Section 24.1 - Reference Standards and Antifade Reagents

24.1 Reference Standards and Antifade Reagents
To obtain accurate and reproducible results from fluorescence
imaging applications, it is essential to maximize the performance
of the optical system. Careful calibration and instrumentation
adjustment are required for high-precision imaging of fluorescent
probes, particularly in multicolor applications that involve multiple exposures, repetitive scans or three-dimensional sectioning.
Molecular Probes offers a variety of microsphere reference
standards designed to facilitate adjustment and calibration of both
conventional fluorescence microscopes and confocal laser-scanning microscopes. Our Reference Dye Sampler Kit (R-14782)
provides ready-made stock solutions of five extensively characterized fluorescence standards for use in spectrofluorometers and
fluorescence microplate readers. Because imaging sensitivity
depends not only on a well-adjusted instrument, but also on the
intensity and stability of the fluorescence signal, Molecular
Probes has developed several antifade reagents to minimize photobleaching of fluorescently labeled specimens. Our popular
FluoCells prepared microscope slides (Figure 24.1, Figure 24.16,
Figure 24.17, Figure 24.18, Figure 24.19, Figure 24.20) are extremely useful products for educational and commercial demonstrations.
FocalCheck products are available in seven different color
configurations, including three suspensions that contain microspheres exhibiting ring stains of two or three different fluorescent
colors:
• Blue-fluorescent and orange-fluorescent ring stains (15 µm,
F-7234)
• Green-fluorescent and dark red-fluorescent ring stains
(15 µm, F-7240)
• Green fluorescent, orange fluorescent and dark red-fluorescent
ring stains (6 µm, F-14806; 15 µm, F-7235)
We also supply four suspensions that contain microspheres
exhibiting a ring stain of one fluorescent color combined with a
stain of a second fluorescent color throughout the bead:
• Green fluorescent ring stain with blue-fluorescent stain
throughout (6 µm, F-14808; 15 µm, F-7237)
FocalCheck Fluorescent Microspheres
FocalCheck Ring-Stained Fluorescent Microspheres
Our patented FocalCheck fluorescent microspheres are specifically designed for examining the alignment, sensitivity and
stability of confocal laser-scanning microscopes.1 They are particularly useful for confirming the optical sectioning thickness
(Z-resolution) in three-dimensional imaging applications. These
polystyrene beads — available in 6 µm and 15 µm diameters —
have been treated using a proprietary method in which a fluorescent dye is allowed to penetrate to only a limited depth within the
microsphere. The resulting beads have a well-defined dye layer
that, when viewed in cross section in the confocal laser-scanning
microscope, appears as a fluorescent ring of varying dimensions
depending on the focal plane (Figure 24.2, Figure 24.3). We refer
to this proprietary staining procedure as ring staining, so as to
differentiate it from routine staining throughout the bead.
FocalCheck microspheres are available in a variety of color
configurations provided by five different fluorescent stains:
• Blue (365/430 nm)
• Green (505/515 nm)
• Orange (560/580 nm)
• Red (575/600 nm)
• Dark red (660/680 nm)
The excitation/emission maxima of the different stains are
well matched to the laser sources and optical filters commonly
used in confocal laser-scanning microscopy and are especially
useful in testing and aligning confocal laser-scanning microscopes with multiple laser lines and detection channels (Figure
24.4). Moreover, because the dyes are localized within the bead
and therefore protected from environmental factors, the FocalCheck microspheres are brighter and much more photostable than
conventional surface-stained beads.
Figure 24.1 FluoCells prepared slide #3 (F-24630) contains a section of
mouse kidney stained with a combination of fluorescent dyes. Alexa Fluor 488
wheat germ agglutinin (W-11261), a green-fluorescent lectin, was used to
label elements of the glomeruli and convoluted tubules. The filamentous-actin
prevalent in glomeruli and the brush border were stained with red-fluorescent
Alexa Fluor 568 phalloidin (A-12380). Finally, the nuclei were counterstained
with the blue-fluorescent DNA stain DAPI (D-1306, D-3571, D-21490). This
image is a composite of three micrographs acquired using filter sets
appropriate for fluorescein, tetramethylrhodamine and DAPI.
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A
B
C
Figure 24.2 Confocal laser-scanning microscope optical cross-sectioning and
alignment with FocalCheck microspheres. A) Serial optical sectioning from top
to bottom along the z-axis of ring-stained microspheres reveals a continuous
pattern of disc-to-ring-to-disc images. B) The diameter of the fluorescent ring
(or disc) seen is dependent on the depth of the optical focal plane. C) In the
confocal laser-scanning microscope, separate light paths exist for UV and
visible wavelengths. Also, fluorescence emitted is detected by different
photomultipliers. Proper optical alignment may be obtained with either of two
types of FocalCheck microspheres. For example, the microspheres with an
orange ring stain that are blue-fluorescent throughout the bead allow UV/
visible wavelength alignment in three dimensions upon aligning the orange
ring with the blue disc. Focal alignment is also possible simultaneously in three
colors by aligning the green, orange and dark-red rings of the FocalCheck
microspheres containing fluorescent green/orange/dark-red ring stains.
Figure 24.3 Images from confocal laser-scanning microscope optical crosssectioning of our 15 µm FocalCheck microspheres that have a dark red-fluorescent ring stain with a green-fluorescent stain throughout the bead (F-7239).
The left panel provides a clear visual representation of poor instrument alignment. Correct image registration has been achieved in the right panel, where
the dark-red ring is aligned with the green disk. Image contributed by Paulette
Brunner, Keck Imaging Center, University of Washington, and Yu-Zhong Zhang,
Molecular Probes, Inc.
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Figure 24.4 Normalized excitation spectra of the dyes contained in the FocalCheck microspheres. Emission lines of several commonly used laser excitation
sources are superimposed on the dyes’ excitation spectra to illustrate the wide
range of usage of these beads as calibration references. Ar = Argon-ion laser.
Kr–Ar = Krypton–argon laser. He–Ne = Helium–neon laser.
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• Green-fluorescent ring stain with dark red-fluorescent stain
throughout (15 µm, F-7238)
• Orange-fluorescent ring stain with blue-fluorescent stain
throughout (15 µm, F-7236)
• Dark red-fluorescent ring stain with green-fluorescent stain
throughout (6 µm, F-14807; 15 µm, F-7239, Figure 24.3)
The sharp ring stains exhibited by the FocalCheck microspheres produce a striking visual representation of instrument
misalignment or other aberrations, making them ideal as reference standards for confocal laser-scanning microscopy. Correct
image registration is indicated when the multiple ring images
of the ring-stained FocalCheck beads (or the ring and disk
images of the combination ring-stained and stained-throughout
FocalCheck beads) are perfectly coincident in all dimensions
(Figure 24.2).
FocalCheck Thin-Ring Fluorescent Microspheres
Our FocalCheck Thin-Ring Fluorescent Microspheres Kit
(F-14791) contains smaller-diameter microspheres that have
spectral and imaging features similar to those of our 6 µm and
15 µm FocalCheck microspheres. Because we prepare these
1.0 µm beads using fluorescent stains that are restricted to the
surface only, they exhibit sharper and thinner fluorescent ring
patterns when viewed in cross section with a confocal laserscanning microscope. The FocalCheck Thin-Ring Fluorescent
Microspheres Kit contains three 200 µL suspensions of 1.0 µm
beads. Each suspension contains beads with a different color
configuration:
• Green-fluorescent (495/515 nm) and red-fluorescent
(575/600 nm) ring stains
• Green-fluorescent (495/515 nm) ring stain with dark redfluorescent (660/680 nm) stain throughout the bead
• Red-fluorescent (575/600 nm) ring stain with blue-fluorescent
(365/430 nm) stain throughout the bead
FocalCheck Microspheres Pre-Mounted on Microscope Slides
In addition to the bead suspensions described above, we offer
FocalCheck microspheres pre-mounted on microscope slides.
The FocalCheck Fluorescent Microsphere Kits feature mounted
samples of three different color configurations, in either the 6 µm
(F-24633) or the 15 µm (F-24634) bead size:
• FocalCheck beads with green-fluorescent, orange-fluorescent
and dark red-fluorescent ring stains
• FocalCheck beads with green-fluorescent ring stain and bluefluorescent stain throughout the bead
• FocalCheck beads with dark red-fluorescent ring stain and
green-fluorescent stain throughout the bead
MultiSpeck, TetraSpeck and Constellation
Fluorescent Microspheres
Our unique MultiSpeck and TetraSpeck fluorescent microspheres greatly facilitate the adjustment and calibration of conventional fluorescence microscopes, confocal laser-scanning
microscopes and associated image-processing equipment for
multicolor applications. These uniform, multiply stained microspheres are useful for co-localizing and focusing different wavelengths of light in the same optical plane, as well as for checking
multicolor image resolution, magnification and sensitivity.
MultiSpeck Multispectral Fluorescence Microscopy
Standards Kit
The 4 µm MultiSpeck microspheres in our MultiSpeck Multispectral Fluorescence Microscopy Standards Kit (M-7901) exhibit three relatively distinct emission bands — blue, green and red
— throughout every particle (Figure 24.5). The spectral characteristics (excitation/emission peaks at 365/405 nm, 520/525 nm
and 580/600 nm) are compatible with optical filter sets designed
for commonly used blue, green and red fluorophores (e.g.,
DAPI, fluorescein and rhodamine or Texas Red dyes or their
Alexa Fluor dye counterparts). The MultiSpeck beads can be
used as external references for comparing images collected with
different optics, on different instruments and in different laboratories, as well as for monitoring routine day-to-day variations
in instrument performance. Furthermore, because a single multispectral microsphere will appear as different colors depending
on the optical filters used for observation, these beads can be
used to assess image registration in two and three dimensions,
allowing the researcher to accurately determine the spatial
relationships of different labels in a multiparameter experiment.
The MultiSpeck Multispectral Fluorescence Microscopy Standards Kit contains:
• A suspension of MultiSpeck multispectral microspheres
• A mixed suspension of separately stained blue-, green- and
red-fluorescent microspheres, which exhibit the same three
excitation/emission bands as the multispectral microspheres
but in separate beads
• Mounting medium
• A slide-mounting protocol
Both suspensions are provided at a ready-to-use density and
can be mounted on slides or incorporated into an experimental
sample. Each kit supplies a sufficient amount of material for
~50 slide preparations using either of the two bead suspensions
provided.
TetraSpeck Fluorescent Microspheres and Sampler Kits
Our TetraSpeck fluorescent microspheres 1,2 expand the multispectral strategy introduced with the MultiSpeck beads in two
important ways. First, the TetraSpeck beads have been stained
throughout with a mixture of four different fluorescent dyes
(Figure 24.6), yielding four well-separated excitation and emission peaks. The excitation/emission maxima of the dyes are
365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange)
and 660/680 nm (dark-red). Second, these microspheres are
available in five nominal sizes (actual bead diameters are indicated on the product labels), spanning the range from subresolution to nearly cell-size particles:
• 0.1 µm (T-7279)
• 0.2 µm (T-7280)
• 0.5 µm (T-7281)
• 1.0 µm (T-7282)
• 4.0 µm (T-7283)
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Each of these products provides a 0.5 mL suspension sample of TetraSpeck microspheres that is sufficient for about 100 slides. We offer the TetraSpeck Fluorescent
Microspheres Sampler Kit (T-7284) and the TetraSpeck Fluorescent Microspheres Size
Kit (T-14792). The TetraSpeck Fluorescent Microspheres Sampler Kit consists of separate suspension samples of our 0.1 µm, 0.5 µm and 4.0 µm TetraSpeck beads, each
sufficient for preparing about 20 slides. The TetraSpeck Fluorescent Microspheres Size
Kit contains six microscope slides; five slides have a mounted sample of the 0.1 µm–,
0.2 µm–, 0.5 µm–, 1.0 µm– or 4.0 µm–diameter TetraSpeck microspheres, and a sixth
slide has a mixture of all five sizes.
Figure 24.5 Multiple exposures of a prepared slide
from our MultiSpeck Multispectral Fluorescence
Microscopy Standards Kit (M-7901). This kit provides fluorescent MultiSpeck microspheres that
exhibit three relatively distinct excitation/emission
bands — blue, green and red — all in the same
particle. Thus, the same microsphere can appear a
different color depending on the optical filter set
used. This photograph was taken through bandpass optical filter sets appropriate for DAPI, fluorescein and Texas Red dyes, with the field of view
shifted slightly between exposures.
Constellation Microspheres for Imaging
Constellation microspheres for imaging (C-14837, Figure 6.3, Figure 24.7) are 3 mL
suspensions of assorted fluorescent microspheres with a variety of sizes and colors.
Designed for use in laboratory tutorials and customer training sessions, they provide
inexpensive and robust test samples for demonstrating filter switching, focus adjustment
and other functional capabilities of fluorescence microscopes. The Constellation microspheres can be stored at room temperature, protected from light.
PS-Speck Microscope Point Source Kit
The fluorescent microspheres in Molecular Probes’ PS-Speck Microscope Point Source
Kit (P-7220) have a diameter of 0.175 ± 0.005 µm, making them ideal subresolution fluorescent sources for calibrating instrument optics. They are particularly useful for measuring
the point-spread function required for computational image deconvolution procedures 3
(Figure 24.8, Figure 24.9). This kit’s four ready-to-use suspensions contain bright, monodisperse particles in the following fluorescent colors (and excitation/emission peaks):
•
•
•
•
Blue (360/440 nm)
Green (505/515 nm)
Orange (540/560 nm)
Deep red (633/660 nm)
The kit also includes mounting medium and a mounting protocol for the user’s convenience. Each suspension provides sufficient material to mount about 100 slides.
InSpeck Microscopy Image Intensity Calibration Kits
Figure 24.6 Four separate exposures of three
TetraSpeck beads (T-7283) photographed using
optical filter sets appropriate for DAPI, fluorescein,
tetramethylrhodamine and the Texas Red dye. The
stage was shifted after each exposure. Note that
the same beads appear blue, green, orange or red,
depending on the filters used.
Figure 24.7 Constellation microspheres (C-14837)
provide an assortment of sizes and colors for use
in fluorescence microscopy demonstrations.
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InSpeck Microscope Image Intensity Calibration Kits provide microsphere standards
that generate a series of well-defined fluorescence intensity levels (Figure 24.10) for
Figure 24.8 One PS-Speck green-fluorescent microsphere (P-7220) used for point-spread function analysis. Shown are individual images, in
pseudocolor, taken in x-y planes with 0.25 µm increments in the z-axis between planes. Image
contributed by Dr. Regina Armstrong, Uniformed
Services University of the Health Sciences, Bethesda, Maryland.
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Figure 24.9 An orthogonal (x-z) display representing a point-spread function of a particular microscope’s optics. The microsphere used is a component of the PS-Speck Microscope Point Source Kit
(P-7220). This pseudocolored image was generated electronically from a series of microsphere images taken in x-y planes. Image contributed by
Jennifer Kramer, Scanalytics.
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constructing calibration curves and evaluating sample brightness. Most of the kits are
offered in a choice of two different microsphere sizes (2.5 µm or 6 µm) and five different
fluorescent colors:
•
•
•
•
•
InSpeck Blue (350/440 nm) Kit, (2.5 µm, I-7221)
InSpeck Green (505/515 nm) Kits (2.5 µm, I-7219; 6 µm, I-14785)
InSpeck Orange (540/560 nm) Kits (2.5 µm, I-7223; 6 µm, I-14786)
InSpeck Red (580/605 nm) Kits (2.5 µm, I-7224; 6 µm, I-14787)
InSpeck Deep Red (633/660 nm) Kit (2.5 µm, I-7225)
Each kit includes six separate suspensions of InSpeck fluorescent microspheres with
relative fluorescence intensities of 100%, 30%, 10%, 3%, 1% and 0.3% (Figure 24.10),
covering the range of intensities commonly encountered in microscopy applications.
Unstained control beads and mounting medium are also supplied. The aqueous suspensions of microspheres may be applied directly to the sample for calibrating fluorescence
intensities or mounted separately in an adjacent well or on another slide. Each suspension
provides sufficient material to prepare about 100 slides.
Figure 24.10 Flow cytometric analysis of the
beads in the 6 µm InSpeck Green Microscope Image Intensity Calibration Kit (I-14785). The microspheres have nominal relative fluorescence intensities of 100%, 30%, 10%, 3%, 1%, 0.3%. For each
lot, actual relative intensities are determined by
flow cytometry and printed on the product labels.
Reference Dye Sampler Kit
Our Reference Dye Sampler Kit (R-14782) provides samples of five extensively
characterized fluorescence standards with emission spectra covering the entire visible
wavelength range. All five fluorescent standards are supplied as 1 mM stock solutions in
1 mL units, sufficient to prepare 500 diluted working samples for spectrofluorometry.
The compositions of the stock solutions are as follows:
•
•
•
•
•
Quinine sulfate in 0.1 M sulfuric acid (H2SO4) (Figure 24.11)
Fluorescein in dimethylsulfoxide (DMSO) (Figure 24.12)
5-Carboxytetramethylrhodamine in DMSO (Figure 24.13)
Sulforhodamine 101 in DMSO (Figure 24.14)
Nile blue perchlorate in DMSO (Figure 24.15)
Spectroscopic data for the five standards are summarized in Table 24.1.
Figure 24.11 Absorption and fluorescence emission spectra of quinine sulfate, dihydrate, a component of the Reference Dye Sampler Kit (R-14782),
in 0.5 M sulfuric acid.
FluoCells Prepared Microscope Slides
In response to requests from educators and instrument manufacturers, Molecular
Probes offers FluoCells prepared microscope slides that contain multi-labeled cell preparations for observation by epifluorescence or confocal microscopy.
• FluoCells prepared slide #1 2 (F-14780, Figure 24.16) shows bovine pulmonary artery
endothelial cells (BPAEC) stained with MitoTracker Red CMXRos for labeling mitochondria, BODIPY FL phallacidin for labeling F-actin and DAPI for labeling the nucleus.
Table 24.1 Spectroscopic data for components of Molecular Probes’ Reference Dye Sampler Kit.
Component
Solvent
Abs (nm) *
Em (nm) *
QY †
Quinine sulfate
Water
347 ‡
455 ‡
0.55 ‡
Fluorescein
Water
490 §
514 §
0.92 §
5-CTMR **
Methanol
542
568
0.68
Sulforhodamine 101
Ethanol
578
605
0.90
Nile blue
Ethanol
636
665
0.27
* Approximate absorbance (Abs) and fluorescence emission (Em) maxima. † Fluorescence quantum yield at
22°C. ‡ Standard values in 0.5 M H2SO4. § Standard values in 0.1 M NaOH. ** CTMR = Carboxytetramethylrhodamine.
Figure 24.12 Absorption and fluorescence emission spectra of fluorescein, a component of the
Reference Dye Sampler Kit (R-14782), in 0.1 M sodium hydroxide.
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Figure 24.13 Absorption and fluorescence emission spectra of 5-carboxytetramethylrhodamine
(5-TAMRA, a component of the Reference Dye
Sampler Kit (R-14782)), in methanol.
• FluoCells prepared slide #2 2 (F-14781, Figure 24.17) again contains BPAEC, but this
time stained with Texas Red-X phalloidin for labeling F-actin, mouse monoclonal
anti–α-tubulin in conjunction with BODIPY FL goat anti–mouse IgG for labeling
microtubules and DAPI for labeling the nucleus.
• FluoCells prepared slide #3 (F-24630; Figure 24.1, Figure 24.18, Figure 24.19) contains a 16 µm cryostat section of a mouse kidney. Green-fluorescent Alexa Fluor 488
wheat germ agglutinin stains the glomeruli and convoluted tubules; red-fluorescent
Alexa Fluor 568 phalloidin labels actin, which is especially prevalent in glomeruli and
the brush border of proximal convoluted tubules; finally, DAPI stains the nuclei with
blue fluorescence.
• FluoCells prepared slide #4 (F-24631, Figure 24.20) contains a 16 µm cryostat section
of a mouse intestine. Alexa Fluor 350 wheat germ agglutinin labels the mucus of goblet
cells with blue fluorescence; the red-fluorescent Alexa Fluor 568 phalloidin labels actin
filaments, which are especially prevalent in the brush border of the intestinal mucosa;
and SYTOX Green nucleic acid stain labels nuclei with green fluorescence.
The multicolor staining in these cell preparations results in stunning, publicationquality images. They are especially useful for setting up microscopes and camera systems and for assessing the capabilities of optical filter sets. When stored properly, these
permanently mounted specimens will retain their bright, specific staining patterns for at
least six months from the date of purchase.
ProLong and SlowFade Antifade Kits
Loss of fluorescence through irreversible photobleaching processes can lead to a
significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. To minimize photobleaching of experimental samples, we have developed the ProLong, SlowFade and
SlowFade Light Antifade Kits, which have been proven to increase the photostability of
Figure 24.14 Absorption and fluorescence emission
spectra of sulforhodamine 101, a component of the
Reference Dye Sampler Kit (R-14782), in ethanol.
Figure 24.15 Absorption and fluorescence emission
spectra of nile blue, a component of the Reference
Dye Sampler Kit (R-14782), in ethanol.
Figure 24.16 FluoCells prepared slide #1 (F-14780) showing bovine pulmonary artery endothelial cells
(BPAEC) that have been labeled with MitoTracker Red CMXRos (M-7512) for mitochondria, and then fixed,
permeabilized and stained with BODIPY FL phallacidin (B-607) for F-actin and DAPI (D-1306, D-3571,
D-21490) for nuclei.
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Figure 24.17 FluoCells prepared slide #2 (F-14781), which shows bovine pulmonary artery endothelial cells (BPAEC) that have been stained
with an anti–β-tubulin mouse monoclonal antibody in conjunction with
BODIPY FL goat anti–mouse IgG (B-2752) for labeling microtubules,
Texas Red-X phalloidin (T-7471) for labeling F-actin and DAPI (D-1306,
D-3571, D-21490) for labeling nuclei.
Figure 24.19 FluoCells prepared slide #3 (F-24630) containing a 16 µm
cryostat section of mouse kidney stained with green-fluorescent Alexa
Fluor 488 wheat germ agglutinin (W-11261), red-fluorescent Alexa Fluor
568 phalloidin (A-12380) and blue-fluorescent DAPI (D-1306, D-3571,
D-21490). The image represents an optical section obtained by simultaneous two-photon excitation of all three dyes at 797 nm using a Bio-Rad
Radiance 2100 multi-photon microscope system. Image acquired at the
2001 3-D Microscopy of Living Cells Course, University of British Columbia, Vancouver, Canada, by John Jordan, Bio-Rad Laboratories; and Iain
Johnson, Molecular Probes, Inc.
Figure 24.18 FluoCells prepared slide #3 (F-24630) contains a mouse
kidney section stained with a combination of fluorescent dyes. Greenfluorescent Alexa Fluor 488 wheat germ agglutinin (W-11261) was used
to label elements of the glomeruli and convoluted tubules. The filamentous actin was stained with red-fluorescent Alexa Fluor 568 phalloidin
(A-12380). Finally, the nuclei were stained with the blue-fluorescent DAPI
(D-1306, D-3571, D-21490). This pseudocolored image was acquired on
a Zeiss confocal microscope located at the Institute of Neuroscience, University of Oregon.
Figure 24.20 FluoCells prepared slide #4 (F-24631) contains a section of
mouse intestine stained with a combination of fluorescent stains. Alexa
Fluor 350 wheat germ agglutinin (W-11263) is a blue-fluorescent lectin
that was used to stain the mucus of goblet cells. The filamentous actin
prevalent in the brush border was stained with red-fluorescent Alexa
Fluor 568 phalloidin (A-12380). Finally, the nuclei were stained with
SYTOX Green nucleic acid stain (S-7020). This image is a composite of
three digitized images obtained with filter sets appropriate for fluorescein,
DAPI and tetramethylrhodamine.
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0
125
250
375
500
Figure 24.21 The relative photobleaching rates of fluorescein and Texas Red fluorophores with
buffer alone (top series) or after treatment with the ProLong antifade reagent (bottom series).
The numbers above each pair of frames represents the duration of continuous excitation in seconds. BPAE cells were fixed, permeabilized and labeled with Texas Red-X phalloidin (T-7471),
which labels F-actin, and with a mouse monoclonal anti–β-tubulin and fluorescein goat anti–
mouse IgG (F-2761), which label microtubules. Images were acquired at appropriate wavelengths
using a cooled CCD camera.
2
Figure 24.22 Bovine pulmonary arterial epithelial (BPAE) cells were labeled with fluorescein
phalloidin (F-432), which labels filamentous actin, and placed under constant illumination on the
microscope with a FITC filter set using a 60× objective. Images were acquired at one-second intervals for 30 seconds. Under these illumination conditions, fluorescein photobleached to about
12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same
illumination conditions when mounted using the reagents in the ProLong Antifade Kit (right,
P-7481).
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Figure 24.23 Bleaching profiles of A) fluorescein
and B) Texas Red dye conjugates in cell samples. In
these photobleaching experiments, human epithelium
(HEp-2) cells were probed with human anti-nuclear
antibodies and then developed for visualization with
fluorophore-labeled secondary reagents. Identical
samples were mounted in ProLong antifade reagent
( : in Kit P-7481), Product X (+) or medium containing no antifade reagent ( ). Although these data
were normalized, we observed little or no quenching
of samples mounted with the ProLong mounting
medium.
Figure 24.24 The fluorescence intensity of fluorescein as a function of illumination time, under the following conditions: in the presence of the SlowFade
Light antifade reagent (in Kit S-7461; green), in
the presence of the SlowFade antifade reagent
(in Kit S-2828; red) and in the absence of an antifade
reagent (blue). In these experiments, we added free
fluorescein directly to a solution and then examined
the mixture in a capillary tube using a 20× lens. In
real samples such as cells and tissues, we find that
the local environment influences the bleaching rate,
yielding results that are sometimes different from
those shown here.
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many of our fluorophores in fixed cells, fixed tissues and cell-free
preparations. The primary function of any antifade reagent is to
sustain dye fluorescence, usually by inhibiting the generation and
diffusion of reactive oxygen species. Strategies for further maximizing the fluorescence signal in both living and nonliving specimens include reducing the excitation light intensity by using
neutral density filters, high–numerical aperture objectives, relatively low magnification, high-quality optical filters and highspeed film or high-efficiency detectors.
ProLong Antifade Kit
The ProLong Antifade Kit (P-7481, Figure 24.21) outperforms
all other commercially available antifade reagents. Each ProLong
Antifade Kit contains:
• ProLong antifade reagent powder
• ProLong mounting medium
• A protocol for mounting samples
Specimens mounted using the ProLong Antifade Kit exhibit
little or no quenching of the fluorescent signal of most dyes.
Bovine pulmonary arterial epithelial (BPAE) cells labeled with
fluorescein phalloidin (F-432) photobleached to about 12% of
the initial value in 30 seconds in PBS, while staying at the initial
value under the same illumination conditions when mounted
using the ProLong Antifade Kit (Figure 24.22). As shown in
Figure 24.23, the ProLong Antifade Kit provides more fluorescence output than a popular p-phenylenediamine–containing
antifade reagent 4 when used to mount fluorescein-stained HEp-2
cells. Furthermore, fluorescence of the Texas Red dye when
viewed through the appropriate Texas Red optical filter (Table
24.6) becomes noticeably brighter upon addition of the ProLong
reagents. The ProLong Antifade Kit reagents also inhibit the
fading of tetramethylrhodamine, as well as the fading of DNAbound nucleic acid stains such as DAPI, propidium iodide and
YOYO-1 (Section 8.1), again without significantly quenching the
fluorescence of these dyes. The compatibility of the ProLong
Antifade Kit reagents with a multitude of dyes and dye complexes makes it an especially valuable tool for multicolor analysis
procedures such as multiplex fluorescence in situ hybridization
(FISH, Section 8.5).5
SlowFade and SlowFade Light Antifade Kits
Our original SlowFade formulation (S-2828) was designed to
reduce the fading rate of fluorescein to almost zero. Because it
provides nearly constant emission intensity from fluorescein, the
SlowFade reagent is especially useful for quantitative measurements and applications that employ a confocal laser-scanning
microscope, in which the excitation intensities can be extreme and
prolonged. The SlowFade reagent can extend the useful fluorescence emission of fluorescein more than 50-fold and can preserve
the signal in cell and tissue mounts for up to two years. However,
the original SlowFade formulation substantially quenches the
fluorescence of fluorescein and almost completely quenches that
of the Cascade Blue and Alexa Fluor 350 fluorophores.
To overcome this limitation, Molecular Probes’ researchers
have developed the SlowFade Light Antifade Kit (S-7461). The
antifade formulation in our SlowFade Light Antifade Kit slows
fluorescein’s fading rate by about fivefold without significantly
reducing fluorescein’s initial fluorescence intensity (Figure
24.24), thereby dramatically increasing the signal-to-noise ratio
in photomicroscopy. Moreover, quenching of the Cascade Blue,
Alexa Fluor 350, tetramethylrhodamine and Texas Red dyes is
minimal. In fact, the SlowFade Light antifade reagent reduces the
fading rate of the Cascade Blue fluorophore to almost zero, while
decreasing its emission intensity by only about 30%.
Each SlowFade or SlowFade Light Antifade Kit contains:
• SlowFade (in Kit S-2828) or SlowFade Light (in Kit S-7461)
antifade reagent in 50% (v/v) glycerol, ready to use and sufficient for at least 200 coverslip-size experiments
• 2× Concentrated SlowFade or SlowFade Light antifade reagent
solution, provided for those applications in which glycerol may
not be compatible
• Equilibration buffer, which raises the pH of the sample, increasing the protection afforded by both SlowFade formulations
• A detailed protocol for mounting samples
We have also formulated the SlowFade and SlowFade Light
Antifade Kits (S-24635, S-24636) to include the blue-fluorescent
nuclear counterstain DAPI (Section 8.6). These kits permit simultaneous staining and protection of the stained sample from photobleaching.
The SlowFade Antifade Kits are especially designed for those
who want a quick and easy solution to photobleaching. Unlike the
ProLong Antifade Kit, the SlowFade Kits require no mixing.
Simply pre-wash fluorescently labeled specimens in the equilibration buffer provided and then apply the mounting medium
directly from the convenient applicator bottle.
References
1. Appl Immunohistochem Mol Morphol 7, 156 (1999); 2. Methods 18, 447
(1999); 3. Biophys J 80, 2455 (2001); 4. J Histochem Cytochem 41, 1833
(1993); 5. Cytometry 32, 163 (1998).
Product List — 24.1 Fluorescence Microscopy and Fluorometry Reference Standards and Antifade Reagents
Cat #
Product Name
C-14837
F-14780
F-14781
Constellation™ microspheres for imaging *mixture of assorted sizes and colors* ..............................................................................................
FluoCells® prepared slide #1 *BPAE cells with MitoTracker® Red CMXRos, BODIPY® FL phallacidin, DAPI* ......................................................
FluoCells® prepared slide #2 *BPAE cells with mouse anti-α-tubulin, BODIPY® FL goat anti-mouse IgG,
Texas Red®-X phalloidin, DAPI* ...........................................................................................................................................................................
FluoCells® prepared slide #3 *mouse kidney section with Alexa Fluor® 488 WGA, Alexa Fluor® 568 phalloidin,
DAPI* ...................................................................................................................................................................................................................
F-24630
Unit Size
Section 24.1
3 mL
each
each
each
887
Product List — 24.1 Fluorescence Microscopy and Fluorometry Reference Standards and Antifade Reagents — continued
Cat #
Product Name
F-24631
FluoCells® prepared slide #4 *mouse intestine section with Alexa Fluor® 350 WGA, Alexa Fluor® 568 phalloidin,
SYTOX® Green* ...................................................................................................................................................................................................
FocalCheck™ Fluorescent Microspheres Kit, 6 µm *mounted on slides* .............................................................................................................
FocalCheck™ Fluorescent Microspheres Kit, 15 µm *mounted on slides* ...........................................................................................................
FocalCheck™ microspheres, 6 µm, fluorescent dark-red ring stain/green throughout .........................................................................................
FocalCheck™ microspheres, 6 µm, fluorescent green/orange/dark-red ring stains ..............................................................................................
FocalCheck™ microspheres, 6 µm, fluorescent green ring stain/blue throughout ................................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent blue/orange ring stains .............................................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent dark-red ring stain/green throughout .......................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent green/dark-red ring stains ........................................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent green/orange/dark-red ring stains ............................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent green ring stain/blue throughout ..............................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent green ring stain/dark red throughout ........................................................................................
FocalCheck™ microspheres, 15 µm, fluorescent orange ring stain/blue throughout ............................................................................................
FocalCheck™ Thin-Ring Fluorescent Microspheres Kit, 1.0 µm *three suspensions* ..........................................................................................
InSpeck™ Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm ....................................................................................................
InSpeck™ Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm ...........................................................................................
InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm .................................................................................................
InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm ....................................................................................................
InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm ...............................................................................................
InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 6 µm ..................................................................................................
InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm ....................................................................................................
InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm .......................................................................................................
MultiSpeck™ Multispectral Fluorescence Microscopy Standards Kit *in suspension* .........................................................................................
ProLong® Antifade Kit ..........................................................................................................................................................................................
PS-Speck™ Microscope Point Source Kit *blue, green, orange and deep red fluorescent beads* .......................................................................
Reference Dye Sampler Kit *five 1 mM solutions, 1 mL each* ............................................................................................................................
SlowFade® Antifade Kit .........................................................................................................................................................................................
SlowFade® Antifade Kit with DAPI ........................................................................................................................................................................
SlowFade® Light Antifade Kit ................................................................................................................................................................................
SlowFade® Light Antifade Kit with DAPI ...............................................................................................................................................................
TetraSpeck™ Fluorescent Microspheres Sampler Kit ...........................................................................................................................................
TetraSpeck™ Fluorescent Microspheres Size Kit *mounted on slides* ................................................................................................................
TetraSpeck™ microspheres, 0.1 µm, fluorescent blue/green/orange/dark red ......................................................................................................
TetraSpeck™ microspheres, 0.2 µm, fluorescent blue/green/orange/dark red ......................................................................................................
TetraSpeck™ microspheres, 0.5 µm, fluorescent blue/green/orange/dark red ......................................................................................................
TetraSpeck™ microspheres, 1.0 µm, fluorescent blue/green/orange/dark red ......................................................................................................
TetraSpeck™ microspheres, 4.0 µm, fluorescent blue/green/orange/dark red ......................................................................................................
F-24633
F-24634
F-14807
F-14806
F-14808
F-7234
F-7239
F-7240
F-7235
F-7237
F-7238
F-7236
F-14791
I-7221
I-7225
I-7219
I-14785
I-7223
I-14786
I-7224
I-14787
M-7901
P-7481
P-7220
R-14782
S-2828
S-24635
S-7461
S-24636
T-7284
T-14792
T-7279
T-7280
T-7281
T-7282
T-7283
Unit Size
each
1 kit
1 kit
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
24.2 Flow Cytometry Reference Standards
Flow cytometers are designed to perform quantitative measurements on individual cells and other particles with high precision, speed and accuracy. As with all high-performance instrumentation, flow cytometers must be calibrated frequently to
ensure accuracy and reliability. The stability, uniformity and
reproducibility of our fluorescent microsphere products make
them ideal reference standards for flow cytometry. Our flow
cytometry reference standards exhibit superior stability and
greater uniformity than other commercially available microparticles, and they are accompanied by a full one-year warranty.
Known numbers of fluorescent microspheres can also be added
to samples to facilitate the estimation of cell numbers,1 as in our
Bacteria Counting Kit 2–4 (B-7277, see below). Unfortunately,
because of high variability in quantum yields of dyes once bound
to an antibody, heterogeneity of antibody labeling, problems with
stoichiometry and accessibility in binding to targets, improper
calibration procedures and several other factors, bead standards
888
containing a known number of fluorophores per bead do not
necessarily provide accurate information on the number of
ligands bound to a cell.5
AlignFlow and AlignFlow Plus Flow Cytometry
Alignment Beads
Due to advances in flow cytometric instrumentation and the
development of fluorescent probes, a scientist can now perform
multiparameter analyses on biological samples. In order to ensure
accurate and reproducible results, flow cytometers should be
checked at least daily for proper performance. Molecular Probes’
AlignFlow and AlignFlow Plus flow cytometry alignment beads
permit the calibration of a flow cytometer’s laser(s), optics and
stream flow without wasting valuable and sensitive experimental
material. These fluorescently stained, polystyrene microspheres are
Chapter 24 — Accessories and Resources
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