PerkinElmer Nuance 3.0.2 User manual

Nuance MULTISPECTRAL IMAGING SYSTEM
User's Manual for Nuance 3.0.2

Notice
The information in this document is subject to change without notice and should not be construed as a commitment by
PerkinElmer, Inc. PerkinElmer assumes no responsibilityfor anyerrors that mayappear in this document. This manual is
believed to be complete and accurate at the time of publication. In no event shall PerkinElmer be liable for incidental or
consequential damages in connection with or arising from the use of this manual.
This manual describes system operation using Nuance version 3.0.2 software.
For more information contact:
PerknElmer, Inc.
68 Elm Street, Hopkinton, MA, 01748, USA
Phone:[phone] or [phone]
Fax: [phone]
Email: [e-mail]
Web site: http://www.perkinelmer.com
US Patent 5,892,612; 5,953,087; 7,655,898; and patents pending.
Document Part No. 130765 Rev. 01

3Contents
Table of Contents
Part I Welcome To Nuance 6
Part II Introduction to Multispectral Imaging 7
...................................................................................................................................... 71 Light...................................................................................................................................... 82 Human Perception of Light Intensity and of Color
...................................................................................................................................... 93 Light Absorbance and Reflection
...................................................................................................................................... 104 Fluorescence
...................................................................................................................................... 125 The Multispectral Solution
...................................................................................................................................... 156 Nuance FLEXMultispectral Imaging
Part III Getting Started with Nuance 17
...................................................................................................................................... 171 Operator and Equipment Safety
...................................................................................................................................... 182 About this Manual
...................................................................................................................................... 193 CE, CSA, and UL Testing and Certification
...................................................................................................................................... 194 Nuance Imaging Module
...................................................................................................................................... 225 Installing & Starting Nuance
...................................................................................................................................... 236 Understanding the Nuance Work Area
...................................................................................................................................... 257 Window Layout
...................................................................................................................................... 278 Specifying Nuance Hardware Settings
...................................................................................................................................... 299 Calibrating Pixel Size
...................................................................................................................................... 3010 Using Low Screen Resolution Mode
...................................................................................................................................... 3011 Reinitializing Nuance Hardware
Part IV Method Development 31
...................................................................................................................................... 311 Methods for Building Spectral Libraries
...................................................................................................................................... 352 Saving Spectral Libraries
...................................................................................................................................... 353 Saving Protocols
...................................................................................................................................... 364 Importing Spectra Into a Library
...................................................................................................................................... 375 Practice Exercise
Part V Brightfield and Fluorescence Imaging 40
...................................................................................................................................... 401 Setting Up Your Microscope for Brightfield Imaging
...................................................................................................................................... 412 Setting Up Your Microscope for Fluorescence Imaging
...................................................................................................................................... 423 About Optical Density Images
...................................................................................................................................... 434 Tips for Obtaining Quantitative Results
...................................................................................................................................... 435 Acquiring Images Using a Saved Nuance Protocol
...................................................................................................................................... 446 Viewing a Live Image Stream
...................................................................................................................................... 447 Selecting Camera Binning and a Region of Interest

Nuance Multispectral Imaging System4
...................................................................................................................................... 458 Specifying the Current Wavelength and Exposure
...................................................................................................................................... 479 Acquiring Cubes in Brightfield
...................................................................................................................................... 4810 Acquiring RGB (Color) Images
...................................................................................................................................... 4911 Acquiring Fluorescence Cubes
...................................................................................................................................... 5212 Acquiring a Mono Image (Snapshot)
...................................................................................................................................... 5213 Assigning Sample IDs and Notes
...................................................................................................................................... 5314 Acquiring Timed Sequences of Image Cubes
...................................................................................................................................... 5415 Saving Images and Image Cubes
...................................................................................................................................... 5516 Viewing Cube Information
...................................................................................................................................... 5617 Extracting an Image Plane from a Cube
Part VI Unmixing Spectral Images 57
...................................................................................................................................... 571 Tips for Accurate Unmixing
...................................................................................................................................... 572 Opening a Spectral Library
...................................................................................................................................... 583 Opening an Image Cube
...................................................................................................................................... 584 Computing and Unmixing Spectra Automatically
...................................................................................................................................... 615 Computing and Unmixing Spectra Manually
...................................................................................................................................... 656 Saving an Unmixed Result Set
...................................................................................................................................... 657 Working With Saved Result Sets
...................................................................................................................................... 668 Checking Your Spectral Library
...................................................................................................................................... 679 Subtracting Spectra from a Cube
...................................................................................................................................... 6710 Using Line Profiles to Analyze Signals
...................................................................................................................................... 6911 Comparing Multiple Images
...................................................................................................................................... 7212 Processing a Batch of Cubes
Part VII Quantifying Results 75
...................................................................................................................................... 751 Measuring Regions
...................................................................................................................................... 802 Ignoring Smaller Regions
...................................................................................................................................... 803 Adjusting Region Transparency and Color
...................................................................................................................................... 804 Understanding Region Measurements
Part VIII Customizing Spectral Displays 83
...................................................................................................................................... 831 Adjusting Brightness and Contrast Levels
...................................................................................................................................... 842 Applying Overlays
...................................................................................................................................... 843 Adjusting a Cube's RGB Mapping
...................................................................................................................................... 854 Changing Components in a Composite Image
...................................................................................................................................... 865 Advanced Display Controls
...................................................................................................................................... 886 Displaying the Intensity Legend
Part IX Macros 89
...................................................................................................................................... 891 Overview of the Macros Dialog Box
...................................................................................................................................... 912 Running Macros

5Contents
...................................................................................................................................... 913 Recording Macros
...................................................................................................................................... 914 Saving Macros
Part X Co-Localization Staining Detection 92
...................................................................................................................................... 921 Opening a Dataset for Co-localization Analysis
...................................................................................................................................... 932 Adjusting Threshold Mask Values
...................................................................................................................................... 943 Selecting Markers for Co-localization
...................................................................................................................................... 954 Saving and Loading Co-localization Settings
...................................................................................................................................... 965 Interpreting the Statistics
...................................................................................................................................... 976 Customizing the Statistics Display
...................................................................................................................................... 977 Drawing Regions of Interest
...................................................................................................................................... 988 Customizing the Composite Image Display
...................................................................................................................................... 999 Copying Images and Data
...................................................................................................................................... 9910 Exporting Images and Data
Part XI FAQs 100
Part XII Troubleshooting 102
Part XIII System Specifications & Dimensions 104
Part XIV Filter Selection Guide 107
Part XV MetaMorph Support 108
...................................................................................................................................... 1091 Installing and Configuring MetaMorph
...................................................................................................................................... 1122 Installing Nuance with MetaMorph Support
...................................................................................................................................... 1133 Import the Journal Suite for MetaMorph
...................................................................................................................................... 1164 Troubleshooting
Part XVI Koehler Aligning the Microscope 117
Part XVII About Legacy Hardware 118
Part XVIII Windows User Management 119
Part XIX Software EULA 123
Index 126

6 Nuance Multispectral Imaging System
1 Welcome To Nuance
Multi-label Imaging Without Cross-talk
As biological imaging becomes more about studying complex
systems rather than single events, Nuance multispectral imaging
systems let you quantitate multiple molecular markers even when
they are co-localized in a single tissue section, producing clear
and accurate images of each individual label on a multi-label
tissue section.
The unique and patented Real Component Analysis (RCA) tool
automatically detects spectral components. View and quantitate
signals as either single images, as overlays, or as a composite
image showing all unmixed labels in high contrast. Nuance
systems offer powerful spectral unmixing capability to remove
autofluorescence, thereby dramatically increasing signal-to-noise
and improving the accuracy of your results.
Getting Started — New Users
Review the Introduction to Multispectral Imaging Theory and Concepts to familiarize yourself
with the concepts behind multispectral imaging technologies.
Study the Getting Started with Nuance section to familiarize yourself with the Nuance
Hardware components and the software.
Make sure you review the Method Development section, which discusses methods for building
accurate and reliable spectral libraries.
You might also find it helpful to run through the Practice Exercise to see how easy it is to
spectrally unmix a counterstain from a target-specific marker.
If desired, see the Practical Multiple Staining Pamphlet on the installation CD for a discussion on
staining and imaging.
7
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Introduction to Multispectral Imaging 7
2 Introduction to Multispectral Imaging
This section provides an introduction to the theory and concepts that enable PerkinElmer's multispectral
imaging systems to function as well as they do.
2.1 Light
Light, as used in this discussion, means the portion of the electromagnetic spectrum that is visible
to the human eye. While the physical description of light can be highly complex, we will restrict this
discussion to the wavelengths of light, and the interaction of that light with physical and biological
materials.
The Electromagnetic Spectrum is illustrated in the figure below from radio to gamma ray
frequencies. We are concerned with the visible wavelength range for purposes of this discussion,
although the Nuance imaging system can operate out to 950 nm, into the so-called near-infrared
wavelength range.
Figure 1: The Electromagnetic Spectrum
Although this illustration of the electromagnetic spectrum(1) suggests that the visible range of light
covers approximately 400 nm to 800 nm, most humans realistically are limited to the range of 400
nm (deep violet) to 700 nm (deep red). Visible light makes up a very small portion of the entire
electromagnetic spectrum.
Light is the transmission of energy. Visible light is associated with an energy level of about one
electron volt per photon. As one moves to shorter wavelengths of light, the energy per photon
increases. In the shorter ultraviolet wavelengths, which approach soft x-rays, the electron energy per
photon increases to 50 to 100 electron volts. This energy content of light is useful when one wishes
to induce a change of energy state in a material (i.e., cause a receptive molecule to undergo a
series of energy additions and then relaxations, resulting in fluorescence).
(1) The Electromagnetic Spectrum illustration was prepared by NASA under Contract NAS5-26555 for the Education Group of the Space
Telescope Science Institute’s Office of Public Outreach. It is used here under public domain in accordance with NASA’s contract.

8 Nuance Multispectral Imaging System
2.2 Human Perception of Light Intensity and of Color
A Highly Adaptive Light Detector
The human eye is a highly adaptive light detector, becoming significantly more sensitive in low light,
and less sensitive under bright light. This adaptive change is not instantaneous, and it takes some
time for the eye to fully adjust to a new illumination level. This is the reason one needs to “dark
adapt” by being in a darkened room for some time before observing weak fluorescence through a
microscope.
While the eye can be very sensitive to low light levels and can also “see” in very bright conditions, it
does not discriminate light levels very well. An individual has no “internal meter” that indicates the
current sensitivity level setting for the eye. The eye also has a limited ability to discriminate levels of
illumination ranging from the lowest level to the highest level at any given sensitivity. US Department
of Defense research indicates that while some people can distinguish as many as 500 levels of gray,
most humans can only distinguish approximately 30 to 35 levels of gray, ranging from black to
white. This is relatively insensitive to the actual total illumination level, although the ability to
discriminate gray levels does degrade in both very dim light and very bright light.
Response to Illumination
The eye’s response to illumination is not a linear response, but more closely approximates a
logarithmic function. The result is that while the human eye interprets differences between gray
levels as “even steps,” to achieve a two-fold brightening of the perceived gray level, the actual
illumination level would need to increase significantly more than a simple doubling.
Contrasting the eye with a digital electronic sensor system, any sensor that has 8-bit resolution can
detect 256 levels of gray. As the number of bits of resolution increase, the number of gray levels also
increases. A 10-bit system gives 1024 levels and 12 bits yields 4096 levels of gray. Digital electronic
sensors are linear in response to light levels.
Ability to Distinguish Colors
While the eye is relatively poor at discriminating gray levels (intensity), it is very good at
distinguishing colors. Most individuals are estimated to be able to discriminate thousands of colors.
The problem is, no two individuals see precisely the same color. In other words, each individual
interprets colors slightly differently when viewing the same color. The basis for this is the way in
which color sensors are arranged in the eye.
The eye contains three different types of color sensors, similar in response to the red, green and
blue detectors in color cameras. Individual colors are composed of some combination of responses
from these three different types of color sensors. While the general arrangement of these color
sensors (cone cells) in the eye is reasonably standard, there are differences in the total number of
each type of cone cell, and in the actual physical arrangement within the detecting cell layer (retina).
These minor variations lead to the differences in perceived color between individuals, as does the
actual way in which the individual’s brain learned to interpret color(s).

Introduction to Multispectral Imaging 9
2.3 Light Absorbance and Reflection
We perceive objects based on the way they interact with incident light (excepting light emitting
objects such as light sources, and fluorescing or phosphorescing objects). Objects transmit,
absorb, and/or reflect light; in most cases they exhibit a combination of transmittance,
absorbance, and, reflection. The perceived color of a non-transmissive object is a direct result of
both absorbance and reflection of light. An opaque object we would perceive as red is one that
absorbs most wavelengths of light, except for red wavelengths that are reflected back to the eyes of
the observer. An object we would perceive as red in transmission is one that transmits primarily red
wavelengths, while absorbing or reflecting other wavelengths.
Generally speaking, absorbance and reflection of light are very similar phenomena. One can say that
the light transmitted through a semitransparent object is the light that is not absorbed by the object.
Note how similar this is to the definition of reflectance. Only the geometry is different. Reflectance is
more complex than simple transmission due to variations in surface texture, transparency of the
reflecting layers, and the characteristics of the opaque surface beneath the reflecting layers.
In brightfield light microscopy we have a controlled geometry, and are observing light that is
transmitted through a specimen. Excepting a few pigments and inclusions, biological specimens are
essentially invisible, unless we employ some absorbing dye, or specific optical arrangement to
impart contrast. It is this need for contrast that led to the initial development of biological stains and
stain protocols and subsequently to phase contrast and other optical contrast enhancing
techniques.
The amount of light absorbed by a dye or stain in a specimen can provide a measurement of the
amount of the absorbing material present. This is the basis of spectrophotometry. A basic law of
spectrophotometry is the Beer-Lambert law, which states that the amount of material present
(absorbance) is defined by the relationship:
Absorbance = -log(transmission) = (molar absorbtivity) x (path length) x (concentration)
For a given material, the molar absorbtivity, also called the molar extinction, is a constant, and
therefore one only needs to determine the percent transmission of light through the specimen and
the path length in order to calculate the concentration. The transmission is the amount of light
without the specimen versus the amount of light with the specimen, and this is easily measured.
Since transmission is based on a ratio, it is not sensitive to actual light level, assuming that the light
does not change between the measurement of the “blank” or 100% level; and the “specimen” or
sample measurement. It is this basic law that is used in solution spectrophotometry to determine
the concentration of absorbing materials in solution.
The Beer-Lambert law has two specific requirements:
The absorbing material must be homogeneous
The absorbing material must be a true absorber of light, not a light scatterer
This first requirement poses problems for spectrophotometry through a microscope. The very reason
one uses a dye on a microscope preparation is to see structure, and by definition, if one can see
structure, then the dyed material is not homogeneous. While this may seem like an insurmountable
obstacle to performing spectrophotometry through a microscope, the solution is simple. Microscope
optics are characterized by their ability to resolve two points as separate. This is the resolution of a
particular lens system. If the light detector element sees a smaller portion of the specimen than the
lens system can resolve as separate points, then by definition, the spot being measured is
homogeneous.
Using modern CCD or CMOS cameras for image collection, accurate spectrophotometry requires

10 Nuance Multispectral Imaging System
that each individual pixel of the sensor see a portion of the specimen that is smaller than the lens
resolution of the particular lens system being used. In practical terms, this means the camera pixels
should be smaller than:
(magnification) x 1.22 x (wavelength) ÷ (NAobjective + NAcondenser)
The result of not having homogeneity of absorption at each pixel is an error called distributional
error. Distributional error can be accurately defined mathematically, but the following illustration will
suffice to understand this principle. Assume you have a light shining through an aquarium filled with
water and some means of measuring the amount of light coming through the aquarium. If you place
a small-capped bottle of ink into the aquarium, the measured amount of light will decrease by only a
small amount. If you reach into the aquarium and remove the cap from the ink bottle and let the ink
diffuse throughout the aquarium, the measured amount of light will decrease significantly. The total
amount of ink in the aquarium is the same in both cases; it is only the distribution of the ink that is
different. In practical microscope systems, distributional error can give errors of up to 40% if the
specimen is not homogeneous at the detector element(s).
The second requirement of the Beer-Lambert law is that the material being measured must be a true
absorber, and not a light scatterer. There are several reasons for this. One is that we cannot
determine a molar extinction (the “constant” in the Beer-Lambert equation) for a sample with mixed
properties. A more understandable rationale is based on the microscope optics. If a material
scatters light, as the concentration of the material increases, more light is scattered, and this
scatter can be outside the capture cone of the objective being used. Obviously, any light that is
scattered in such a way as to not be seen by the objective cannot be measured. The consequence
of this is that the detected signal is non-linear as the concentration of the mixed absorber-scatterer
changes.
Assuming a specimen with a true absorbing chromogen, and an appropriate magnification for the
detector being used, the Beer-Lambert law provides for true brightfield microscopy quantitation. After
an image is converted to optical density or absorbance, each pixel is a true measure of light
transmission at that pixel. This is because the background illumination is taken into account in the
conversion. Absorbance images are therefore not constrained by total illumination level, and if the
same specimen is imaged through identical optical systems at two different illumination levels, the
resulting absorbance images should be identical.
There are several requirements that need to be met to achieve the accuracy inherent in optical
density or absorbance measurements. It is necessary that the illumination be even over the entire
image field. For microscope systems, this may not be the case, if the microscope illumination
system is not carefully aligned.
Accurate measurements depend on some mechanism to perform a “flat field correction” for
illumination inconsistencies. A common way to do this is to collect an image of the illumination field
with no specimen in place (a blank field image). To ensure accurate data, any quantitative system
should be able to meet the “test of the five positions”. This involves selecting some object in a field of
view, and then taking images of this object when placed in the center of the image field, and when
placed at the top, bottom, left and right of the image field. In each of these five positions, the object
should yield identical measurement results.
2.4 Fluorescence
Many biological and natural materials give off light of a particular color when exposed to light of
another color. This property is a type of luminescence. If the emitted light occurs rapidly after
illumination (around one-millionth of a second), the luminescence is called fluorescence. If the light
emission takes longer than one-millionth of a second, the luminescence is called
phosphorescence. Materials that exhibit fluorescence have proven extremely useful as labels or

Introduction to Multispectral Imaging 11
indicators in many biological systems.
Fluorescence light emission is different than light absorption. Each fluorescent molecule generates
light. We measure the total amount of light generated, and are not dependent on the interaction of
the light with another material, such as a dye. While it would seem that fluorescence is much more
amenable to accurate measurement than absorbed light, there are a number of factors that
complicate such measurements. Fluorescence is emitted by a molecule in all directions, and most
imaging systems are designed to capture light coming from a particular direction only. Therefore,
there is no way to capture all of the light emitted by a fluorescent molecule with such a system.
Additionally, fluorescence emission is influenced by the local environment, in particular by pH. The
total amount of fluorescence will therefore depend on these local conditions of pH, as well as other
surrounding molecules that may either enhance or quench some of the fluorescence energy. There
is also the problem of obtaining identical excitation of all fluorescence molecules in a specimen, and
this can be exceedingly difficult to achieve.
Fluorescence is an extremely sensitive technique, as it is much easier to visually assess or
measure light emission against a dark background than it is to see a decrease in light intensity from
absorption by a dye. Regardless of the sensitivity of fluorescence, the difficulty in establishing
uniform excitation, and controlling for local environment effects makes quantitation of fluorescence
emission difficult in biological preparations.
Stoke’s Shift
Materials that fluoresce always emit light at a longer wavelength than the wavelength of the exciting
light. As an example, rhodamine isothiocyanate can be excited by green light, and then emits red
light. This difference between the wavelength of the exciting light and the emitted light is called the
Stoke’s Shift and is based on Stoke’s Law.
A range of excitation wavelengths will excite fluorescence. This range of wavelengths is known as
the absorption spectrum. The emitted light also covers a range of wavelengths, and this is known
as the emission spectrum. Since the Stoke’s Shift for most materials is not that great, there is
generally some overlap between the excitation and the emission spectra. We will return to this point
shortly as it does impact choice of emission and excitation filters in fluorescence systems. This
figure contains an example excitation and emission spectra, showing Stoke’s Shift and the overlap
of the spectra.
Figure 2: Stoke's Shift

12 Nuance Multispectral Imaging System
Autofluorescence
Many biological materials are naturally fluorescent. In particular, many vitamins, some hormones,
and a variety of biological enzymes and structural proteins are naturally fluorescent. These materials
often fluoresce strongly enough to interfere with specific fluorescence labeling studies. Because of
this unwanted background, or autofluorescence, both excitation light sources and emitted light
paths are highly filtered in fluorescence systems.
On continued stimulation (illumination at the excitation wavelength), most fluorescent materials fade.
While some specific preparation methods can reduce the rate of fading, and different fluorescent
materials fade at different rates, all fluorescent materials eventually fade, and this effect is
irreversible. For this reason, specimens should be illuminated only while aligning and focusing, and
during actual image collection. At other times, the excitation light should be closed off.
Excitation and Emission Filters
Filters that are used for fluorescence excitation and emission are specifically constructed to have
very narrow pass bands. They pass only a limited range of wavelengths of light. Restricting the
excitation light wavelengths may reduce the amount of autofluorescence. Restricting the wavelength
range of the emitted light helps minimize the amount of autofluorescence light that interferes with
observing and measuring the desired specific fluorescence.
Figure 3: Excitation Filter Types
Excitation filters should be chosen to match the excitation maximum of the fluorescence label being
used. The emission filter should match the emission maximum. In practical terms, the filter maxima
may be slightly different from the ideal case, simply due to limitations of filter manufacturing, or to
assist with autofluorescence reduction.
Specific excitation and emission filter combinations are available for most commonly used
fluorescence dyes or labels. Nevertheless, regardless of how carefully one matches the excitation
and emission filters to a given label, there will be some background autofluorescence, and this will
reduce the perceived contrast between the “real” or actual label fluorescence and the specimen
background.
2.5 The Multispectral Solution
The Nuance imaging system offers a unique solution to the problem of autofluorescence and
selection of emission filters. Multispectral analysis is based on the fact that all fluorescent materials

Introduction to Multispectral Imaging 13
produce a unique spectral emission. In other words, if you excite a material, and then examine the
emitted fluorescence over a range of wavelengths, and record the intensity of emission at each point
along the plotted curve of those wavelengths, you can generate an “emission spectrum” (like the
green emission spectrum shown in the figure illustrating Stokes Law ). This spectrum is different
for each specific fluorescent material.
Overlapping Emission Spectra
The complication is that for many fluorescent labels of biological interest, the emission spectra
overlap significantly, and these emission spectra may also be obscured by autofluorescence from
other constituents of the specimen. Often, autofluorescence is a strong (bright) broad signal that
may obscure the specific fluorescence that the investigator wishes to see.
Figure 4: Overlapping Excitation and Emission Spectra
In this example of overlapping excitation and emission spectra, green is MitoTracker® Green Fn,
Blue is Acridine Orange, and Red is Fluorescein isothiocyanate (FITC). The Cyan line at 488
represents the illumination light. Dotted lines are excitation spectra, and solid lines are emission
spectra. (Illustration derived from Invitrogen™ Spectraviewer.)
There are three points to note in the graph shown above.
1. The excitation spectra overlap significantly with the emission spectra. This is why one needs to
carefully select excitation filters. The goal is to prevent as much excitation light as possible from
appearing in the emission spectra.
2. Even with distinct emission spectra, there is significant overlap in the emission spectra of these
three dyes. Visual examination of such a mixture of fluorescence spectra would be unable to
distinguish these three dyes as individual “colors.” They would be seen as some combination of
yellow and green by most observers.
3. The graphical display of spectra is normalized, and in actual practice, some fluorescent
materials are much brighter than others.
As the graph illustrates, many labels of biological interest have emission spectra that are so similar
that separation using narrow band filters is difficult or impossible. Multispectral analysis provides the
solution to this problem and reduces the need for multiple expensive narrow-band emission filters. A
single long-pass emission filter can replace a large collection of emission filters.
11

14 Nuance Multispectral Imaging System
Multispectral Analysis
Multispectral analysis generates the spectral curves for the various fluorescent dyes or materials in
a specimen. In addition, it generates a spectral curve for the autofluorescence that almost always is
present to some degree. Using sophisticated algorithms, the contribution of autofluorescence to the
image can be removed, and the individual fluorescence spectra separated. The result is a set of
images representing each spectrum that contributes to the final image.
In other words, as illustrated in the graph above, multispectral analysis yields (1) an
autofluorescence image, (2) a MitoTracker Image, (3) a Acridine Orange image, and (4) a FITC
image. By removing the autofluorescence contribution to the image, the actual signals from the
applied labels (MitoTracker, Acridine Orange and FITC) can be readily seen. If these individual
images are recombined using highly contrasting colors to represent the location of each of the
labels, a composite image of high contrast and readily observable colors can be generated.
The Nuance Imaging System
The Nuance imaging system’s combination of unique hardware and sophisticated software makes
powerful multispectral analysis possible. In a multispectral analysis, a series of images is captured
at specific wavelengths. The range of wavelengths captured should cover the spectral emission
range of the labels present in the specimen. The number of images within that range should be
chosen to adequately define the emission spectral curve for each label. The result will be a series of
images, called an “image cube,” taken at specific wavelengths. The data within the image cube is
used to define the individual spectra of both autofluorescence and specific labels.
Figure 5: Mouse Intestine with Five Unmixed Fluorescent Elements
This figure shows a specimen (courtesy of Molecular Probes, Inc.) with poor contrast between the

Introduction to Multispectral Imaging 15
five different fluorescent elements. A conventional color image is shown in the upper left. By
acquiring a multispectral data cube and using the Nuance system’s unmixing tools, a new
Composite Unmixed Image can be created (shown on the right). Separate grayscale images
representing each unmixed component are also created.
As the figure illustrates, many labels of biological interest have emission spectra that are so similar
that separation using narrow band filters would be difficult or impossible. Multispectral analysis
provides the solution to this problem, and in addition reduces the need for multiple, expensive and
very narrow band emission filters. A single long pass emission filter can replace a large collection of
emission filters. Multispectral analysis is able to separate all of these autofluorescence signals from
the specific labels applied to the specimen, and provides the ability to localize each material
present, and to detect weak specific labeling even in the presence of strong autofluorescence.
2.6 Nuance FLEX Multispectral Imaging
Nuance FXand EXimaging systems have the unique ability to narrow the bandwidth of the Liquid
Crystal Tunable Filter by half. Flex technology lets you use the system in broad or narrow mode,
which greatly increases the system’s multiplexing capabilities for brightfield and fluorescence
imaging. This figure shows LCTF transmission with and without Flex technology at 700 nm and 800
nm.
Figure 6: Comparison of LCTFtransmission with and without FLEX
Use the system in narrow mode to obtain better resolution of closely spaced and overlapping
emission spectra. Narrow mode can also increase the number of detectable chromogens or
fluorophores in your sample with overlapping spectra. This can increase the dynamic range of your
multiplexed signals. This figure illustrates the higher spectral resolution obtained with Flex
technology.

16 Nuance Multispectral Imaging System
Figure 7: Comparison of 605 nm Qdot™ with and without FLEX

Getting Started with Nuance 17
3 Getting Started with Nuance
PerkinElmer’s Nuance multispectral imaging systems are high-performance multispectral imaging
systems that can be installed on an upright or inverted microscope's C-mount camera port, on a copy
stand, or on a camera tripod for field use. Properly configured Nuance systems can be used for
applications as diverse as biomedical research, materials QA/QC, forensic analysis, and archeology.
(Nuance systems are not intended for clinical or diagnostic use at this time.)
The patented liquid crystal (LC) tuning element functions as a high-quality interference filter that enables
the transmitted light to be electronically tunable. This allows rapid, vibrationless selection of wavelengths
in the visible or NIR range, and digital images (called image cubes) are captured at the specified
wavelengths. The intuitive Nuance acquisition and analysis software performs spectral classification and
unmixing of overlapping dyes or stains that may look indistinguishable to the naked eye but have
differing spectral signatures.
The Nuance product family includes one general purpose model and two fluorescence-optimized models:
The Nuance VX for visible wavelengths operates on or off the microscope in the wavelength
range of 420-720 nm.
The Nuance FX and Nuance EX operate on or off the microscope in either the 420-720 nm range
(FX) or the 450-950 nm range (EX) and have electronically selectable bandwidths.
The topics in this section provide an introduction to the Nuance multispectral imaging system. This
section includes a brief description of each of the system’s hardware components and software
features.
3.1 Operator and Equipment Safety
It is the responsibility of the purchaser to ensure that all persons who will operate the imaging system
are aware of the following cautionary statements. As with any scientific instrument, there are important
safety considerations, which are highlighted throughout this User’s Manual.
General Cautionary Statements
READ AND UNDERSTAND THIS USER’S MANUAL BEFORE ATTEMPTING TO
OPERATE, TROUBLESHOOT, OR MAINTAIN THE NUANCE IMAGING SYSTEM.
READING THIS MANUAL FIRST MAKES IT EASIER AND SAFER TO OPERATE AND
MAINTAIN THE SYSTEM.
Operate the system on a flat, stable surface.
Do not drop the imaging module.
Do not expose the imaging system to prolonged heat above 40 °C.
Do not operate the system in an environment with explosive or flammable gases.
Do not subject the imaging system or its components to intense light from laser, focused arc or Hg
lamp sources.
Do not operate the system in places where it may be splashed with liquid.
Use only a properly grounded power cable appropriate for the site where the system is installed.

18 Nuance Multispectral Imaging System
Some cables and adapters supplied with the system have proprietary specifications. Do not connect
components supplied by PerkinElmer using unqualified cables or adapters. Doing so could result in
damage, and voids the Warranty.
Use only a properly grounded power outlet when connecting the system to power.
If you are using third-party mechanical components for the Nuance system, consult the System
Specifications & Dimensions topic.
Clean the Nuance imaging system periodically. This will help ensure optimal performance over years
of use. See Cleaning the Nuance Imaging Module .
Caution: Service should be performed by PerkinElmer authorized and trained personnel only. Power
must be disconnected from the system before servicing.
For Technical Assistance
If you experience any difficulty setting up, operating, or maintaining your imaging system, please
refer to the Troubleshooting section of this documentation. If that does not resolve your problem,
contact your PerkinElmer representative. Office hours are 8:00 a.m. to 8:00 p.m. (Eastern Standard/
Daylight Time), Monday through Friday.
Telephone: [phone] or [phone]
Fax: [phone]
E-Mail: [e-mail]
3.2 About this Manual
This manual describes the use and functionality of the Nuance multispectral imaging system and the
Nuance version 3.0.2 software. Operating instructions, functional descriptions, troubleshooting,
illustrations, and other relevant information are contained in this manual.
Design Change Disclaimer
Due to design changes and product improvements, information in this manual is subject to change
without notice. PerkinElmer reserves the right to change product design at any time without notice
to anyone, which may subsequently affect the content of this manual. PerkinElmer will make every
reasonable effort to ensure that this User’s Manual is up to date and corresponds with the shipped
Nuance multispectral imaging system.
Reproduction Disclaimer
This User’s Manual is solely for the use of the owner and operator of the PerkinElmer Nuance
multispectral imaging system. Any reproduction of this publication in part or in whole without the
express written consent of PerkinElmer is strictly prohibited. Neither may this publication be made
available for electronic download without the express written consent of PerkinElmer.
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Getting Started with Nuance 19
Help Menu in the Nuance software
The Help menu in the Nuance software provides quick access to the Nuance Help Topics. You can
also view the Filter Selection Guide and the Quick Start Guide, both in PDF format. The Help >
About menu item displays the current Nuance software version number as well as other important
system information.
3.3 CE, CSA, and UL Testing and Certification
The Nuance multispectral imaging system has been tested by an independent CE
testing facility, and bears the appropriate CE mark.
The Nuance system has been awarded the right to display the CSA mark.
The Nuance system has undergone tests to meet UL standards.
For more information, contact PerkinElmer.
3.4 Nuance Imaging Module
The Nuance Imaging Module contains all of the principal imaging components in a single compact
enclosure:
High-resolution, scientific-grade CCD imaging sensor
Solid-state liquid crystal (LC) wavelength tuning element
Spectrally optimized lens and internal optics
Industry-standard C-mount (compatible with 1x C-mount camera tube)
Industry-standard threaded mounting hole for securing the module to a camera tripod or other
holder.

20 Nuance Multispectral Imaging System
Figure 8: Nuance Imaging Module (front view)