C H R O M A

T E C H N O L O G Y

C O R P

H A N D B O O K of
O P T I C A L F I LT E R S
for F L U O R E S C E N C E
MICROSCOPY
by J A Y R E I C H M A N

HB2.0/June 2017

C H R O M A
H A N D B O O K of
O P T I C A L F I LT E R S
for F L U O R E S C E N C E
MICROSCOPY
by J A Y R E I C H M A N

T E C H N O L O G Y

C O R PAN INTRODUCTION TO
FLUORESCENCE MICROSCOPY

2

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

8

Terminology
Available products					

Colored filter glass
Thin-film coatings
Acousto-optical filters
Liquid Crystal Tunable Filters

DESIGNING FILTERS FORFLUORESCENCE MICROSCOPY

14

Image Contrast
Fluorescence spectra
Light sources
Detectors
Beamsplitters
Optical quality
Optical quality parameters
Optical quality requirements for wide-field microscopy

FILTER SETS FOR SUB-PIXEL REGISTRATION24

FILTERS FOR CONFOCAL MICROSCOPY

25

Optical quality requirements
Nipkow-disk scanning
Laser scanning
Spectral requirements
Nipkow-disk scanning
Laser scanning

FILTERS FOR MULTIPLE PROBE APPLICATIONS

29

REFERENCES

30

GLOSSARY31

Fluorescence microscopy requires optical filters that have demanding spectraland physical characteristics. 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, optimally designed filters can produce quite dramatic benefits, so it
is useful to have a working knowledge of the principles of optical filtering as
applied to fluorescence 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 fluorescencemicroscopes and applications, including wide-field microscopes, confocal
microscopes, and applications involving simultaneous detection of multiple
fluorescent probes. Also included is information on the terms used to describe
and specify optical filters and practical information on how filters can affectthe optical alignment of a microscope.
Finally, the handbook ends with a glossary of terms that are italicized or inboldface in the text.
For more in-depth information about the physics and chemistry offluorescence, applications for specific fluorescent probes, sample-preparation
techniques, and microscope optics, please refer to the various texts devoted
to these subjects. One useful and readily available resource 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 andmanufacture of optical filters and coatings 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 experience in both coating design and opticalfabrication. 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 systemand selection of suitable filtering components.

© 2000-2010 Chroma Technology Corp An Employee-Owned Company
10 Imtec Lane, Bellows Falls, Vermont 05101-3119 USA
Telephone: 800-8-CHROMA or 802-428-2500 Fax: 802-428-2525 E-mail: sales@chroma.com
Website: www.chroma.com

1

AN INTRODUCTION TO FLUORESCENCE MICROSCOPY
Fluorescence is a molecular phenomenon in which a substance absorbs lightof some color and almost instantaneously 1 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-organic,
exhibit some fluorescence. In the early days of fluorescence microscopy (atthe turn of the century) microscopists looked at this primary fluorescence, orauto­fluorescence, but now many dyes have been developed that have very
bright fluorescence and are used to selectively stain parts of a specimen. Thismethod 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 fluorescent probes or
fluorophores. (These various terms are often used interchangeably.) There
are now fluorochromes that have characteristic peak emissions in the nearinfrared as well as the blue, green, orange, and red colors of the spectrum.
When indirect fluorescence via fluorochromes is used, the autofluorescenceof a sample is generally considered undesirable: it is often the major sourceof unwanted light in a microscope image.

EXCITATION AND EMISSION SPECTRA
Figure 1 shows a typical spectrum of the excitation and emission of
a fluorochrome. These spectra are generated by an instrument called

Arbitrary Unitsa spectrofluorimeter, which is comprised of two spectrometers: anilluminating spectrometer and an analyzing spectrometer. First the

Excitation
Spectradye sample is strongly illuminated by a color of light that is foundEmissionSpectrato cause some fluorescence. A spectrum of the fluorescent emission
is obtained by scanning with the analyzing spectrometer using this
fixed illumination color. The analyzer is then fixed at the brightest
emission color, and a spectrum of the excitation is obtained byscanning with the illuminating spectrometer and measuring the
Wavelengthvariation in emission intensity at this fixed wavelength. For the
purpose of designing filters, these spectra are normalized to a scale of

FIGURE 1
Generic excitation and emission spectra
for a fluorescent dye.

relative intensity.
These color spectra are described quantitatively by wavelength of light. The
most common wavelength unit for describing fluorescence spectra is thenanometer (nm). The colors of the visible spectrum can be broken up into theapproximate wavelength values (Figure 2):
violet and indigo

400 to 450 nm

blue and aqua 		

450 to 500 nm

green 			500 to 570 nm

yellow and orange

570 to 610 nm

red 			

610 to approximately 750 nm

1 The time it takes for a molecule to fluoresce is on the order of nanoseconds
9(10 - seconds). Phosphorescence is another photoluminescence phenomenon,2with a lifetime on the order of milliseconds to minutes.

On the short-wavelength end of the visible spectrum is thenear-ultraviolet (near-UV) band from 320 to 400 nm, and on
the long-wavelength end is the near-infrared (near-IR) bandfrom 750 to approximately 2500 nm. The broad band of lightfrom 320 to 2500 nm marks the limits of transparency ofcrown glass and window glass, and this is the band most often
used in fluorescence microscopy. Some applications, especiallyin organic chemistry, utilize excitation light in the midultraviolet band (190 to 320 nm), but special 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 havewell-defined bands of excitation and emission. The spectra

FIGURE 2
Color regions of the spectrum.

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, thespectral distribution 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 conjugation to other substances. Several dyes (FURA-2 and Indo-1,
for example) are useful expressly because they have large shifts in their
excitation or emission spectra with changes in concentration of ions such as
H + (pH level), Ca 2+, and Na + . Lastly, there are photochemical reactions thatcause the fluorescence efficiency of a dye to decrease with time, an effectcalled 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) thedye concentration within stained sections of the specimen, and the thicknessof 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 wavelengthdependent absorption characteristics indicated by the excitation 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 allowed in the sample,
thus reducing the overall amount of light actually absorbed by the stained
specimen.

2 The emission spectrum might change “shape” to some extent, but this is an insignificant effect for mostapplications. See Lakowicz (1983) for an in-depth description of the mechanism of fluorescence.

3

The quantum efficiency, which is the ratio of light energy absorbed tofluorescence 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 specimens
have very small amounts of stained material in the observed field of view,
gives a ratio of emitted fluorescence intensity to excitation light intensity ina typical application of between 10 -4 (for very highly fluorescent samples) and10 -6. Current techniques (e.g. fluorescence in situ hybridization), which utilize
minute amounts of fluorescent material, might have ratios as low as 10 -9
or 10 -10.
Thus, in order to see the fluorescent image with adequate contrast, thefluorescence microscope must be able to attenuate the excitation lightby as much as 10-11 (for very weak fluorescence) without diminishing thefluorescence signal. How does the fluorescence microscope correct for thisimbalance? Optical filters are indeed essential components,
but the inherent configuration of the fluorescence microscopealso contributes greatly to the filtering process.
Eyepiece

CameraField
Stop
FilterSliderAperature
Stop

Illumination Path

THE FLUORESCENCE MICROSCOPE

Imaging Path

Figure 3 is a schematic diagram of a typical epifluorescencemicroscope, which uses incident-light (i.e., episcopic)

Heat
Filterillumination. This is the most common type of fluorescencemicroscope. Its most important feature is that by illuminatingwith incident light it need only filter out excitation light

Fluorescence
FilterCube

scattering back from the specimen or reflecting from glass
surfaces. The use of high-quality oil-immersion objectives(made with materials that have minimal autofluorescence
FilterSliderObjective

Collector
LensesArc Lamp

and using low-fluorescence oil) eliminates surface reflections,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 reflects the excitation light into theSpecimen Stage

objective, filters out the back-scattered excitation light byanother factor of 10 to 500. (The design of these beamsplittersis described below.)

FIGURE 3
Schematic of a wide-field epifluorescence microscope,
showing the separate optical paths for illuminating
the specimen and imaging the specimen:
illumination pathimaging path

An epifluorescence microscope using oil immersion, but without any filters
other than a good dichroic beamsplitter, can reduce the amount of observable
excitation light relative to observed fluorescence to levels ranging from 1(for very bright fluorescence) to 10 5 or 10 6 (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 observed
excitation light by as much as 10 -6 or 10 -7 (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 10) that are able to meet these stringent requirements.

4

TYPES OF FILTERS USED IN
FLUORESCENCE MICROSCOPY
The primary filtering element in the epifluorescence microscope is the setof three filters housed in the fluorescence filter cube (also called the filter

Ocularblock): the excitation filter, the emission filter, and the dichroic beamsplitter.

Emission
FilterA typical filter cube is illustrated schematically in Figure 4.

Beamsplitter

1) The excitation filter (also called the exciter) transmits only thosewavelengths of the illumination light that efficiently excite a specific dye.

Excitation
FilterCommon filter blocks are named after the type of excitation filter:
UV or U 	Ultraviolet

excitation

for

dyes

such

as

DAPI
Light
Sourc
e

and Hoechst 33342
B 		

Blue excitation for FITC and related dyes

GGreen excitation for TRITC, Texas Red ® , etc.

Although shortpass filter designs were used in the past, bandpass filter designs
are now used almost exclusively.
2) The emission filter (also called the barrier filter or emitter) attenuates allof the light transmitted by the excitation filter and very efficiently transmits

Objective

FIGURE 4
Schematic of a fluorescence filter cube

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 orpale yellow in the U-block, green or deep yellow in the B-block, and orangeor red in the G-block.
3) The dichroic beamsplitter (also called the dichroic mirror or dichromatic
beamsplitter3 ) is a thin piece of coated glass set at a 45-degree angle tothe optical path of the microscope. This coating has the unique ability
to reflect one color, the excitation light, but transmit another color, theemitted fluorescence. Current dichroic beamsplitters achieve this with great
efficiency, i.e., with greater than 95% reflectivity of the excitation along withapproximately 95% transmission of the emission. This is a great improvement
over the traditional gray half-silvered mirror, which reflects only 50% andtransmits only 50%, giving only about 25% efficiency. The glass (called thesubstrate) 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 fourindividual filter cubes. It must be noted that the filters in each cube are amatched set, and one should avoid mixing filters and beamsplitters unless thecomplete spectral characteristics of each filter component are known.

3 The term “dichroic” is also used to describe a type of crystal or other material that selectively absorbs light dependingon the polarization state of the light. (Polaroid ® plastic film polarizer is the most common example.) To avoid confusion,
the term “dichromatic” is sometimes used.

5

Other optical filters can also be found in fluorescence microscopes:
1) A heat filter, also called a hot mirror, is incorporated into the illuminatorcollector 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 theintensity of illumination.
3) Filters used for techniques other than fluorescence, such as color filters fortransmitted-light microscopy and linear polarizing filters for polarized lightmicroscopy, are sometimes installed.

THE EVOLUTION OF
THE FLUORESCENCE MICROSCOPE 4
The basic configuration of the modern fluorescence microscope describedabove is the result of almost 100 years of development and innovation. By
looking at its development over the years, one can gain a better understandingof the function of these various components.
The first fluorescence microscopes achieved adequate separation of excitationand 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 of Wood’s solution (nitrosodimethylanilinedye) in gelatin, a chamber of liquid copper sulfate, and blue-violet coloredfilter glass. This first excitation filter produced a wide band of near-UV lightwith relatively little visible light, enabling the microscopist to observe theinherent primary fluorescence of specimens. Microscopists were aided by thefact that most substances will fluoresce readily when excited by UV light. In
1914 fluorochromes were first used with
SPECIMENSLIDEOBJECTIVE

GLASS/LIQUID
EXCITATIONFILTER

this type of microscope to selectively

QUARTZCOLLECTOR
LENS

stain different parts of cells, the firstutilization of secondary fluorescence.6

CONDENSEREYEPIECE
CARBON ARC
LAMP

These first fluorescence microscopes,
illustrated schematically in Figure 5,
used diascopic (i.e., transmitted light)
illumination.

FIGURE 5
Schematic of an early transmitted-lightfluorescence microscope.
(After Kasten, 1989.)

Bothbrightfieldanddarkfield oil-immersion condensers wereused, but each had certain important disadvantages. With the brightfield
condenser, the maximum intensity of illumination was severely limitedby the capabilities of the optical filters that were available at the time. The
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, butsome of the early fluorescence microscopes might have lacked even this.

6

6 Several fluorescent dyes were synthesized in the eighteenth century for other purposes, including use as chemo-

therapeutic drugs that stained parasitic organisms and sensitized them to damaging rays.

darkfield condenser, which directed the excitation light into a cone of lightat oblique angles, prevented most of the excitation light from entering theobjective lens, thus reducing the demands on the optical filters. However,
the efficiency of the illumination was greatly reduced, and the objective lensrequired a smaller numerical aperture, which resulted in a further reductionin brightness as well as lower resolution.7
The most important advance in fluorescence microscopy was the developmentof episcopic illumination for fluorescence microscopes in 1929. Episcopic
illumination was first utilized to observe the fluorescence of bulk and opaquespecimens. These first epifluorescence microscopes probably used halfsilvered mirrors for the beamsplitter, with a maximum overall efficiency of
25%, but important advantages were 1) the ability to use the high numericalaperture objective as the condenser, thus achieving greater brightness;2) the fact that the intensity of excitation light that reflects back into
an oilimmersion objective is, as discussed above, roughly 1% of theincident light;8 and 3) ease of alignment. The development of dichroic
mirrors, introduced by E. M. Brumberg in 1948 for ultraviolet excitationand independently developed by J. S. Ploem in the 1960’s to enable visible
excitation, increased the efficiency of beamsplitters to nearly 100% andfurther improved the filtering capabilities of the microscope. Further advancesin the optics of epi-illuminators by Ploem included the introduction of narrowband interference filters for blue and green excitation and the development of
the filter-cube, which permitted an easy exchange of filters and beamsplittersfor multiple fluorochromes.9 These advances led to the commercialization of
the epifluorescence microscope.
Other important technical advances during this historical period were 1)
the development of compact mercury-vapor and xenon arc lamps (1935);
2) advances in the manufacture of colored filter glasses, which enabled the useof fluorochromes that were efficiently excited by visible light (thus allowing,for example, the use of simple tungsten filament light sources); 3) advancesin microscope objective design; and 4) the introduction of anti-reflection
coatings for microscope optics (c. 1940).
More recent technological developments have enabled fluorescencemicroscopy to keep pace with the remarkable advances in the biological andbiomedical sciences over the years. These include ultrasensitive cameras10,
laser illumination, confocal and multi-photon microscopy, digital image
processing, new fluorochromes and fluorescent probes, and, of course, great
improvements in optical filters and beamsplitters.

7 Abramowitz (1993).
8 Assuming nonmetallic specimens.
9 Information and sequence of events confirmed through communication with Dr. Bas Ploem, 2006.
10 Inoué (1986) is an excellent text detailing the use of video imaging and microscopes in general.

7

A GENERAL DISCUSSION OF OPTICAL FILTERS
Before describing in detail the design of optical filters for fluorescent
microscopy, 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 fordescribing 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 forfluorochrome spectra described previously. Note that the perceived colorof a filter depends on the bandwidth (described below) as well as specificwavelength designation. This is especially noticeable when looking throughfilters in the range of 550 to 590 nm: a filter with a narrow band will lookpale 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.
FIGURE 6A) Nomenclature for transmission
characteristics
B) Nomenclature for blocking1) Bandpass filters are denoted by their center wavelength (CWL) andbandwidth (FWHM).11 The center wavelength is the arithmetic mean of the
wavelengths 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 havea very sharp slope (see next page) are often callededge filters. The average transmission is calculated
over the useful transmission region of the filter,
rather than over the entire spectrum. (Please notethat the use of terms “highpass” and “lowpass” arediscouraged because they more accurately describe
frequency rather than wavelength.)3) The attenuation level, also called blocking level,
and attenuation range, also called blocking range, arenormally defined in units of optical density (OD):
OD = – log(T) or OD = – log(%T / 100)

FIGURE 7
Cross-talk level of two filters in series.

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

11 Full Width at Half of Maximum Transmission

8

ways other than absorption. For example, thin-film interference filters%Tblock primarily by reflection, and acousto-optical filters block bydiffraction. Therefore, the term “optical density” is more precise. (Bothof these filter products are described in detail in the section beginningon page 10.)
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 transition fromtransmission to blocking. Figure 8 illustrates two sets of filters withthe same bandwidth or cut-on point but different slope. In the Figure
note that although the bandpass filters look very similar on a 100%

FIGURE 8
Filter sets with varying slope, shown in A) percent
transmission and B) in optical density.

transmission scale, the slopes as indicated on the optical density scale are
significantly different. Slope can be specified by stating the wavelength atwhich a particular filter must have a specified blocking level.
5) The angle of incidence (AOI) is the angle between the optical axisof 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,” which means that the characteristic performancechanges with angle. (These products are described in greater detail in the

FIGURE 9
Schematic illustration of terms used to describe polarization.
The normal axis is the axis perpendicular to the surface of thecoating, and the plane of incidence is defined by the normal
axis and the direction vector of the incident light beam.

next section.) If a filter or beamsplitter is to be used at any angle other thanFilterthe usual zero- or 45-degree angle, it must be specified explicitly.
One consequence of angle sensitivity is that the half-cone angle of theLight Cone

Half-cone
Angle

incident light might need to be specified if the filter is to be used in aconverging or diverging beam (Figure 10). The half-cone angle can also be
described in terms of the f-number, or the numerical aperture (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

FIGURE 10
Illustration of half-cone angle of divergent or convergent
incident light.

that is used at non-normal angles-of-incidence) will cause some amountof polarization (see Glossary), the precise effect varying greatly withwavelength and with the particular coating design. Some relevant terms
are illustrated in Figure 9: P-plane (also called TM-mode, i.e., transverse
magnetic) is the component of the electric field vibration that is parallel tothe plane of incidence of the beamsplitter, and S-plane (also TE-mode, i.e.,transverse electric) is the component of the electric field vibration that isperpendicular to the plane of incidence of the beamsplitter. The polarizing
effect of a typical dichroic beamsplitter is illustrated in Figure 11.
FIGURE 11
Polarizing effect of a typical dichroic mirror. Thisparticular coating is designed for reflecting the 488 nm
linearly polarized argon-ion laser line in the S-plane.

9

AVAILABLE PRODUCTS
The two main types of filter technology used in fluorescence analysis arecolored filter glass and thin-film coatings. In addition, acousto-optical tunablefilters are finding increased use in special applications. These products aredescribed below. Other products, such as holographic filters and liquid-crystal
tunable filters, are available, but they are used infrequently in fluorescencemicroscopy.

Colored Filter Glass
Colored filter glass, also called absorption glass, is the most widely used typeof filter in fluorescence analysis, particularly the yellow and orange sharpcut
glasses and “black” glasses that transmit the UV and absorb the visible. Filter
glass attenuates light solely by absorption, so the spectral performance isdependent on the physical 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 thickness value must be determined. Stock
thicknesses offered by the glass manufacturers represent a thickness valuethat is typical for the general uses of the glass, but other thicknesses might bebetter for a specific application.
Following are some advantages of filter glass:
1) It is relatively inexpensive;2) It is stable and long-lived under normal conditions;123) 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 peaktransmittance;3) There is less flexibility in the specification of filter thickness
because of the dependence of spectral performance on thickness;
4) Most of the longpass filter glasses have high auto-fluorescence;5) Since absorption converts most of the radiant energy into heat,
untempered filter glass might crack under conditions of intense
FIGURE 12
Spectra of near-IR blocking glass (Schott® BG-39) at
1 mm and 2 mm thickness, shown in A) percent internaltransmission and B) optical density.
(From Schott Glasswerke catalogue.)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 neutraldensity coatings described below).

10

12 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.)Thin-Film Coatings

NOT TO SCALE!!!

Two widely used categories of thin-film coating are 1) metallic coatings

Incident
light

for making fully reflective mirrors and neutral-density filters; and 2) thin-

Reflected Light,
modified by interference

film interference coatings, which are the main component of interference
filters. The main advantage of thin-film interference coatings is thetremendous flexibility of performance inherent in the way they work.
As shown in Figure 13, interference coatings are composed of a stackThin FilmlayersThickness
Glass Substrateof microscopically thin layers of material, each with a thickness on theTransmitted light, modified byinterference

order of a wavelength of light (usually around a quarter of a wavelengthof light—approximately 1/10,000 of a millimeter in thickness). Althougheach material is intrinsically colorless, the reflections created ateach interface between the layers combine through wave interference
to selectively reflect some wavelengths of light and transmit

FIGURE 13
Schematic illustration of a thin-film interference coating100

others. A common natural example of thin-film interference is theformation of swirls of color on a soap bubble. Interference occurs
between the reflections from the inner and outer surfaces of the bubble,%Tand the colors follow contours of constant thickness within the single
layer of soap.13
Almost any filter type can be designed using interference coatings,
including bandpass, shortpass, and longpass filters, and dichroic
beamsplitters. By adjusting the number of layers in the stack and thethickness of each layer, one can control to high precision the nominal
wavelength, the bandwidth, and the blocking level. One can also
create filters with greater complexity than the standard bandpass,

0
400

500

600

700

Wavelength (nm)

FIGURE 14
Spectra of a triple-band filter designed for DAPI, FITC, andTexas Red ® emissions. (Chroma Technology Corp P/N 69002
emission filter)

longpass, or shortpass. For example, filters with multiple bands
produced using the modified magnetron sputter deposition process,illustrated in Figure 14, have several highly transmissive bands andare valuable tools in fluorescence microscopy.
There are several limitations to thin-film interference coatings,
including the following:1) In traditional interference filter design, the characteristic
blocking performance only holds within a finite wavelength range.
This coating technology has been the industry standard for many
decades, using dielectric materials in a sealed, laminated format.
Additional components, both interference coatings and absorption
glass, can be added to increase blocking range, but with reduced
transmission and increased physical thickness. An example of afilter with no added blocking (often called an "unblocked" filter),and the same filter blocked with added components, is shownin Figure 15. For applications such as fixed cell stains, plant
imaging with autofluorescence, flow cytometry, plate readers, andanywhere there is an adequate signal this type of filter performs
excellently.

13 But unlike a soap bubble, the optical thin-films are of solid material (either polycrystalline oramorphous), and the coating layers are extremely uniform in thickness.

FIGURE 15
An example of a blocked filter: A) spectra of an unblocked filter andblocking components, including a wide-band “blocker” and infraredblocking blue filter glass; and B) the spectrum of the blocked filter.
The blocking range in the infrared (not shown) is determined by therange of the blue glass, approximately 1.2 microns.

11Due to advances in coating technology, some of these limitations
have been greatly reduced. All interference coatings, unlike metal
coatings or absorption glass, have a finite blocking range, but highenergy coating technology maintains transmission while extending
the blocking region. Filters produced by modified magnetron sputter
technology, for example, have broader ranges of extinction by usingsophisticated blocking strategies incorporated into the coating
design.

The extended blocking region using this coating process

has a negligible effect on the transmission through the bandpass.
The signal-to-noise ratio that this type of filter offers allow for the
FIGURE 16
Reflectivity vs. absorptance. This dichroic
beamsplitter sample has high absorption in the UV.

acquisition of high quality data in many photon-critical applications.
2) In traditional filter technology, the coating materials are limited intheir range of transparency. Outside of this range, coatings become highly
absorbing rather than highly transmissive or reflective. Some of the coating
materials that are ideal for visible filters have excessive absorption in the UV,
so materials with different and less-than-ideal characteristics must be used
for UV filters. As a result, UV filters tend to have more limited performanceand less design flexibility. Another consequence is that one cannot always
calculate the reflectivity from a transmission spectrum by assuming zeroabsorption in the wavelength range shown in the spectrum. Figure 16 is an
example of a dichroic beamsplitter designed for high reflectivity in the visible
only. The drop in transmission in the UV is a result of absorption insteadof reflection. In general, one cannot assume that a beamsplitter designed toreflect the visible will also reflect the UV. Filters produced with newer highenergy coating technology use materials with broader ranges of transparency
in the violet and near UV.
3) As noted on page 9, interference coatings are sensitive to angle of incidence.
As the angle is increased, the spectral characteristics of the coating shift toshorter wavelengths, i.e., the spectrum is “blue-shifted.”

14

In addition thepolarizing effect of coatings at oblique angles of incidence is undesirable
in most applications. Although the coating designer can minimize thepolarization, it cannot be eliminated entirely. This effect is, however, utilized
FIGURE 17
Schematic representation of an acousto-opticaltunable filter. (From Brimrose Corp.)

advantageously in some special applications.

Acousto-Optical Filters
The acousto-optical tunable filter (AOTF), shown schematically in Figure 17,
is most often used for filtering excitation light, especially laser excitation.
This filter works by setting up radio-frequency acoustical vibrations in an
appropriate crystal and creating, in effect, a bulk transmission diffraction
grating. By varying the frequency, one can rapidly tune the filter to diffract
out with high precision any wavelength of light within the useful range. A
typical AOTF will accept incident light with a maximum half-cone angle ofapproximately 5 degrees. The AOTF is an electronically controlled device
that uses an external control unit.

12

14 The effective physical thickness of the thin-film coating does indeed increase with increasing angle of incidence,but the difference in path length of the interfering reflected light rays decreases.

Advantages of the AOTF include:
1) the ability to change to any wavelength within microseconds, perform
wavelength scanning, and generate multiple-band filtering by mixing multiple
radio frequencies; and2) the ability to rapidly vary the intensity by changing the amplitude of theacoustic vibrations.
Some disadvantages include:1) limited FWHM (approximately 2 nm in the visible), which restricts theavailable light output from white light sources when the AOTF is used as an
excitation filter;2) limited physical dimensions such as a small aperture (approximately 10 mmor less) and a large overall thickness of approximately 25 mm; and3) linearly polarized output, giving a maximum of 50% T when using
unpolarized incident light.

Liquid Crystal Tunable Filters*
The liquid crystal tunable filter (LCTF) is another electronically controlled
device that is finding increasing use as an emission filter because it works ona principle that enables imaging-quality filtering with an ample clear apertureand an in-line optical path. At the heart of the LCTF are a series of waveplates,
each composed of a layer of birefringent material paired with a liquid crystal
layer and sandwiched between linear polarizers. The birefringence of the liquid
crystal layer, and thus the total magnitude of the waveplate, is fine-tuned byvarying the voltage applied to transparent conductive coatings adjacent to theliquid crystal layer.
Briefly, the birefringence of the waveplate induces a wavelength-dependent
rotation of the incoming polarized light. The second polarizer then attenuates
the polarized and rotated light to varying degrees, in effect converting therotation into an amplitude variation which is also wavelength-dependent. The
LCTF designer is able to control filtering parameters by varying structural
aspects such as the number of waveplates in series and the birefringence
qualities of each waveplate.
Characteristics of LCTFs include:
1) a speed of wavelength selection on the order of milliseconds;
2) no wavelength-dependent image-shift;3) variable attenuation capabilities; and4) a choice of bandwidths (FWHM), spectral range of tunability, and blocking
level, although these parameters are somewhat interdependent.
LCTFs are polarizing optical components, so the maximum peak transmission
for unpolarized light is fifty percent. Existing devices can have significantly
less than fifty percent, depending on wavelength and blocking level. However,the use of special polarizing beamsplitters in an epi-fluorescence microscope
can mitigate the overall effect of these losses. The maximum blocking levelcommercially available is around 10 -5.

* See references on page 31

13

A)

100

FITC
Excitation

DESIGNING FILTERSFOR FLUORESCENCE MICROSCOPY%

FITCEmission

The primary goal of filter design for fluorescence microscopy is tocreate filter sets (typically an excitation filter, emission filter, anddichroic beamsplitter) that maximize image contrast and maintain
0
300

400

500

600

Wavelength (nm)B)

image quality.

100

Image contrast is a combination of several factors: 1) the absolute
Excitation
Filter%Emission
Filterbrightness of the image; 2) the brightness of the fluorescing substance
relative to the background, known as signal-to-noise ( S/N ); and 3) to alesser extent in the case of visual or photographic observation, the color
balance as perceived by the eye. Filters must have high throughput (i.e.,
wide bandwidth combined with high transmittance), as well as low

0
300

400

500

600

Wavelength (nm)cross-talk, because no amount of improvement in S/N will improve
the image contrast if the image is not bright enough to be adequately

100

detected. In addition, the best way to achieve maximum contrast often
Emission
Filter

Bandpass
Excitation
Filter%C)

depends on the specific application or technique, so the filter designer
should have a conceptual understanding of these various applications.
How the filter designer incorporates the available information regarding
a particular application is described in detail below.

0
300

D)

400

500

600

100

TRITC
Emission

TRITC
Excitation

Image quality is maintained by ascertaining the optical quality
requirements at each point in the microscope where a filter is

Wavelength (nm)inserted, and ensuring that each filter is manufactured to the correct
specifications. In order to ascertain these requirements, one must be%

well grounded in the fundamentals of microscope optics and havean understanding of the requirements of particular applications
and techniques. This is especially true today, with the growing
number of applications that are taking advantage of the newest
technologies: lasers for both illumination and sample manipulation,

0
300

E)

400

500

600

Wavelength (nm)100

Bandpass
Emission

Bandpass
Excitation

digital image processing, computerassisted positioning and controls,
and ultrasensitive detection devices.
In addition, the filter designer must be aware of all the physical%dimensions required by the various microscope brands and models.

IMAGE CONTRAST
The general process by which a filter set is optimized for a particular

0
300

400

500

600

Wavelength (nm)fluorochrome can be illustrated by taking as an example a specimen
stained with the dye FITC and explaining how filters are designed for

FIGURE 18
A) FITC excitation and emission spectraB) Perfect filter pair for FITC, overlaid on excitation andemission spectraC) Filter pair with bandpass excitation filter specific to FITC
D) TRITC excitation and emission spectra, overlaid on
FITC spectra
E) Bandpass filter pair for FITC (Chroma Technology Corp P/N
49002), overlaid with excitation/emission spectra for FITC
and TRITC

14

this dye.

Fluorescence spectra
The single most important parameter for designing a filter set is the
spectral characteristic of the dye, with excitation and emission spectra
shown in Figure 18A. If this were the only parameter to be considered,one would illuminate the specimen using a shortpass excitation filter thattransmits all of the excitation spectrum and observe the fluorescence using
a longpass emission filter that transmits the entire emission spectrum. A
pair of filters for FITC having these characteristics is shown in Figure 18B.
These represent “ideal” shortpass and longpass filters: real filters would needa wider separation between the cut-on and cut-off because of slope limitationsof filters, and the shortpass excitation filter would have a cut-off point
somewhere in the UV.
But in a real specimen there are other considerations. Many substances inthe specimen are likely to autofluoresce if a shortpass excitation filter isused, especially one that transmits UV light. Tissue specimens for pathology
are especially prone to autofluorescence. Also, the presence of UV light,
which has higher intrinsic (i.e., quantum) energy, might increase the rateof photobleaching of the fluorochrome and/or cause photodamage to thespecimen. Therefore, one should limit the band to a region where the FITC
excitation is at a maximum, but still wide enough to allow adequate intensity,using a bandpass excitation filter as shown in Figure 18C.
If a second fluorochrome is included in the specimen, for example TRITC
with excitation and emission spectra as shown in Figure 18D, there is low but
significant excitation efficiency in the blue for this dye. If a longpass emission
filter is used for the FITC, a small but noticeable orange emission from TRITC
might be seen. This is usually considered an undesirable effect, especiallywhen imaging with a monochrome camera that does not distinguish between
colors. In this case, one should restrict the FITC emission filter to a narrowerband (Figure 18E) that is more specific to the band of peak emission for FITC.
The filters in Figure 18E are examples of Chroma's 49-series ET filters.
For cytometric applications where image brightness is not critical, even morenarrow bands are often used in order to maximize the selectivity between
fluorochromes.

Light sources
So far, a hypothetical light source having an equal output in allcolors—a pure white light—has been assumed. Real light sources
can have a wide variety of spectral output, and in many cases it isimportant to design filters with the type of light source in mind.
Following is a list of light sources often used for fluorescencemicroscopy.
1) Mercury arc lamps are the most common light source forfluorescence microscopy, chosen for its high brightness (known
technically as luminance or radiance) in the ultraviolet andvisible spectrum. The spectrum of this light source (Figure 19) is
far from continuous; most of its light output is concentrated ina few narrow bands, called lines, and each line is approximately

FIGURE 19
Spectrum of a mercury arc lamp. (Mid-UV output below 300
nm is not shown.)

15

10 nm wide. Most general-purpose filter sets should have
100 00

400

500

600

Mercury
Spectrumexcitation filters that transmit one or more of these lines, butthere can be noticeable exceptions, one of which is illustrated in
the following example. Figure 20 shows the effect of modifying the%excitation spectrum of FITC by the output of a mercury arc
illuminator. A wide-band excitation filter that included the light at
436 nm would provide an emission signal significantly brighter than
the filter that excludes this line. But for most specimens a reduction
in overall S/N is to be expected because the increase in noise from
0
300

400

500

600

autofluorescence will outweigh the increase in fluorescence signal.
However, for certain applications involving extremely low absolute

Wavelength (nm)levels of fluorescence, or for specimens in which the FITC spectrum

100

Wide-Band
Excitation
Filter

has been blue-shifted15, a wide-band excitation filter that includes
the 436 nm line might provide improved detection. Note that thesame emission filter would be used regardless of excitation band%

because the emission spectrum would not be significantly altered.
2) Xenon arc

lamps (Figure 21), havea relatively continuous

spectrum in the visible. They are preferred in systems where the0300

spectral characteristics of dyes and/or specimens are being analyzed
400

500

600

Wavelength (nm)quantitatively, but they are not as bright as a mercury lamp ofequivalent wattage. Even in the region of FITC excitation (between

FIGURE 20
A) FITC excitation spectrum unmodified, and modified
by the mercury arc lamp spectrum (normalized to 100%
relative peak T).
B) Modified FITC excitation spectrum overlaid withstandard and wide-band excitation filters.

450 and 500 nm) where the mercury lamp is relatively weak,
the xenon arc lamp is only marginally brighter. There are twomain reasons for this. First, the light-producing arc of the xenon
lamp is larger than the arc in the equivalent mercury lamp, which
reduces the amount of available light that can be focused onto
the specimen using a typical microscope configuration. Somerelevant data concerning mercury and xenon arc lamps are given in
Table 1. Note the differences in arc size between the lamps. Second,
the xenon arc lamp has proportionally much greater output in the
Near-IR as seen in Figure 21, as well as the IR.
This high IR output poses another disadvantage of the xenon arc
lamp. Excitation filters must be designed to have excellent blocking
in the Near-IR (>800 nm,) and depending on the location of the filter,they must be designed to withstand higher thermal loads.

FIGURE 21
Spectrum of a xenon arc lamp.

3) Metal-halide short-arc lamps are used increasingly for fluorescencemicroscopy. Spectrally, there is significant variation in output
between models. In general they are similar to mercury arc lamps,
but with greater relative intensity between the main emission lines.
In this respect they are similar to standard xenon arc lamps. Metal
halide sources have significant advantages over traditional mercury
and xenon arc lamps: a) they have a useful life up to 2000 hours,
compared to 200 hour of the typical mercury illuminators and 400

15 This shift can occur, for example, under conditions of low pH values (pH less than 6) (Haugland, 1992).

16

LAMP TYPE		RATED POWER(watts)

LUMINOUS FLUX
(lumens)

AVERAGE BRIGHTNESS(candela/mm2)

ARC SIZE
w x h (mm)RATED LIFE
(hours)

Mercury
HBO 50W/3
HBO 100W/2
HBO 200W/2

50		
100
200		

1300		
2200		
10000		

900		1700		
400		

0.20 x 0.35
0.35 x 0.25
0.60 x 2.20

200
200
400

Xenon
XBO 75W/2
XBO 150W/1

75		
150		

1000		
3000		

400		
150		

0.25 x 0.50
0.50 x 2.20

400
1200

TABLE 1
Data for xenon and mercury arc lamps. Boldface entries indicate most common sizes for fluorescence microscopes. (From Abramowitz, 1993)

hours of the xenon lamps; and b) they are usually configured in a
precisely machined housing with an integral elliptical reflector, making
possible easy and stable alignment.
The main disadvantage is that due to larger arc size (the smallest size
being approximately 1.2 mm) and limitations of optics of the ellipticalreflector, the average brightness is similar to the xenon arc lamps. And
like xenon arc lamps, they generally have higher output in the IR thanthe mercury arc lamp.
4) Light-emitting diode (LED) sources are also gaining in popularity,
particularly in less expensive microscopes used in schools. LEDs
are uniquely different than arc lamps in that they emit light withina relatively narrow spectral bandwidth, with FWHMs ranging fromapproximately 10 to 40 nm. Figure 22 shows spectra for sometypical LED sources. This is an attractive feature for fluorescence100

microscopy because the need for an excitation filter to have an
extended blocking range is eliminated. In some applications
might not even be necessary. However most applications use%

using fluorochromes with large Stokes shifts an excitation filter
fluorochromes with narrow Stokes shifts and still require an
excitation/emission filter pair.
LED's have other significant advantages: a) LEDs are typically0

rated for at least 10,000 hours, with many lasting upwards of300

their output (both on/off and intensity) can be controlled very
rapidly, on the order of a millisecond.

500

600

700

800

900

Wavelength (nm)50,000 hours; b) their output intensity remains relatively constantover the entire rated life; and c) since they are solid-state devices,

400

FIGURE 22
Spectra for typical LED sources. Nominal wavelengths:
365 nm, 390 nm, 455 nm, 470 nm, 505 nm and 535 nm.
(390 nm LED data from Nichia Corp.)

A main drawback of LED sources that has limited their use influorescence microscopy has been their brightness. However, recent
advances in LED technology have led to LED's that are approximately
as bright as the arc lamps within the narrow spectral band emitted by
the LED.

17

The main disadvantage now is, ironically, the narrow spectral emission
band. This is for two reasons: a) for multi-channel applications, the output
of several diffferent colors of LED must be combined, necessitating relatively
complex optical schemes that can limit overall brightness; and b) there are
only a limited number of spectral bands available, especially one in the green
spectral region that can match the very useful 546 nm mercury line.16 One
must also be aware that for any single LED color, there can be significant
variability in peak wavelength from batch to batch (up to 20 nm,) as well
as a wavelength dependence on the temperature and magnitude of the drive
current used to power the LED (up to 5 nm.)5) Lasers have become almost ubiquitous in confocal fluorescencemicroscopy because of there extremely high brightness over narrow(usually monochromatic) spectral bands. However, they are rarely used
for wide-field fluorescence microscopy because the coherent natureof laser light makes it difficult to achieve a uniform illumination
over the sample field. A detailed discussion of filter design for laser
illumination can be found on page 25.

Detectors
FIGURE 23
Typical spectrum of an argon-ion laser. (Data from Spectra-physics
Lasers, Inc.)

The excitation filter must be designed to block any out-of-band lightthat can be picked up by the detector. Arc lamps and filament lamps
have output throughout the near-UV, visible, and IR, so the filter
must have adequate attenuation over the whole range of sensitivity ofthe detector. But for laser illumination, the blocking range of excitation
filters need only cover the range of output of the laser. For example, IR

1 00

blocking is not required for the argon-ion laser (Figure 23).

50
10Figure 24 shows some sensitivity spectra of important detectors.

%

5

Not shown is the sensitivity of unintensified silicon photodiodes or1
0.5

CCDs, which have sensitivity to 1100 nm, falling to zero by 1200 nm.
Note that silicon detectors that are intensified with, for example, a

0.1

300

500

700

Wavelength (nm)

FIGURE 24
Sensitivity of various detectors. Each spectrum isnormalized to peak sensitivity. (Video and PMT
data from Hamamatsu Corp.)

900

microchannel plate, have sensitivity ranges similar to the intensified
video spectra shown in Figure 23.
In general it is preferable to block out-of-band light with the excitation

filter instead of the emission filter for three reasons: 1) the specimen will be
exposed to less radiation; 2) fewer components in the emission filters generally
improve its optical imaging quality; and 3) many microscopes have shallow
cavities for holding emission filters, so it is beneficial to eliminate componentsthat add to the finished thickness. There are certain cases, for example UV
excitation, where the process of extending the blocking of the excitation filter
greatly reduces the peak transmission of the filter. In these cases it would beappropriate to provide extended IR blocking in the emission filter instead.

18

16 The recent use of conversion phosphors has helped to overcome this limitation. Conversion phosphors are coat-ings that are added to LED systems that absorb the LED light and emit light at a longer wavelengths. Phosphorshaving output spectra that better match the excitation spectra fluorochromes can be selected.

When doing visible photographic work, it is important to have IR blocking
because some built-in light meters have IR sensitivity that could affectexposure times.

Beamsplitters
The final stage in the design of a filter set is to select a dichroic beamsplitterthat matches the spectra of the excitation and emission filters. Beamsplitters
having both high reflectance of the excitation band and high transmittanceof the emission band not only maximize the signal but also improve contrast
by further reducing background noise. Reflectance of greater than 95% of
the excitation band, with average transmission greater than 95% in theemission band, can now be achieved with advanced coating technology suchas modified magnetron sputtering.
At the relatively high angle of incidence of 45 degrees, the coatings canbe highly polarizing, so the designer must work to minimize this effect.
Beamsplitters for imaging systems, such as microscopes, usually consist of
one or two coatings applied directly onto the glass substrate, so extra care inhandling is advised. The surface that is designed to face the light source andspecimen is called the front surface of the beamsplitter. Determining whichis the front surface can be difficult, so the manufacturer usually provides
some type of marking to indicate the correctorientation.
Figure 25 shows a completed design for a
filter set for FITC, including excitation filter,emission filter, and dichroic beamsplittermatched to these filters.

FIGURE 25
Completed filter set design for FITC (Chroma Technology Corp P/N 49002)

19

OPTICAL QUALITY
The optical quality requirements for an optical filter depend very muchon such factors as the type of filter (especially whether it is a componentof the illumination optics or the imaging/detection optics), the type of
microscope, and the intended application. For example, the optical quality
specifications for an emission filter used for quantitative image analysis witha laser scanning confocal microscope are much different from those for an
excitation filter used for qualitative visual observation with a standard widefield microscope. Although specific requirements for every type of filter andevery application cannot be described here, by introducing the key opticalquality parameters and analyzing a few basic microscope configurations, one
can develop a set of general guidelines that can be applied to most situations.

Optical quality parameters

SURFACE FLATNESSThe following is a list of important parameters used to define the overall
image quality of a filter or beamsplitter. The first three parameters are

incident
wavefront

illustrated in Figure 26.
1) Surface flatness is a measure of the deviation of the surface of an
optical element from a perfect plane, usually measured in fractions orreflected

wavefront

multiples of a wavelength of visible light (usually 550 or 630 nm, butsometimes using the CWL for a bandpass filter). The actual reflected

TRANSMITTED WAVEFRONT DISTORTION

wavefront distortion (RWD) that a plane wave of light undergoes whenincident
wavefront

transmitted
wavefront

reflected from the surface is twice the value of the surface flatness.17
It should be noted, however, that specifications using the term "RWD"
are usually interpreted as the surface flatness of the substrate at normal
incidence, because this is the standard configuration when measuring

WEDGE (PARALLELISM)

with a commercial interferometer. If the specification must truly be for
RWD and not flatness, then this should be clearly noted.

incident
wavefront

deviated
wavefront

wedge angle

The RWD of light reflected off a beamsplitter or mirror is solely
determined by the surface flatness of the reflecting surfaces, usually justthe front surface.

FIGURE 26
Illustration of the effects of surface flatness,transmitted wavefront distortion, and wedge.

2) Transmitted wavefront distortion (TWD) is a measure of the distortiona plane wave of light undergoes when transmitted through an optical element,
also measured in fractions or multiples of a wavelength of light, the same
as for surface flatness, described above. The surface flatness of the outersurfaces of the element and, to a lesser degree, internal structures that cause
inhomogeneity of the refractive index, combine to make up the overall TWDof the optical element.
3) Wedge (also called parallelism) is a measure of how parallel are the outersurfaces of an optical element. Wedge is usually measured in arc-minutes
or arc-seconds of angle. The main effect of wedge is to induce an angular
17 This is strictly true only for light reflected at normal incidence. The value for light reflected at non-normal incidence

is modified by a cosine factor. For example, the reflected wavefront distortion at 45 degrees angle of incidence isapproximately 1.4 times the surface flatness.

20

deviation in the direction of a light beam, causing, for example, image shifting.
The amount of angular deviation is about one-half the wedge angle for a typicalfilter.18 The wedge of internal coating surfaces, while not contributing greatly
to beam deviation, can cause noticeable ghost images as a result of off-axis
internal reflections.
4) The clear aperture of optical filters should not restrict the overall apertureof the microscope. It is also of critical importance that there is no leakage ofunfiltered light around the edge of the clear aperture. Filter manufacturers
usually sell filters in rings to ensure there is no leakage. If a filter is purchased
without a ring, it is usually the responsibility of the customer to install thefilter in a way that eliminates leakage.
5) Scratches and digs are measured in terms of mil-spec standards; e.g., “80/ 50
scratch/dig.” The term digs includes such things as particles and small bubbles
embedded inside a filter and macroscopic inclusions in exposed coatings.
6) Pinholes are small breaks in the coating of an interference filter, usuallycaused by the presence of dust particles on the substrate during coating.
Pinhole size must be measured against standard maximum-tolerance pinholes
under specific conditions using a high-intensity illuminator.

Optical quality requirements for wide-fieldmicroscopes with Köhler illumination
Most fluorescence microscopes are wide-field epifluorescence systems thatutilize Köhler illumination (illustrated in Figure 3, on page 4). Following is abrief description:
In Köhler illumination, two adjustable diaphragms are provided for control ofthe light beam: the aperture diaphragm that gets imaged onto (i.e., is conjugateto) the back aperture of the objective, and the field diaphragm that gets imaged
onto the in-focus specimen plane. The field diaphragm is positioned near thelens that images the aperture diaphragm onto the objective back aperture. The
collector lens assembly focuses the arc lamp onto the aperture diaphragm andthus onto the objective back aperture. Three essential effects are achieved withthis configuration: 1) the image of the arc lamp is completely out of focus at
the specimen plane, thus creating a uniform field of illumination; 2) the imageof the field diaphragm appears in sharp focus on the specimen, and it can be
adjusted to precisely fill the field of view or isolate any part of it; and 3) thenumerical aperture of the illuminating beam can be adjusted (by adjusting theaperture diaphragm) without affecting the size of the illuminated field.19
With this in mind, some observations can be made regarding optical quality
requirements for this type of microscope.

18 For small angles of incidence, the angular deviation = ( N - 1) α, where N is the refractive index of the glass in thefilter, and α is the wedge angle. Most filters use optical glass with a refractive index of approximately 1.5.
19 For an in-depth description of Köhler illumination optics, please refer to Inoué (1986) or literature from the micro-

scope manufacturers.

211) The illumination optics of the microscope are designed for uniform
illumination of the specimen with minimal flare, and not necessarily forfaithful imaging of the light source or apertures. Therefore, excitation filters
do not require transmitted wavefront distortion of imaging quality,20 but
only what is generally considered the industry standard of optical quality.
Likewise, the surface flatness of the dichroic mirror does not require imaging
quality.
2) Variations in the alignment of the beamsplitter inside the filter cube
assembly will produce noticeable shifts in the position of the illumination
spot (i.e., the image of the field diaphragm on the specimen plane) whenswitching filter cubes. For typical applications, an alignment of 45 degrees
plus or minus three arc-minutes is considered adequate. (Because the angleof the reflected beam is always equal to the angle of the incident beam, theoverall angular deviation caused by this alignment tolerance would be twice
this, i.e., plus or minus six arc-minutes.) When filter cubes are suppliedwith the beamsplitter premounted, it is the responsibility of the supplier toproperly align the beamsplitter. In addition, care must be taken to ensure
proper alignment when the filter cube is installed into the microscope.
In contrast, variations in alignment have little effect on the apparent positionof the image in the field of view, known as registration shift.
3) Similarly, the effect of angular deviation induced by wedge in the excitation
filter will be a shift in the position of the illumination spot, but there will
be no registration shifts of the image itself. Wedge must be controlled only
to within alignment tolerances of the dichroic mirror mounted in the filter
block assembly.
4) Filters in the image path, including the dichroic beamsplitter as well as
the emission filter, generally do require transmitted wavefront distortion ofimaging quality.
5) The effect of wedge in the emission filter and beamsplitter will be aregistration shift of the image when switching filters or cubes. Wedge must
be controlled in these components.
In addition, variations in the thickness of the beamsplitter might cause
registration shifts. The extent of this thickness-related shift depends on the
microscope: epifluorescence microscopes with standard tube-length optics
have a light beam in the imaging path that is not collimated.21 They are
therefore more sensitive than microscopes with infinity-corrected optics that
have, in principle, collimated light in the image path.

20 The term "imaging quality" denotes a level of optical quality that will not degrade the overall performance of a spe-22cific optical system. For a typical microscope, a TWD of 1 wave per inch is generally considered adequate for opticalfilters and beamsplitters.
21 Most
modern microscopes of this type have relay lenses in the filter-block housing that improve the collimation

inside the cube. The sensitivity is reduced but not eliminated entirely.

The wedge and, to a lesser extent, the thickness of the dichroic beamsplittersand the emission filters must be tightly controlled for filter sets that require
sub-pixel registration (SPR). The optical-quality requirements for SPR filter
sets is discussed in more detail on page 24.
6) Since the excitation filter is usually positioned near the field diaphragm (apoint conjugate to the image plane in Köhler illumination), pinholes in the
excitation filter are very noticeable and must be eliminated.
7) Autofluorescence of the dichroic beamsplitter must be minimized because
this is the one element that is both fully illuminated by excitation light andpart of the optical path of the image.
It is important to note that the beamsplitter requires separate and independent
specifications for surface flatness and transmitted wavefront distortion.
Based on the above observations, some typical optical quality specifications
for filters used in wide-field epifluorescence microscopes are listed in Table 2.
					
OPTICAL QUALITY
PARAMETER		EXCITATION		
Surface flatness			
Transmitted wavefront		Wedge			Scratch/dig			
Pinholes			
Autofluorescence		

no specification		no specification		<6 arc-minutes		
80/50			none			no specification		

FILTER TYPE
EMISSION		BEAMSPLITTER

no specification		
1 wave per inch		
1 arc-minute		
60/40			minimal			
moderate control		<10 waves per inch
1 wave per inch
1 arc-minute
40/40
no specification*
minimalTABLE 2
Typical optical quality specifications for filters in a wide-field epifluorescence microscope.

*Measured as a dig using scratch/dig specifications.

23

FILTER SETS FOR SUB-PIXEL REGISTRATION
A common method for imaging specimens with multiple fluorochromes
is to record images of each fluorochrome separately, using a highresolution monochrome CCD camera. The separate images are thenpseudo-colored and overlaid into a single image. Images are obtainedusing a different filter "cube" for each fluorochrome. The matched set
of filter cubes are installed in a multi-position slider or turret.
Some applications, in particular those that involve co-localization
analysis, require the separate images to be registered to within one
pixel. Filter sets that are manufactured to have this level of image
registration are called sub-pixel registration (SPR) filter sets.
As described above, the primary cause of a shift in image registration is thewedge of the dichroic beamsplitter and the emission filter. Although thespecific requirements for achieving SPR depend on the optical layout of the
microscope and the type of CCD detector, typical specifications for wedge are
2 arc-seconds for beamsplitters and 10 arc-seconds for emission filters. The
wedge specification for the beamsplitter is typically lower for two reasons:1) the angular deviation induced by a substrate at 45 degrees AOI is greaterthan at normal incidence, and2) beamsplitters are always composed of a single substrate that can becontrolled readily for wedge.
A secondary cause of image shift can be the thicknesses of the emission
filter and dichroic beamsplitter. As noted above, the extent of this thicknessrelated shift depends on the microscope: epifluorescence microscopes withstandard tube-length optics have a light beam in the imaging path that is notcollimated, and therefore are more sensitive to thickness differences than
microscopes with infinity-corrected optics, which have more collimated lightin the image path where the filter cube is located. Thickness variations of
0.2 mm or less between emission filters in a set, and likewise 0.2 mm or lessbetween beamsplitters, should eliminate this effect.22
Another secondary cause of image shift can be the flatness of the dichroic
beamsplitter. Thin-film interference coatings in have intrinic mechanicalstress that will cause a substrate to warp. The amount of stress is heavily
dependant on the coating process. Some processes such as magnetron andion-beam sputter deposition, make coatings with very high stress, enoughto noticeably warp a thin substrate. If the light beam in the imaging path isnot collimated, this warpage can induce a significant image shift. This effectmay be noticeable if all dichroic beamsplitters in the sets that are installed in
multi-position slider or turret are not made by the same deposition process.

24

22 This assumes that the refractive indices of the glass substrates used in the filters are similar.

FILTERS FOR CONFOCAL MICROSCOPY
When designing filters for use in confocal fluorescence microscopes(CFMs), one must consider primarily the optical configuration of the
microscope. Almost every make and model of CFM has a uniqueconfiguration and therefore a unique set of optical specifications,but most systems can be grouped into one of two basic
categories: CFMs that utilize a Nipkow-disk mechanism

Illumination Path

CameraImaging Pathfor scanning the specimen, and those that scan with a laser
beam. The optics used in each of these scanning methods arebriefly described below by comparing them to the optics of astandard widefield microscope. For a more detailed analysis
of CFM optics, such as depth of field, resolution, and image

Aperture
Stop

Field
Stop
Fluorescence
Filter Cube

generation techniques, please refer to the literature available

Relay
Opticsfrom the CFM manufacturers, as well as articles published inscientific journals associated with microscopy or laboratory

Arc Lamp

Nipkow Disk
Relay
Opticstechniques for biology and genetics.

Confocal Section
Objective

OPTICAL QUALITY REQUIREMENTS FORCONFOCAL FLUORESCENCE MICROSCOPES
Nipkow-Disk Scanning CFMs
Figure 27 is a schematic illustration of a CFM with broad-band arc

Specimen Stage

FIGURE 27
Schematic of a confocal fluorescence microscopewith Nipkow-disk scanning.

lamp illumination, a Nipkow-disk scanning mechanism, and an
image detector, either a camera or direct visual observation.
This microscope has an optical path similar in principle to standard wide-field
scopes (illustrated in Figure 3). The disk is set in a position conjugate to thespecimen plane and must be uniformly illuminated over the entire field ofview, usually with Köhler illumination. Therefore the transmitted wavefront
requirements for the excitation filter and the surface flatness requirement
for the dichroic beamsplitter are the same as for the wide-field microscope.
Similarly, the transmitted wavefront requirements for the emission filterand beamsplitter are the same as for the wide-field microscope. The wedge
requirements for the various filters are, in general, also unchanged, althoughvariations in the focal length of specific CFM designs might necessitate
modifications of these specifications.

25

Laser Scanning CFMs
Illumination Path
Imaging Path

Detectors
With Apertures

moving-mirror scanning mechanism, and photometric detection such as aphotomultiplier tube (PMT). The optical paths in this microscope design

Emission Filters
Dichroic Mirror
For Emission

Figure 28 is a schematic illustration of a CFM with laser illumination, aSpatial Filters(Pinholes, Slits)

are significantly different from those in wide-field microscopes. The two
major differences are:Laser1) the pinhole (or slit) in the illuminator is imaged onto the specimen

Excitation/Emission
Beamsplitter

Laser

plane via critical illumination, with the laser spot being directly focused
onto the sample; andScanner
Objective

Excitation Filters
Dichroic Mirror
For Excitation

Specimen

FIGURE 28
Schematic of a laser scanning confocal fluorescence microscope.

2) the image is formed by synchronously matching the train of electricalsignals generated by the detector to the position of the laser beam on thespecimen, similar to the image-forming mechanism of a television set.
The electrical signals from the detector correspond to the variations in
the intensity of the fluorescence signal that passes through the conjugate
pinhole (or slit) aperture.
In this configuration the laser beam must remain undistorted in order toachieve the best focus at the specimen plane, and there must be minimal
angular deviation from the true optical path. In addition, most CFMs ofthis type have relatively long optical path lengths that can magnify any
angular deviations introduced by a filter. Therefore, the excitation filters andbeamsplitters require a degree of optical quality and registration equal to or
greater than that required for emission filters and beamsplitters. Most systems
have transmitted wavefront distortion and surface flatness specifications on
the order of one wave per inch for excitation filters and beamsplitters. The
wedge specifications are similarly tighter, typically one arc-minute or less, inorder to minimize alignment adjustments when switching filters.
In the emission optics, any components that are located between the objective
and the detection aperture must be of imaging quality. In the example
illustrated, this includes all of the emission filters and beamsplitters shown.
Some systems might use a single aperture that would be located between themain excitation/emission beamsplitter and the detection unit. In this case,the optical requirements for the filters within the detection unit might be lesscritical.

SPECTRAL REQUIREMENTS FOR CONFOCALFLUORESCENCE MICROSCOPES
Any unique spectral requirements generally stem from the type of illuminationand detection in the microscope. Since the illumination and detection
systems of Nipkow-disk scanning CFMs differ from those of laser-scanning
CFMs, most can again be grouped into one of these two categories.

Nipkow-Disk Scanning CFMs
Since the light source and the detection system of this configuration (describedabove and illustrated in Figure 26) are the same as for standard wide-field

26

EFMs, the spectral requirements are basically the same, with one exception:
because of reduced signal intensity and the addition of extra optical elements,
each of which produces undesirable reflections and scattering, excitation andemission filter pairs must have very low cross-talk.23 In addition, extra care isrequired in the quality control of pinholes in the filters.

Laser Scanning CFMs
Filters and beamsplitters for this configuration (described above and illustrated
in Figure 27) are designed to work optimally for the specific laser being used.
The most common lasers are now the solid state lasers, including laser-diodes(LDs) and diode-pumped solid-state (DPSS) lasers. There is a wide selectionof laser wavelenghts now available throughout the visible spectrum. Moretraditional lasers, such as the argon-ion, illustrated in Figure 22, the heliumneon. and argon-krypton are also used. When designing filters for use withlasers, the following considerations must be made:1) Excitation filters must have a blocking range that covers the entire output
spectrum of the laser. While filters that block to 800 nm are suitable fortraditionalthe laser sources , the DPSS lasers often have residual output at the
NIR wavelengths used in the laser, particularly 1064 nm, that may fall within
the response range of the detector. One special consideration regarding thefiltering of laser excitation is that excitation filters for lasers should attenuate
as much as possible by reflection in order to reduce the thermal load on thefilter. For example, UV excitation filters for argon-ion lasers should have highreflectivity in the blue-green region of the visible spectrum where most of thelaser power is concentrated.
2) The lasers used in most CFMs are polarized, so knowledge of the polarization
conditions is very useful when designing beamsplitters for optimum
performance. The dichroic beamsplitter illustrated in Figure 9, for example,
was optimized for use in S-plane orientation with the 488 nm argon-ion laserline, enabling the beamsplitter to have greater throughput in, for example, aband of fluorescent emission that cuts on at 500 nm. Note that fluorescentemissions are largely unpolarized even when the excitation light is polarized,
so the beamsplitter must efficiently transmit the fluorescence in both planesof polarization. Knowledge of the polarization conditions is especially usefulwhen designing multiple-band polychroic beamsplitters, which must have very
narrow reflection bands in order to maximize the transmission of the emission
signals.
3) Emission filters must have a blocking range on the short wavelength sideof the band that includes all laser lines that might be used with the particularfilter. Blocking on the long wavelength side is recommended for applications
that use multiple probes, to improve selectivity between the probe signals.
Here the range of blocking only needs to cover the range of the detector, which
can be as short as 700 nm for some PMTs (Figure 23).

23 Some systems utilize polarized optics to reduce the effect of these reflections.

27

TABLE 3
Methods for applications using multiple probes.

ME T H O D
1. Separate single-band filter sets.

COMPONENT S
Standard microscope.

ADVANTAGESNo extra equipment is necessary.

DI SAD VA N TA G ES
Simultaneous imaging is
not possible.

Brightest images.
Imprecise imaging of combined
images.
2. Multi-band filter sets.

Standard microscope.

Simultaneous visual observation
with no registration errors.
No extra equipment is required(other than special filter set).

Reduced brightness.
Not recommended with xenon arc
illumination.
Color balance is fixed.

3. Multi-band beamsplitter andemitter, single-band exciter.

Microscope with filter wheel orslider in the illumination path.

Sequential visual observation withno registration errors.

Photo camera, or electronic
camera with image processing.

Precise registration of combined
images; adjustable color
balance.

Extra equipment (a filter wheel orslider) is necessary.

Optimized exciters offer brighter
image than method 2.
4. Multi-band beamsplitter,
single-band emitters andexciters, single camera.

5. Multi-band beamsplitter,
single-band emitters andexciters, multiple cameras.

Microscope with filter wheels or
sliders in both the illumination
path and imaging path.

Exciters and emitters can be
designed to have brightness
similar to method 1.

Photo camera, or electronic
camera with image processing.

Reduction in registration error(by eliminating movement of thebeamsplitter).

Microscope with filter wheel orslider in the illumination path.

In addition to advantages inmethod 4:

Beamsplitter assembly for separating channels, each channel
having a separate emission filter.

Registration errors from emitterscan be eliminated;

Extra equipment (two filter wheels
or sliders) is necessary.
Registration errors between
emitters might still be present.

More complicated apparatus
requiring additional beamsplittersand cameras.

Additional applications are supported (e.g., ratio imaging).

6. Neutral beamsplitter, replacing
multi-band beamsplitter inmethods 4 and 5.

28

Any valid combination of exciterand emitter can be used.

Brightness can be reduced by
as much as 80%, so special lightsources are recommended (e.g.,laser illumination).

FILTERS FOR MULTIPLE PROBE APPLICATIONSWhen making a multiple-exposure photograph or multiple electronic
images of specimens stained with several fluorochromes, using separate
filter cubes in a standard microscope, there will be unavoidable
registration shifts between exposures. Varying amounts of wedge in theemission filter and beamsplitter, variations in thickness and alignmentof the beamsplitter, and mechanical vibration that occurs when the
cubes are switched all contribute to this shift. Although for some
applications these effects can be reduced to acceptable levels, manyothers require more sophisticated filter designs and optical apparatus.
Listed in Table 3 are some available methods for applications involving multiple
probes. These include the multi-band filter sets introduced earlier, illustrated
in Figure 29, and various configurations that utilize both multi-band and singleband filter components. All of these methods are designed to eliminate theaforementioned registration errors. Method 2 offers the ability to visually observe
up to three colors simultaneously. (Methods 3 through 6 can also do this if multiband filters are added to the filter sets.) It should be noted that this list is only aguide, not an exhaustive list of all possible methods and configurations.

Emission Filter

Excitation Filter

Dichroic Beamsplitter
100

%T

0

400

500

600

700

Wavelength (nm)
FIGURE 29®
Spectra of a triple-band filter set designed for the dyes DAPI, FITC, and Texas Red .
(Chroma Technology Corp P/N 69002)

29

REFERENCES
Abramowitz, M. (1993) Fluorescence Microscopy: The Essentials. Olympus
America, Inc.
Brimrose Corp., Baltimore, MD. AOTF Spectroscopy (March 1993 catalogue).
Ealing Electro-optics (1992) Product Guide, Holliston, MA.
Haugland, R.P. (1992) Handbook of Fluorescent Probes and Research
Chemicals, 5th ed. Molecular Probes, Eugene, OR.
Inoué, S. (1986) Video Microscopy. Plenum Press, New York.
Kasten, F. H. (1989) The origins of modern fluorescence microscopy andfluorescent probes. Cell Structure and Function by Microspectrophotometry(E. Kohen and J. G. Hirschberg, eds.). Academic Press, San Diego, CA.
Lakowicz, J. (1983) Principles of Fluorescence Spectroscopy. Plenum Press,
NewYork.
Schott Glasswerke. Mainz, Germany. Optical Glass Filters (catalogue).

References for Liquid Crystal Tunable Filters (p.13)
Cambridge Research and Instrumentation, Inc., Cambridge, MA. Varispec
Tunable Imaging Filter (catalogue).
Morris, Hannah R., Clifford C. Hoyt, Peter Miller, and Patrik J. Treado (1996)
Liquid crystal tunable filter Raman chemical imaging. Applied Spectroscopy
50:806-808.
Hoyt, Clifford (1996) Liquid crystal tunable filters clear the way for imaging
multiprobe fluorescence. Biophotonics International July/August.

30

GLOSSARY

with page references
Acousto-optical tunable filter (AOTF) (p. 12)
An active crystal device that works by setting up radio-frequency acousticalvibrations in the crystal and creating, in effect, a bulk transmission diffraction
grating. By varying the frequency, one can rapidly tune the filter to diffract out
a desired wavelength of light and transmit this wavelength out of the device.

Angle of incidence (AOI) (p. 9, fig. 9)
The angle between the optical axis of the light incident on the surface of afilter and the axis normal to this surface.

Angular deviation (p. 21)
A shift in the direction of light beam from the true optical axis of the system,measured in units of angle such as arc-minutes (1/60 of a degree) or arc-seconds(1/60 of an arc-minute).

Aperture diaphragm (p. 21)
An adjustable diaphragm located in the illumination optics which controls
the numerical aperture of the illuminating beam and affects the brightnessof the beam.

Attenuation level (p. 8, fig. 6)
Also Blocking level. A measure of the out-of-band attenuation of an opticalfilter over an extended range of the spectrum. The attenuation level is often
defined in units of optical density (see optical density).

Autofluorescence (p. 2)
In fluorescence microscopy, any fluorescence from substances otherthan fluorochromes, including primary fluorescence from the specimen,
fluorescencefrom immersion oils and other media, and fluorescence from glass opticalcomponents within the microscope.

Average transmission (p. 8, fig.6A)
The average calculated over the useful transmission region of a filter rather
than over the entire spectrum. For a bandpass filter, this region spans the
FWHM of the transmission band.

Background (p. 4)
Any detectable light that is not a desired primary or indirect fluorescentemission. Sources of background include cross-talk between excitation andemission filters, light leaking through pinholes in filters, and electronic noisein cameras, as well as autofluorescence.

31Bandpass (p. 8, fig. 6)
An optical filter that has a well-defined short wavelength cut-on and longwavelength cut-off. Bandpass filters are denoted by their center wavelengthand bandwidth.

Bandwidth (p. 8, fig. 6A)
Also FWHM (Full width at half of maximum transmission). For optical
bandpass filters, typically the separation between the cut-on and cut-off
wavelengths at 50% of peak transmission. Sometimes a bandwidth at, forexample, 10% of peak transmission is specified.

Barrier filter
See Emission filter.

Blocking level
See Attenuation level.

Blocking range (p. 8, fig. 6B)
The range of wavelengths over which an optical filter maintains a specified
attenuation level.

Blocker (p. 11, fig. 15)
A thin-film interference coating that is incorporated into a bandpass
interference filter to extend the blocking range of the primary coating in
the filter. A blocker is usually a very wide-band bandpass filter having hightransmission in the band of the primary filter.

Brightfield (p. 6)
A kind of diascopic illumination in which the field of view is illuminateddirectly. Also, the type of condenser used for this kind of illumination.

Center wavelength (CWL) (p. 8, fig. 6A)
For optical bandpass filters, the arithmetic mean of the cut-on and cut-off
wavelengths at 50% of peak transmission.

Clear aperture (p. 21)
The surface area of an optical filter which is free of any defects or obstructions.
On interference filters the clear aperture is often delimited by an annulus of
metal or opaque material.

Critical illumination (p. 26)
A type of illumination optics in which the image of the light source is focused
onto the specimen plane, in contrast to Köhler illumination optics. See also
Köhler illumination.

32

Cross-talk (p. 9, fig. 7)
The minimum attenuation level (over a specified wavelength range) of two
filters placed together in series. The transmission spectrum of the beamsplitteris sometimes included in this evaluation.

Darkfield (p. 6)
A kind of diascopic illumination in which the specimen is illuminatedobliquely, i.e., at angles that will not enter the objective directly. Also, thetype of condenser used for this kind of illumination.

Diascopic illumination (p. 6)
Illumination using light transmitted through the specimen, using a condenser
to focus the light.

Dichroic beamsplitter (p. 5, 7 footnote, fig. 4)
Also Dichroic mirror, Dichromatic beamsplitter. A special mirror housedin the filter cube that selectively reflects the excitation light filtered by the
exciter and transmits the emitted fluorescence. Dichroic beamsplitters canalso be found in any other part of a microscope system where light needs tobe split or combined by wavelength.

Edge filter (p 8)
Another term for either a shortpass or longpass optical filter. The term
usually denotes a filter with a very sharp cut-on or cut-off.

Emission filter (p. 5, fig. 4)
Also Barrier filter, Emitter. A color filter that attenuates all of the lighttransmitted by the excitation filter and very efficiently transmits any
fluorescence emitted by the specimen.

Emitter
See Emission filter.

Epifluorescence microscope (p. 4)
A fluorescence microscope that illuminates the specimen episcopically (i.e.,with light reflected onto the specimen).

Episcopic illumination (p. 7)
Also Incident-light illumination. Illumination with light reflected onto thespecimen rather than transmitted through the specimen. The illuminating
light is reflected through the objective by means of a beamsplitter that iseither partially reflective or a dichroic.

Excitation and emission (p. 2)
See Fluorescence.

33

Excitation filter (p. 5, fig. 4)
Also Exciter. A color filter that transmits only those wavelengths of theillumination light that efficiently excites a specific dye. See Emission filter.

Exciter
See Excitation filter.

Extinction coefficient (p. 3)
A measure of the absorption characteristics of a fluorochrome.

FadingSee Photobleaching.

Field diaphragm (p. 21)
An adjustable diaphragm located in the illumination optics which controls
the area of illumination on the specimen.

Filter block
See Filter cube.

Filter cube (p. 5, fig. 4)
A removable cube-shaped unit that holds a matched set of fluorescence filters.
This set usually includes an excitation filter and emission filter but always
includes a dichroic beamsplitter.

Filter glass (p. 10)
Also Absorption glass. Colored glass that is manufactured for technical andscientific applications. The most common types of filter glass used in fluorescence microscopy are UV-transmitting “black glass” filters; IR-absorbing
heat filters; and yellow, orange, and red sharp-cut longpass filters. Filter glass
selectively attenuates light by absorption, so the spectral performance isdependent on the thickness of the glass.

Fluorescence (p. 2)
A molecular phenomenon in which a substance absorbs light, then radiatespart of this absorbed energy as light of another color, one of lower energy andthus longer wavelength. This process is known as excitation and emission.
Fluorescence is distinguishable from other types of luminescence in that theprocess of excitation and emission occurs nearly instantaneously (i.e., on theorder of nanoseconds).

Fluorescent probe (p. 2)
Also Fluorophore. A fluorochrome that has been conjugated to an active
substance, such as a protein, antibody, or nucleic acid, in order to selectively
stain a targeted substance within the specimen.

34

Fluorochrome (p. 2)
A fluorescent dye used either directly as a specimen stain or conjugated to an
active substance to make a fluorescent probe.

Fluorophore
See Fluorescent probe.

Front surface (p. 19)
The side of a beamsplitter that is designed to face the incident light. In afilter cube this is the side that faces both the light source and the specimen.
Beamsplitters generally perform better when oriented correctly.

FWHM
See Bandwidth.

Half-cone angle (p. 9)
The angle between the most oblique ray of a convergent or divergent lightbeam and the optical axis of the beam. See also Numerical aperture.

Heat filter (p. 6)
An optical filter that attenuates infrared radiation but transmits the visible.
Attenuation is achieved by means of absorption (using filter glass), reflection(using a thin-film interference coating, often called a hot mirror), or a
combination of the two.

Hot mirror
See Heat filter.

Indirect fluorescence (p. 2)
Also Secondary fluorescence. In fluorescence microscopy, fluorescenceemitted by fluorochromes introduced into a specimen as a stain or probe. See
Primary fluorescence.

Infinity-corrected optics (p. 22)
An optical configuration employed by some microscopes in which theobjective forms an image at infinity, and a secondary tube lens forms animage atthe intermediate image plane. (This intermediate image is focused in turnby the ocular.) This configuration allows for a flexible distance between theobjective and ocular because the distance between the objective and the tube
lens can be varied without affecting the image-forming characteristics of the
microscope. Note that objectives designed for infinity-corrected optics are
not interchangeable with objectives designed for standard tube-length optics.

35

Interference filter
See Thin-film interference coating.

Köhler illumination (pp. 21)
The type of illumination optics, usually used in wide-field epifluorescencemicroscopes, in which the image of the light source is completely out of focus
at the specimen plane.

Longpass (LP) (p. 8)
An optical filter that attenuates shorter wavelengths and transmits longer
wavelengths over the active range of the spectrum (which depends on thespecific application). LP filters are denoted by the cut-on wavelength at 50%of peak transmission.

Luminance
See Radiance.

Nanometer (nm) (p. 2)
A unit of length commonly used for measuring the wavelength of light: 1 nm
= 10 angstroms (A) = 10-9 meters; and 1000 nm = 1 micron (µ) = 10-6 meters.

Near-infrared (NIR) (p. 3)
The region of the electromagnetic spectrum ranging in wavelength fromapproximately 750 to 2500 nanometers.

Neutral-density (ND) filter (p. 6)
An optical filter that attenuates light by an amount independent of the
wavelength within the useful range of the filter. Metal-coated filters typicallyhave a wider neutral range than glass filters and are more heat-tolerant.

Normal incidence (p. 9)
An angle of incidence of zero degrees.

Numerical aperture (NA) (p. 7, 19)
In the microscope, a measure of the effective cone-angle of light focused onto
the specimen (NA of the condenser) or light captured by the objective (NAof the objective). A high value of NA improves both the brightness and theresolution of the image. (NA = N sin(θ), where N is the refractive index of themedium surrounding the specimen and θ is the half-cone angle of the light.)

Optical density (OD) (p. 8)
A logarithmic unit of transmission: OD = -log10(T), where T is the transmission(0≤T≤1).

Parallelism
See Wedge.

36

Photobleaching (p. 3)
Also Fading. A photochemical reaction that causes the intensity of fluorescenceto decrease with time.

Pinholes (p. 21)
Small breaks in the coating of an interference filter, usually caused by thepresence of dust particles on the substrate during coating.

Polarization (p. 9)
Restriction of the orientation and phase of the electromagnetic field vibrations
caused by propagating light. These vibrations are transverse to the directionof propagation of the light, and can be oriented at some angle around the axisof propagation. When the orientation and the phase of the vibrations change
rapidly and randomly in time, the light is said to be unpolarized. When thevibrations are restricted to one particular orientation angle over an extended
length of time, the light is said to be plane-polarized. Light can be partially aswell as totally plane-polarized. When the relative phase of the vibrations varies
with angle in a regular periodic fashion over an extended length of time, thelight is said to be elliptically polarized. Circular polarization is a special casein which the amplitude of the vibrations are equal for all angles. When lightis elliptically polarized, the orientation of the vibrations rotates around the
propagation axis at the frequency of the light.
When light strikes a specular surface at non-normal incidence, the componentof the electric field vibrations parallel to the plane of incidence of the surface(P-plane) behaves differently than the component perpendicular to the plane ofincidence (S-plane). This causes a polarizing effect that is aligned orthogonallyto the orientation of the surface.

Polychroic (p. 27)
A name for a dichroic beamsplitter that has multiple reflection bands andtransmission regions.

P-planeSee Polarization.

Primary fluorescence (p. 2)
In fluorescence microscopy, fluorescent emissions from the specimen itself
rather than emissions from any fluorochromes present in the specimen. See
also Autofluorescence.

Quantum efficiency (p. 3)
A measure of how efficiently a fluorochrome converts absorbed radiation into
emitted fluorescence.

37

Quenching (p. 4)
Any chemical process which reduces the quantum efficiency of a fluorochromein situ.

Radiance (p. 15)
A measure of the radiometric brightness of a light source. Technically, theradiance is the radiant flux emitted per unit solid angle per unit area of thelight source. A common unit is watts per steradian per square centimeter.
Luminance is a measure of the brightness of a light source as perceived by thehuman eye (i.e., a photometric measure), commonly measured in candelas per
square centimeter.

Registration shift (p. 22)
A shift in the apparent position of the specimen that occurs when an opticalelement is inserted or removed, adjusted, or switched with another element.

Scratch/dig (p. 21)
A set of specifications for defining the maximum allowable size and numberof scratches and digs on an optical surface. The scratch/dig values (e.g.,
80/50) specify the scratch width in microns and the dig diameter in tensof microns, respectively. Although extensive evaluation procedures exist if
rigorous standards must be maintained (military specification MIL-F-48616,
for example), a qualitative visual assessment of the scratches and digs usually
suffices.

Secondary fluorescence (p. 6)
See Indirect fluorescence.

Shortpass (SP) (p. 8)
An optical filter that attenuates longer wavelengths and transmits shorter
wavelengths over the active range of the spectrum (which depends on thespecific application). SP filters are denoted by the cut-off wavelength at 50%of peak transmission.

Signal-to-noise (S/N) (p. 14)
A measure of the brightness of the desired fluorescence (the signal) relative tothe brightness of the background (the noise).

Slope (p. 9)
A measure of the sharpness of the transition from the transmitting region to
the blocking region of a color filter.

Spectrofluorimeter, also spectrofluorometer (p. 2)
An instrument used for measuring the excitation and emission spectra of afluorescent substance.

38

S-planeSee Polarization.

Standard tube-length optics (p. 22)
An optical configuration employed by most microscopes in which theobjective forms an image at the intermediate image plane. (This intermediate
image is focused in turn by the ocular.) The distance between the nosepiecethat holds the objective and the barrel that holds the ocular is fixed at astandard length of 160 mm so that objectives can be interchangeable between
microscopes. See also Infinity-corrected.

Stokes shift (p. 3)
The shift in wavelength between the peak excitation intensity and peakemission intensity of a fluorochrome.

Sub-Pixel Registration (SPR) (p. 24)
A term used to describe filter sets that are manufactured to a level of precisionthat eliminates variations in image registration when switching filters or
filter-cubes.

Substrate (p. 5)
The ground and polished piece of optical glass that is used as a base for thebeamsplitter coating.

Surface flatness (p. 20)
A measure of the deviation of the surface of an optical element from a perfectplane, measured in fractions or multiples of a wavelength of visible light(usually 550 or 630 nm).

TE-modeAnother term for S-plane polarization. (Short for “transverse-electric” mode.)
See Polarization.

Thin-film interference coating (p. 11)
A type of optical coating composed of a stack of microscopically thinlayers of material. Although each material is intrinsically colorless, thereflections created at each interface between the layers combine throughwave interference to selectively reflect some wavelengths of light andtransmit others. Thin-film interference coatings are the main component ofinterference filters, which consist of one or more coatings separated by glass
substrates and frequently one or more layers of filter glass.

TM-modeAnother term for P-plane polarization. (Short for“transverse-magnetic” mode.) See Polarization.

39

Transmitted wavefront distortion (TWD) (p. 20)
A measure of the distortion a plane wave of light undergoes when transmitted
through an optical element, measured in fractions or multiples of a wavelength of visible light (usually 550 or 630 nm).

Ultraviolet (UV) (p. 3, fig. 2)
The region of the electromagnetic spectrum ranging in wavelength fromapproximately 100 to 400 nanometers. Three distinct bands are: 1) near-UV,
from 320 to 400 nm; 2) mid-UV, from 190 to 320 nm; and 3) vacuum-UV(VUV), below 190 nm. The terms UV-A and UV-B denote bands with distinct
physiological effects: 320 to 380 nm and 280 to 320 nm, respectively.

Wedge (p. 20)
Also Parallelism. A measure of the deviation of the outer surfaces of an
optical element from perfect parallelism, usually measured in arc-minutes or
arcseconds of angle.

Wide-field (p. 21)
An epifluorescence microscope in which the full field of view is illuminated,in contrast to a confocal epifluorescence microscope. The term brightfield isalso used, but this might be confused with traditional diascopic brightfield
illumination.

40

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Voice +49 8142 2847526
europe@chroma.com

Voice +86 0592 5062089
china@cn.chroma.com

Voice +81 045 285 1583
japan@chroma.com

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