Review Imaging technologies for the detection of multiple stains in
Transcription
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: [email protected] 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