development of hundreds of fluorochromes and fluorescent probes, and, of
`course, great improvements in the capabilities of optical filters.
`
`8 Assuming nonmetallic specimens.
`9 Inoué (1986) is an excellent text detailing the use of video imaging and microscopes
`in general.
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`
`A GENERAL
`DISCUSSION OF
`OPTICAL FILTERS
`
`Figure 6
`A) Nomenclature for transmission
`characteristics
`B) Nomenclature for blocking
`
`Before describing in detail the design of optical filters for fluorescence mi
`
`croscopy, it is worthwhile to introduce some of the terms used to specify
`filter performance, and describe the characteristics of available products.
`
`TERMINOLOGY
`Although color designations such as U, B, and G are often adequate for de-
`scribing the basic filter sets, it is useful to be familiar with the terms used for
`more precise descriptions of filters, especially when dealing with special sets
`for unusual dyes and probes. The most common unit for describing filter
`performance is the wavelength of light in nanometers, the same as for fluoro-
`chrome spectra described previously. Note that the perceived color of a filter
`depends on the bandwidth (described below) as well as specific wavelength
`designation. This is especially noticeable when looking through filters in the
`range of 550 to 590 nm: a filter with a narrow band will look pale green while
`a filter with a wide band, especially a longpass filter, will look yellowish or
`even bright orange.
`
` Some of the important terms used to describe the spectral performance of
`optical filters are defined below. Please refer to the illustrations in Figures 6
`through 9.
`1) Bandpass filters are denoted by their center wavelength (CWL) and band-
`width (FWHM).10 The center wavelength is the arithmetic mean of the wave-
`lengths at 50% of peak transmission. The FWHM is the bandwidth at 50% of
`peak transmission.
`
`2) Longpass and shortpass cut-on filters (LP and SP)
`are denoted by their cut-on or cut-off wavelengths at
`50% of peak transmission. LP or SP filters that have a
`very sharp slope (see next page) are often called edge fil-
`ters. The average transmission is calculated over the useful
`transmission region of the filter, rather than over the entire
`spectrum. (Please note that the use of terms “highpass”
`and “lowpass” are discouraged because they more accu-
`rately describe frequency rather than wavelength.)
`
`3) The attenuation level, also called blocking level, and
`attenuation range, also called blocking range, are nor-
`mally defined in units of optical density (OD):
`
`OD = – log(T) or OD = – log(%T / 100)
`Example: OD 4.5 = 3 x 10-5 T (0.003 %T)
`
`Optical density uses the same logarithmic units as the
`quantity absorbance, which is a measure of absorption,
`but filters can attenuate light in various ways other than
`absorption. For example, thin-film interference filters
`block primarily by reflection, and acousto-optical filters
`block by diffraction. Therefore, the term “optical den-
`sity” is more precise. (Both of these filter products are
`described in detail in the section beginning on page 13.)
`
`10 Full Width at Half of Maximum Transmission
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`A term related to attenuation level is cross-talk (Figure
`7), which describes the minimum attenuation level (over
`a specific range) of two filters placed together in series.
`This value is important when matching excitation filters
`with emission filters for a fluorescence filter set.
`4) The term slope describes the sharpness of the transi-
`tion from transmission to blocking. Figure 8 illustrates
`two sets of filters with the same bandwidth or cut-on
`point, but different slope. In the Figure, note that al-
`though the bandpass filters look very similar on a 100%
`transmission scale, the slopes as indicated on the optical
`density scale are significantly different. Slope can be
`specified by stating the wavelength at which a particular
`filter must have a specified blocking level.
`5) The angle of incidence (AOI) is the angle between
`the optical axis of the incident light and the axis normal
`to the surface of the filter, as illustrated in Figure 9.
`Most filters are designed to be used at zero degrees
`angle of incidence, called normal incidence, but for
`beamsplitter coatings the usual angle is 45 degrees. It
`should be noted that most types of filters, such as thin-
`film interference coatings and acousto-optical crystal
`devices, are “angle-sensitive” — the characteristic per-
`formance changes with angle. (These products are de-
`scribed in greater detail in the next section.) If a filter or
`beamsplitter is to be used at any angle other than the
`usual zero or 45 degree angle, it must be specified ex-
`plicitly.
`
`One consequence of angle sensitivity is that the half-
`cone angle of the incident light might need to be speci-
`fied if the filter is to be used in a converging or diverging
`beam (Figure 10). The half-cone angle can also be de-
`scribed in terms of the f-number, or the numerical ap-
`erture (NA) of the light beam, which equals the sine of
`the half-cone angle.
`
`6) Dichroic beamsplitters (and, in fact, any thin-film
`interference coating that is used at non-normal angle of
`incidence) will cause some amount of polarization, the
`exact effect varying greatly with wavelength and with
`the particular design. Some relevant terms are illustrated
`in Figure 9: P-plane (also called TM-mode,
`i.e., “transverse magnetic”) is the component of the light
`beam’s electric field that is parallel to the plane of inci-
`dence of the beamsplitter, and S-plane (TE-mode,
`i.e., “transverse electric”) is the component of the light
`
`FIGURE 9
`Schematic illustration of terms used to describe polariza-
`tion. The normal axis is the axis perpendicular to the
`surface of the coating, and the plane of incidence is
`defined by the normal axis and the direction vector of the
`incident light beam.
`
`CHROMA TECHNOLOGY CORP.
`
`FIGURE 7
`Cross-talk level of two filters in
`series.
`
`FIGURE 8
`Filter sets with varying slope,
`shown in A) percent transmission,
`and B) in optical density.
`
`Incident beam
`
`Normal axis
`
`AOI
`
`Plane of incidence
`
`Beamsplitter coating
`on glass substrate
`
`E-field in
`S-plane
`
`E-field in
`P-plane
`
`12
`
`0014
`
`

`
`beam’s electric field that is perpendicular to the plane of incidence of the
`beamsplitter. The polarizing effect of a typical dichroic beamsplitter is illus-
`trated in Figure 11.
`
`AVAILABLE PRODUCTS
`The two main types of filter technology used in fluorescence analysis are
`colored filter glass and thin-film coatings. In addition, acousto-optical tun-
`able filters are finding increased use in special applications. These products
`are described below. Other products, such as holographic filters and liquid-
`crystal tunable filters, are available, but they are used infrequently in fluores-
`cence microscopy.
`Colored Filter Glass
`Colored filter glass, also called absorption glass, is the
`most widely used type of filter in fluorescence analysis,
`particularly the yellow and orange sharp-cut glasses and
`“black” glasses that transmit the UV and absorb the
`visible. Filter glass attenuates light solely by absorption,
`so the spectral performance is dependent on the physi-
`cal thickness of the glass. Increasing the thickness will
`increase the blocking level, but also reduce the peak in-
`band transmission (Figure 12), so an optimum thick-
`ness value must be determined. Stock thicknesses
`offered by the glass manufacturers represent a thick-
`ness value that is typical for the general uses of the
`glass, but other thicknesses might be better for a spe-
`cific application.
`
`Filter
`
`Half-cone angle
`
`Light cone
`
`Figure 10
`Illustration of half-cone angle
`of divergent or convergent
`incident light.
`
`Wavelength (nm)
`
`600
`
`FIGURE 11
`Polarizing effect of a typical dichroic mirror. This
`particular coating is designed for reflecting the 488 nm
`linearly polarized argon-ion laser line in the S-plane.
`
`Following are some advantages of filter glass:
`
`1) It is relatively inexpensive.
`
`2) It is stable and long-lived under normal conditions.11
`
`3) Its spectral characteristics are independent of angle
`of incidence, except for slight changes due to increased
`effective thickness.
`
`Disadvantages of filter glass include the following:
`
`1) There is a limited selection of glasses.
`
`2) The bandpass types have poor slope and often low
`peak transmittance.
`
`3) There is less flexibility in the specification of filter
`thickness because of the dependence of spectral per-
`formance on thickness.
`
`4) Most of the longpass filter glasses have high auto-
`fluorescence.
`
`5) Since absorption converts most of the radiant en-
`
`11 Some minor exceptions are 1) sharp-cut longpass filter
`glasses have a shift in cut-on of approximately 0.1 to 0.15 nm /
`°C temperature change; and 2) some types of filter glass can be
`affected by unusual environments such as intense UV radiation
`("solarization") or high humidity. (Schott Glasswerke catalogue.)
`
`FIGURE 12
`Spectra of near-IR blocking glass (Schott® BG-39) at 1 mm
`and 2 mm thickness, shown in
`A) percent internal transmission and B) optical density.
`(From Schott Glasswerke catalogue.)
`
`BRATTLEBORO VERMONT 05301 USA
`
`13
`
`0015
`
`

`
`ergy into heat, untempered filter glass might crack un-
`der conditions of intense illumination.
`
`Included in the category of filter glass are polymer-
`based filters, which are sometimes used as longpass
`barrier filters because they have low autofluorescence
`compared to an equivalent filter glass, and a type of
`neutral-density glass (not to be confused with thin-film
`neutral-density coatings described below).
`Thin-Film Coatings
`Two widely used categories of thin-film coating
`are: 1) metallic coatings for making fully reflective mir-
`rors and neutral-density filters, and 2) thin-film inter-
`ference coatings, which are the main component of
`interference filters. The main advantage of thin-film interference coatings is
`the tremendous flexibility of performance inherent in the way they work. As
`shown in Figure 13, interference coatings are composed of a stack of micro-
`scopically thin layers of material, each with a thickness on the order of a
`wavelength of light (usually around a quarter of a wavelength of light —
`approximately 1/10,000 of a millimeter in thickness). Although each
`material is intrinsically colorless, the reflections created at each interface
`between the layers combine through wave interference to selectively reflect
`some wavelengths of light and transmit others. A common natural example
`of thin-film interference is the formation of swirls of color on a soap bubble.
`Interference occurs between the reflections from the inner and outer sur-
`faces of the bubble, and the colors follow contours of constant thick