Review Imaging technologies for the detection of multiple stains in

Proteomics 2003, 3, 1097±1108
DOI 10.1002/pmic.200300428
1097
Review
Kenji Miura
Fuji Photo Film,
Tokyo, Japan
Imaging technologies for the detection of multiple
stains in proteomics
Laser-based scanners and charge-coupled device (CCD) camera systems are evolving
to have greater functional capabilities for capturing images from a range of staining technologies used in gel electrophoresis and electroblotting. Digitizing Coomassie Brilliant
Blue (CBB) stained gels and silver stained gels has now become possible using a laserbased gel scanner, the FLA-5000 fluorescent image analyzer system. Also, a simultaneous dual fluorescent imaging function has been incorporated into the FLA-5000
system, utilizing dichroic mirrors with both the optical system and the emission filter. In
the workflow of routine proteomics research, the relationship between SYPRO dye staining and fluorescent detection using the FLA-5000 system have become symbiotic. Additionally in many cases, subsequent staining of the gel with CBB is useful for future research, and thus imaging instruments should be able to handle both staining formats.
Digitizing the CBB stained gel can now be easily performed by the FLA-5000 fluorescent
image analyzer system using a fluorescent board as an epi-illumination background. A
cooled CCD camera system has the potential of imaging not only chemiluminescent
membranes but also digitizing molecular weight markers and fluorescent detection of
SYPRO dye-stained gels. With Multi Gauge software version 2.0 it is now a simple task
to combine two images into one, as commonly required in dual detection experiments.
The LAS-3000 system was designed to capture chemiluminescent images and to digitize
the images automatically. Thus, new capabilities added to gel imaging systems make
them capable of detecting and displaying multiple signals more conveniently.
Keywords: Cooled charge-coupled device camera / Fluorescent image analyzer / Luminescent
image analyzer / Multiple staining / Scanner / Review
PRO 0428
Contents
1
2
3
4
5
6
7
8
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparison of the laser-based scanner and
the CCD camera system . . . . . . . . . . . . . . . . . .
Logarithmic and linear conversion of data . . . .
Multiple fluorescence detection and digitizing
of stained gels by the FLA-5000 system . . . . . .
Use of the FLA-5000 system in proteomics
research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Imaging using the LAS-3000 cooled CCD
camera system . . . . . . . . . . . . . . . . . . . . . . . . . .
The future of imaging . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Introduction
1097
1099
1101
1103
1104
1106
1107
1108
Correspondence: Kenji Miura Ph.D., Science Systems Group,
Industrial Materials and Products Division, Fuji Photo Film Co.
Ltd., 2-26-30, Nishiazabu, Minato-ku, Tokyo, 106-8620, Japan
E-mail: kmiura@tokyo.fujifilm.co.jp
Fax: +81-3-3406-2158
Abbreviation: LED, light-emitting diode
ã 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Imaging systems have become more sophisticated in
recent years, developing from single to multiple function
instruments [1±3]. For example, a fluorescence scanner
system, such as the FLA-3000 instrument (Fuji Photo
Film, Tokyo, Japan) has both fluorescence detection capabilities and radioisotope detection capabilities, when
used with phosphor imaging plates. For many proteomic
applications, however, the detection of CBB and silver
stained gels is often necessary, and to date fluorescence-based laser scanners have not been able to image
these gels. Thus, a methodology for the imaging of CBB
and silver stained gels has been investigated and new
functions have been incorporated into the FLA-5000 system for the first time (Fig. 1). This imaging methodology
utilizes a fluorescent board as an epi-illumination background (Fig. 2). The stained gel is placed onto the glass
platen of the Fluor-stage, the board is placed onto the
gel, the stage is slid into the scanner. It is lowered and
scanning is performed. For CBB stained and silver stained gels the optimal excitation source is the 532 nm laser.
0173-0835/03/0707±1097 $17.501.50/0
1098
K. Miura
Proteomics 2003, 3, 1097±1108
Figure 1. FLA-5000 fluorescent
image analyzer. Three types of
removable stages, namely IPstage, Fluor-stage and Multistage are available. The imaging
area is 40646 cm.
Figure 2. Principle of digitization using a fluorescent board.
The sample can be any kind of
stained gel, film or other media,
having absorbance at the excitation and emission wavelengths.
Also, multiwavelength excitation and detection functions
have been incorporated into the mechanics of the FLA5000 system utilizing dichroic mirrors. The excitation laser
beams pass through the dichroic mirrors and follow the
same optical path (Fig. 3). The different emitted fluorescent signals of the multiple fluorophores are partitioned
by the dichroic mirror to either the first photomultiplier
tube (PMT) or to the second PMT (Fig. 4). Since the FLA5000 unit is a modular system, various types of configurations can be specified by the customer. If only one PMT is
incorporated into the unit, the images can be acquired
consecutively (Fig. 5) but if the instrument is equipped
with two PMTs the two images can be acquired at the
same time (Fig. 6).
The CCD camera-based imaging systems are also capable of capturing fluorescent signals and performing
image digitization, if equipped with a proper light source
and emission filters. A cooled CCD camera system
enables long exposure times to capture weak luminescent signals. For example, the detection of chemiluminescent signals in Western blotting, Southern blotting and
Figure 3. Schematic diagram of the excitation lasers'
optical path in the FLA-5000 instrument. Up to four lasers
can be installed inside the instrument.
Northern blotting is conveniently and efficiently achieved
using the LAS-3000 cooled CCD camera system (Fig. 7)
This system is equipped with a newly developed F0.85
Proteomics 2003, 3, 1097±1108
Imaging technologies for the detection of multiple stains
1099
images consecutively. The CCD camera used in the LAS3000 instrument utilizes a 3.2 million pixel octagonallyshaped CCD at the size of advanced photo system (APS)
film size [4].
In the imaging of 2-DE gels, as often required for the first
step of proteomics research, a scanner system such as
the FLA-5000 instrument is probably the best choice.
The imaging of chemiluminescent signal from Western
blots, as part of the functional analysis of proteins, is
best achieved using a highly efficient cooled CCD camera
system such as LAS-3000 (Fuji Photo Film). The characteristics of these two types of systems are discussed in
greater detail below.
Figure 4. Schematic diagram of the filter unit in the
FLA-5000 instrument. This capability allows for simultaneous detection of two fluorophores, such as Cy3 and
Cy5 dye.
lens, a filter wheel (Fig. 8), and various light sources such
as blue light-emitting diode (LED) and white light LED
for epi-illumination, as well as UV and white LED transilluminators for transillumination. A new function of ªImage
acquire and digitizeº was instituted to capture the chemiluminescence signals generated in luminol-based Western blotting and the digitized molecular weight marker
2 Comparison of the laser-based scanner
and the CCD camera system
Laser-based scanner and CCD camera systems are
the two most widely used imaging methods employed
in modern biomedical research laboratories [1±3]. The
methodological principles that ensure the optimal basic
performance of these two systems as analytical systems
differ, as summarized in Table 1.
Sensitivity is often defined as the detection limit at S/N = 2.
So, the reduction of noise level is important to increase
the S/N and lower the detection limit. In scanners, the
Figure 5. The image reader screen for ª1-Laser 1-Image Cyclicº settings. Up to four image captures
can be performed consecutively according to the set conditions.
1100
K. Miura
Proteomics 2003, 3, 1097±1108
Figure 6. The image reader screen for ª2-Laser-2-Image-Cyclicº settings. A second PMT and the
appropriate filter is required for this function to be operative.
Table 1. Comparison of the scanner and CCD camera
systems
Criteria
Scanner system
CCD camera
system
Sensitivity
High sensitivity by
using PMT, but
trade off with
resolution
High sensitivity
with long time
exposure. Cooling is necessary
to decrease
noise
Resolution
Primarily defined by
the pixel size set
by the software
when reading
Defined by the
sample size and
pixel numbers of
CCD
Quantitative Suitable because of
analysis
even excitation light
intensity over the
whole area
Dynamic
range
Dark frame and
flat frame correction is necessary
Depends on the PMT's Depends on the
CCD's pixel size
performance
,4 to 5 digits
,5 digits
A/D conver- Logarithmic conversion Linear response in
sion
shows more details
nature
at lower range
Figure 7. LAS-3000 luminescent image analyzer. USB
connection to PC or Mac is used for controlling the system and capturing images.
Proteomics 2003, 3, 1097±1108
Imaging technologies for the detection of multiple stains
1101
size is defined by the number of pixels of the CCD chip
and the imaged sample size. So, the sample size in the
image can be easily changed by modifying the distance
between the CCD camera and the sample or by zooming
the lens. For image analysis, a ruler should be imaged
under the same conditions as the sample for accurate
calibration purposes.
Figure 8. Specially designed F0.85 high sensitivity lens
with remote focus and remote iris function and the filter
wheel. Up to 5 filters of 77 mmf can be set. An F-mount
Nikon lens can be used instead of the high sensitivity
lens.
stray light from the excitation light source through the filter
can be the main source of background noise. Interference
filters have sharp and narrow transmission spectra and
low transmittance at other wavelengths to effectively
reduce the noise. However, in the actual imaging system,
the interference filter reduces not only the background
noise but also the signal. So, long pass filters which transmit all the light having longer wavelength than a defined
value can have higher signal strength and a fairly good
S/N, which is the reason why they are widely employed
in instruments. The amount of light reaching the PMT is
affected by the pixel size. If the pixel size is bigger, the
amount of light is greater and the S/N becomes higher. In
the CCD camera system, noise comes from the CCD
itself. The electronic noise of a CCD camera is reduced
by cooling the unit. A Peltier cooler is often used to cool
the CCD device. Also, heat from the amplifier circuit can
be reduced by stopping it during very long exposure
times, such as with overnight exposures.
Resolution is primarily affected by the pixel size of the
image. So, the pixel size value is often quoted as a representation of the resolution of a system. In the laser scanner system, pixel size is defined by the design of the
machine and the researcher cannot modify it. From the
image analysis point of view, the physical length of a
scanned object is easy to measure in the case of the laser
scanner image. In the case of the CCD camera, the pixel
For quantitative image analysis, the background optical
density of the image should be uniform if the sample has
a flat, even surface, such as can be achieved with a fluorescent plastic board. Scanners are suitable for obtaining
a flat image because the excitation light intensity is
designed to be the same at every position in the imaging
area. In the CCD camera system, the light is collected
through a lens, which inevitably causes a phenomenon
where the imaged field has a brighter center and darker
surrounding perimeter. Furthermore, in the case of fluorescence and digitizing, the incident light (light box) is
another cause of unevenness in the CCD camera system.
Using the flat frame correction function compensates for
these artifacts.
The dynamic range of scanners is defined by the performance of the PMT and the A/D conversion circuit. Logarithmic conversion and linear conversion are defined by
the device used and the circuit design. The dynamic
range of CCD cameras are primarily defined by the physical size of the pixels. The data conversion of a CCD chip
is linear in nature.
3 Logarithmic and linear conversion of
data
Precision of the data can be defined by the data conversion in the case of laser scanners. As shown in Fig. 9, the
data of five digits from 0.1 to 10 000 can be converted to
8-bit (256 levels) gray levels by either logarithmic conversion or linear conversion. It is the same in principle when
working with a 16-bit image. In the case of logarithmic
conversion, dividing the 0.1 to 10 000 by 256 steps means
the first, second and the third data points are 0.1, 0.1046
and 0.1094, respectively. In the case of linear conversion,
the first, second and third data points are 0, 39 and 78.
The value of 39, which is the second gray level in the linear
conversion series is the 133rd gray level in the logarithmic
conversion. The logarithmic conversion is superior in describing the low level signals near background. In the
FLA-5000 instrument, both logarithmic conversion types
of data and linear conversion tagged-image file format
(TIFF) files can be generated. Logarithmic data is stored
using Fuji file format, having a combination of file name
xxx.img, which is the digital data of the image itself and
1102
K. Miura
Proteomics 2003, 3, 1097±1108
Figure 9. Explanation of logarithmic conversion and linear
conversion for the FLA-5000
instrument. In this figure, the
relationship between the gray
scale in the X-axis and the gray
levels between black and white
on the Y-axis is shown as a linear relationship.
Figure 10. Fluorescent image
of a SYPRO Ruby dye stained
2-DE gel taken using the logarithmic conversion file format.
Details of very low level spots
with intensity values near background can be observed.
the xxx.inf file, which is the information regarding the
image. A SYPRO Ruby dye (Molecular Probes, Eugene,
OR, USA) stained 2-DE gel was imaged by the FLA-5000
instrument and the generated images were compared
for logarithmic conversion (Fig. 10) and linear conversion
(Fig. 11). The histograms of the data by both conversions
are shown in Fig. 12. If one examines the spots in the
images that represent lower concentrations of proteins,
it is readily apparent that the logarithmic conversion displays more spots near the background density.
Proteomics 2003, 3, 1097±1108
Imaging technologies for the detection of multiple stains
1103
Figure 11. Fluorescent image
of a SYPRO Ruby dye stained
2-DE gel taken using the linear
conversion TIFF file. The image
is contrast controlled to show
as many of the lowest intensity
spots as possible.
Figure 12. Comparison of the histograms of the images
in Fig. 10 (logarithmic; upper) and Fig. 11 (linear; lower).
The vertical lines show the upper and lower limit of the
contrast control.
4 Multiple fluorescence detection and
digitizing of stained gels by the FLA-5000
system
As discussed in Section 1, the FLA-5000 scanner not only
has fluorescence detection capabilities but also other
functions such as the ability to digitize CBB or silver
stained gels. The principle of the digitization is based
upon using a fluorescence board positioned to the upperside of the gel. Imaging is achieved by using the fluorescence of the board as background and the decrease in
the fluorescence arising from the opacity of the stain is
measured as signal. Then, the grayscale image data is
inverted to show high signal for the stained regions and
low signal for the background. The fluorescence detection is utilizing the laser light source as the excitation light.
Multiple lasers are used in the FLA-5000 for excitation, as
shown in Fig. 3. In such an optical system, simultaneous
multiple wavelength excitation can be performed. When
performing simultaneous multiple wavelength detection,
the optical system must have multiple PMT detectors. In
Fig. 4, the emission light from two fluorophores, excited
by two wavelengths of light are detected separately by
dividing the emission light using dichroic mirrors. In this
case, PMT1 and PMT2 have different characteristics of
sensitivity to the wavelengths and the sensitivity to longer
wavelength is higher with PMT2. So, the dichroic mirror
used here reflects longer wavelength and transmits
shorter wavelength. A gel electrophoresed with three prelabeled proteins, such as carbonic anhydrase labeled
with FITC, BSA labeled with Cy3 and lysozyme labeled
with Cy5 were imaged by the FLA-5000 instrument using
the dual wavelength excitation and detection function.
Images of Cy3-BSA (Fig. 13) and Cy5-lysozyme (Fig. 14)
are shown. The image reader software of the FLA-5000
system can show either separate images or a superimposed image of the two channels. Furthermore, the
1104
K. Miura
Figure 13. Cy3 dye-labeled BSA imaged with the FLA5000 instrument using the ª2-Laser-2-Imageº mode. The
amount of BSA-Cy3 applied was 0, 62.5, 31.3, 15.6, 7.8,
3.9, 2.0, 1.0, 0.5 and 0 ng from left to right.
Proteomics 2003, 3, 1097±1108
Figure 15. Superimposed image of Fig. 13 and Fig. 14
created by applying green and red pseudo-color to each
image. MultiGauge V2.0 software was used to produce
the image.
5 Use of the FLA-5000 system in proteomics
research
Currently, the most widely accepted strategy towards
proteomic analysis of gels is schematically depicted in
Fig. 16. The first step of the process is separating the
proteins by 2-DE and detecting the proteins using a gel
scanner after staining with CBB, silver, SYPRO Ruby,
Figure 14. Cy5 dye-labeled lysozyme imaged with the
FLA-5000 instrument using the ª2-Laser-2-Imageº mode.
The amount of lysozyme-Cy5 applied was 0, 356.0, 178.0,
89.0, 44.5, 22.3, 11.1, 5.6, 2.8 and 0 ng from left to right.
contrast control of the two images can be adjusted separately during imaging. The resulting images can then be
pseudocolored and composed into one image using the
Multi Gauge V2.0 software (Fig. 15).
Figure 16. Schematic diagram outlining the standard
processes employed in routine proteome analysis.
Proteomics 2003, 3, 1097±1108
Imaging technologies for the detection of multiple stains
SYPRO Orange or some other fluorescent stain [2±3]. The
selected spots are then further analyzed by Edmanbased amino acid sequencing or by a mass spectrometry
method, such as MALDI-TOF MS. However, silver staining is not very suitable for MALDI-TOF MS or amino acid
sequence analysis. The ideal staining method for such
purposes uses either CBB or SYPRO dye stains. SYPRO
dye stains are known to have high sensitivity similar to
silver stains [2, 3]. However, the SYPRO dye stained spots
are invisible to the unaided eye and require a fluorescent
scanner to perform imaging for visualization. If the gel is
to be dried and kept for further analysis in the future, the
relationship between the SYPRO dye stain and the actual
gel should be able to be traced. For this purpose, CBB
staining after SYPRO dye staining is often suitable. The
gel can be used for further analysis by MALDI-TOF MS
1105
even after CBB staining and drying of the gel. The relatively poor sensitivity of the CBB stain can be compensated for by using higher concentrations of the protein in
the sample solution or by using the more abundant spots
in the CBB stained gels as reference points to excise the
less abundant ones, using the original SYPRO image as
a reference template. In this way, the targeted spot in
the fluorescence image can be traced using multiple
reference marker spots from the CBB stained gel. As an
example, the Sake yeast sample was electrophoresed
according to the method mentioned in the Fuji Application
Note No. 10 [5]. The SYPRO Orange stained gel was
imaged at excitation wavelength of 473 nm (Fig. 17) and
after CBB staining was subsequently imaged by the digitizing function of the FLA-5000 instrument at the excitation wavelength of 532 nm (Fig. 18). Furthermore, a silver
Figure 17. A wet 2-DE gel
stained with SYPRO Orange
dye. Imaging was performed
using the FLA-5000 instrument
and the logarithmic file format.
Figure 18. A CBB stained and
dried gel digitized by the FLA5000 instrument. Excitation:
532 nm; emission: O575 (long
pass green filter); voltage:
250 HV.
1106
K. Miura
Proteomics 2003, 3, 1097±1108
Figure 19. Silver stained and
dried gel digitized by the FLA5000 instrument. Excitation:
532 nm; Emission: O575 (long
pass green filter); voltage:
250 HV.
stained and dried gel was imaged using the same conditions as the CBB stained gel, for comparison's sake.
(Fig. 19).
6 Imaging using the LAS-3000 cooled CCD
camera system
Multiple functional capabilities are also the main concept
behind newer cooled CCD camera systems. Not only
chemiluminescence but also fluorescence and digitization are necessary functions for detection of the target
protein and the molecular weight markers on a Western
blotted membrane. The ªImage acquire and digitizeº
function of the LAS-3000 instrument (Fig. 20) allows automatic capturing of the chemiluminescence signal and
digitization of the image. To superimpose the two images,
the new Multi Gauge V2.0 software is equipped with the
compose function. It has a basic density measurement
function suitable for measuring the spots' density when
the region of interest (ROI) is restricted. The Multi Gauge
software is now available only on the Microsoft Windows
PC platform. Another method to visualize the molecular
weight markers can be marking their positions using a
luminescent pen. Recently, a luminescent pen using the
phosphorescent material from Nemoto and company [6]
was made as a trial. This is illustrated for the fluorescent
detection of a SYPRO Ruby dye stained gel by epi-illumination using the blue LED illuminator (Fig. 21).
The binning function is often used in CCD camera systems. This method combines several pixels into one large
assumed pixel to increase the area of a pixel and increase
the sensitivity of the system. In the LAS-3000 system, the
Figure 20. A portion of
the image reader software screen showing the
ªImage acquire & Digitizeº function. This appears only in Lite software with Chemiluminescent mode.
binning function was pursued not only to gather many pixels but also to generate smoother images by extrapolating
from the binned image. The exposure time of a Southern
blot using CDP-star (Applied Biosystems, Foster City, CA,
USA) as a chemiluminescent substrate required 120 s exposure by standard mode but needed only 8 s to capture
the image by the super mode (Fig. 22). The relationship between the binning mode and image resolution are schematically depicted in Fig. 23.
Proteomics 2003, 3, 1097±1108
Imaging technologies for the detection of multiple stains
1107
Figure 21. Fluorescence detection of a SYPRO Ruby dye
stained 2-DE gel using the LAS3000 instrument. Excitation:
Epi-blue LED illumination. Exposure time: 30 s.
Figure 22. Comparison of exposure times using Standard
mode and Super mode (binning). Sample: Slot blotted DNA
on a membrane detected by
CDP-Star chemiluminescent reagent. Exposure time: Standard
mode 120 s (upper image);
Super mode 8 s (lower image).
7 The future of imaging
The development of new fluorophores is welcomed to
expand the usage of the wider wavelength capabilities
of scanners. Newer instruments are being equipped with
the capability to use more excitation wavelengths, as
demonstrated with the four laser FLA-5000 system. The
fourth laser at 670 nm enables imaging of longer wavelength dyes, such as the Alexa Fluor 750 dye (Molecular
Probes, Eugene, OR, USA). We have discovered that by
using 670 nm for excitation with an appropriate emission
filter, the background fluorescence of blotting membranes can be eliminated. The Alexa Fluor 750 dye can
be excited using the 635 nm laser as well, but membrane
fluorescence is still problematic at this wavelength. The
development of laser scanner systems with broader
wavelength capacity that have the ability to image longer
wavelength dyes, as well as the shorter wavelength dyes,
1108
K. Miura
Proteomics 2003, 3, 1097±1108
Figure 23. Explanation of various binning modes. High, Super and Ultra images are processed
by High binning, Super binning and Ultra binning to the same image size as the Standard mode.
An example of a luminescent image of the alphabet in the luminescent ruler is displayed in order
to demonstrate the image size and resolution.
is still on the horizon, and relegated to developments in
the future. Multiwavelength imaging instrumentation plays
a central role in the fluorescence multiplexing technologies
of proteomics research.
The author greatly appreciates Mr. Yoshihiro Yamamoto,
Kiyoo Hirooka and Nobuo Tsutui (Applied Fermentation
Lab, Kyoto Municipal Institute for Industrial Research)
for kind permission to use their samples and images, the
numerous discussions with them were also very useful.
The author appreciates Mr. Hidetaka Yamamura (Technical Frontier Co.) and the staff for offering us their techniques in gel electrophoresis and staining. The author
thanks Ms. Akiko Nagahama and Ms. Makiko Nagashima
(Fuji Photo Film Co., Ltd.) for their assistance and discussions related to various experiments of imaging.
8 References
[1] Miura, K., Electrophoresis 2001, 22, 801±813.
[2] Patton, W., Electrophoresis 2000, 21, 1123±44.
[3] Patton, W., Biotechniques 2000, 28, 944±957.
[4] Yamada, T., Kim, Y.-G., Wakoh, H., Toma, T. et al., IEEE SolidState Circuit, 2000, 35, 110±111.
[5] Yamamoto, Y., Hirooka, K., Tsutui, N., Science Imaging Systems Application Note No.10, Fuji Photo Film Co., Tokyo
1998.
[6] Matsuzawa, T., Aoki, Y., Takeuchi, N., Murayama, Y., J. Electrochem. Soc. 1996, 143, 2670±2673.
Received December 23, 2002
Per informazioni:
IMMAGINI & COMPUTER Snc
Via Don Carlo Riva 4 – Bareggio MI
Tel. 02.90.36.40.90
www.immaginiecomputer.it