the box and the stage for the gel have to be painted mat black to prevent reflections. ... into the cam- era. At the other end, a male IS-pin D-shell connector to plug.
Technical Report
Video Imaging of Ethidium Bromide-Stained DNA Gels with Surface UV Illumination
Marc Solioz University of Berne, Berne, Switzerland
ABSTRACT .
We describe here the use oj surface UV illumination to record ethidium bromide-stained DNA gels with a video camera. This mode oj illumination allows the use oj a standard video camera equipped with a red jilter and results in a high signal strength. The assembly oj a low-cost video system on this basis is described. It uses the public domain software called Image on a Macintosh® computer and PostScript™ laser printer or a thermal printer to generate hard copies. The setup is sensitive enough to detect 500 pg oj DNA on an ethidium bromide-stained DNA gel. The UV illumination method described here can also greatly improve the sensitivity oj existing video recording equipment.
INTRODUCTION Preparing a permanent record of an ethidium bromidestained DNA gel by Polaroid® instant photography is very costly and of limited use for quantitative interpretation of the data. Therefore, many different systems employing scanners or video cameras for recording such images have become commercially available in recent years. Also, Southerland et al. described an excellent home-made electronic imaging system (4). However, for reasons outlined below, these are very costly solutions to the problem. We therefore devised a setup that can be assembled at considerably lower cost, yet offers a number of advantages. A substantial part of the cost of commercial imaging systems is from the purchase of the software. We thus based our system on the public domain software called Image that was developed for Macintosh® computers and is periodically up1130 BioTechnioues
dated at the National Institutes of Health (NIH) in Bethesda, MD, USA. Other public domain software described below can be used to expand the capabilities for image analysis still further. A second major source of expense is the need for a highsensitivity video camera, usually equipped with a cooled detector and long exposure capability. In the procedure generally used to capture an image of an ethidium bromide-stained DNA gel with a video camera, the gel is laying on top of a UV transilluminator and viewed with the camera from above, as is done in Polaroid instant photography. However, due to the spectral characteristics of a video camera, a background image of the fluorescent tubes of the UV transilluminator can only be prevented by using an interference filter. The narrow bandwidth of such a filter allows only a small fraction of the ethidium bromide fluorescence emission to reach the camera. This dramatic loss of fluorescence intensity has to be compensated for by using a special camera. We therefore devised a surface UV illumination that allows the use of a regular red filter in place of an interference filter. The much stronger fluorescence signal in tum permits the use of an inexpensive video camera and short exposure times (live images). The system described here requires only limited machine shop work to construct a support for the UV light source(s) and to possibly assemble several cables. A UV transilluminator will already be present in a laboratory where DNA gels are run and a Macintosh computer is often available. Under these conditions, the setup described here can be assembled for as little as $2300, while the cost of comparable commercial systems range from $12000 to $20000. COMPONENTS AND ASSEMBLY All the components that are required in addition to a UV transilluminator and a computer are summarized in Table 1, and the suppliers and approximate prices are given. The components are described in detail below.
Support for UV Transilluminators Figure I shows the box-like support for the UV transilluminators. It was constructed from 6-mm PVC panels; however, plywood or metal is equally suitable. A hole in one of the side panels allows access to the interior. The box permits Vol. 16. No. 6 (1994)
Table 1. Supplementary Materials Required for Video Imaging Setup
Item
Supplier
Image software
NIH
Support for UV transilluminators
home made
Video camera and lens
Cohu
Red filter
photo shops
Frame-grabber card
Scion
Frame-grabber card (alternative)
Data Translation
1295
Cables and plugs (for self-assembly)
computer shops
20
Cable to frame-grabber card (optional)
Approximate Cost($) free
60 1300 20
Computer Image software requires a Macintosh with at least 4 megabytes of memory and a monitor able to display 256.colors or shades of gray. There are different versions of Image for computers with or without a floating-point co-processor (see below). Image runs very well on a Macintosh IIci or any of the current models (such as Centris or Quadra).
A
895
UV transilluminator
Data Translation
125
Second UV transilluminator (optional)
scientific suppliers 1100
Thermal printer (optional)
Mitsubishi
Visible light box (optional)
photo shops
o o
200 r - --
0'
o o o
1500 100
working in room light. If a camera lens different from the one specified here is used, the height of the box should be adjusted so that the red filter is near the camera lens. The position of the UV transilluminator(s) is critical to avoid reflections on the gel surface. The lower surface of the top panel of the box and the stage for the gel have to be painted mat black to prevent reflections. Into the gel stage, a light box can be integrated for imaging stained protein gels or Western blots with transmitted white light.
220
--- ", Gel (6 mm thick)
Stage Box
B
Camera and Filter We use a Cohu 4812 black-and-white, charge-coupled device (CCD) video camera (Cohu, San Diego, CA, USA) equipped with an ALl 6 lens with l6-mm focal length and a manual iris. CCD cameras are available with two types of output signals: RS-170 (US standard) or CCIR (European standard). CCIR signals are preferable since they offer higher vertical resolution (576 vs. 488 picture elements). The sensitivity of this camera is 0.07 lux, but a camera with 30% to 50% of that sensitivity can also be used. The Image software . offers the possibility to sum up individual picture frames, thus simulating longer exposure times. The same type of red filters suitable for Polaroid instant photography of DNA gels are employed. We use a 5x red filter, type 090 (B+W, Bad Kreuznach, FRG). Frame-Grabber Card The Image software supports two different frame-grabber cards for digitizing the video signal for input into the computer: the Scion LG-3 card from Scion Corp. (Frederick, MD, USA) and the QuickCapture Card DT2255 from Data Translation (Marlboro, MA, USA). We used the latter without any particular reason. The cards must be factory-set for either RS170 or CCIR input. Vol II> No I> IIQQ4)
Figure 1. A) Drawing of the cross-section of the box supporting the UV transilluminatoTS. Measurements are given in mrn. The access hole in one side panel is indicated by the dashed line. B) Photograph of the camera setup with the box supporting the UV transilluminators. The right UV transilluminator has been tipped back to expose the inside of the box, where the stage for the gel (actually a small light box for transillumination with visible light) is visible. The DNA gel is placed on a black panel that can be removed. B ioTechnioue.< I 131
Printer It is possible to reproduce the halftone images on a regular laser printer. Best results are obtained with a high-resolution laser printer (600 dots per inch [dpi)), fitted with a signal converter. However, true halftone images of photographic quality can only be generated with a thermal printer. A suitable thermal printer is the Mitsubishi Model P-68 E (Tokyo, Japan). The size of the printed images is 7.5 by 10 cm.
UV Transilluminator Two standard UV transilluminators for DNA gel photography containing six 15-W fluorescence tubes and equipped with a 312-run UV filter plate are used. They are available from many scientific suppliers. For further savings, the setup can be operated with one UV transilluminator only and a polished metal sheet (such as is used for drying conventional photographic paper to obtain glossy prints) as a reflector in piace of the second UV transilluminator. The resultant illumination is only very slightly uneven and suffices for most applications.
Cables The electrical connections of the hardware components for setups with a PostScript™ laser printer or a thermal printer are illustrated in Figure 2. Cable C I is an accessory to the frame-grabber card, but is very expensive (part EP20S) and cables C2 and C3 are nonstandard. All the cables can readily be assembled; the necessary plugs and cables are available in computer supply shops. Cable C I is a standard coaxial monitor cable with a BNC male connector that plugs into the camera. At the other end, a male IS-pin D-shell connector to plug into the QuickCapture card is added. The core of the coaxial cable is connected to pin 8 (video IN - channel I) and the shielding of the cable to the metal housing of the plug. For the connection of the computer's monitor output to the
W
thermal printer (C2), a shielded three-pole cable is fitted with a male IS-pin D-shell plug on the computer side and a male IS-pin HD-sub-D connector that plugs into the "RGB in" input of the printer. Pins 3 (composite H and V sync), 5 (monochrome video) and II (CSYNC ground) on the computer side are connected to pins 13 (Hs/Cs), 2 (G video) and the plug housing (ground), respectively, on the printer side. The shielding of the cable is also connected to the plug housings. The cable that leads from the thermal printer port "RGB out" to the monitor (C3) similarly connects pins 2 and 13 of a IS-pin male HD-sub-D plug on the printer side to pins 5 and 3, respectively, of a female IS-pin D-shell plug that links up to the Macintosh monitor. If a PostScript laser printer is used, the printer and the monitor are connected to the computer in the usual fashion and with standard cables PI and MI, respectively.
Hardware Settings On the QuickCapture card, the jumper W4 (chrominance filter) has to be removed to obtain a sharp image. The thermal printer has to be set to "Analog" data input/output. Other printer settings are chosen according to the printer manual. We found that the quality of prints of DNA gels is optimal if the user-selectable black and white levels of the thermal printer are set to -8 and +26, respectively (see printer manual). No adjustments have to be made on a PostScript laser printer.
Software The NIH Image software (current Version 1.54) can be downloaded by anonymous file transfer protocol (ftp) from zippy.nimh.nih.gov (128.231.98.32). After a connection to this host is established, one can log in with the user name "anonymous" and ones own EMail address as the password. The Image software is found in the directory /pub/image as
12345678910
12345678910
Pos",,"pl Pnme. .....23
P1 :
..... 9.4 _6.6 _4.4
I I
_2. 3 -2.0
I I
._0.56
I
Camera
Computer
Thermal Printer
Monitor
Figure 2. Wiring diagram of the imaging system. The camera output is fed into the frame-grabber card installed in the computer by connection C I. If a tbermal printer is employed, the monitor output of the computer is fed into the thermal printer by connection C2 and then to the computer monitor by C3. Details of the pin connections are given under Components and Assembly. If a PostScript laser printer is used, the standard connections P I and M I are made instead (dashed lines). The various power lines are omitted. I 132 BioTechniques
Figure 3. Polaroid print (left) and video print (right) of the same gel. The lanes contain the following DNA samples: (lanes 1-5) 10,5,2.5, I and 0.5 ng, respectively, of uncut pUC 18 plasmid DNA; (lanes 6-10) 800, 400, 200, 100 and 50 ng, respectively, of !..DNA standard. The numbering on the side of the gel gives the sizes of the ADNA fragments in kbp. VoL Hi.
0
(i
I1QQ4)
file nih-image 154fpu.hqx for computers with a floating-point co-processor or nih-imageI54nonfpu.hqx for Macintosh computers without it. A manual in Word™ format is contained in the file nih-imageI54_docs.hqx. The program can also be obtained by mail at a cost of $100 from National Technical Information Service, 5285 Port Royal Road, Springfield, VA, USA, 22161. Additional public domain software that contains further useful tools for gel analysis is available from the National Center for Supercomputing Application (NCSA), Champaign, IL, USA. It can also be downloaded by anonymous ftp from zaphod.ncsa.uiuc.edu. We are not aware of corresponding free software for the IBM® PC and there are no plans to develop it at the NIH.
Biochemical Methods DNA gels contained 0.6% agarose (type I; Sigma Chemical, St. Louis, MO, USA), 2 Ilg/mL of ethidium bromide (Boehringer Mannheim GmbH, Mannheim, FRG), 40 roM Tris, 20 roM acetic acid, 1 roM EDTA and were run in the same buffer, essentially as described (3). ADNA cut with HindIII was used as DNA size marker. pUCl8 plasmid DNA was prepared by the method of Humphreys et al. (2) and the concentration determined by absorption measurement at 260 nm. RESULTS AND DISCUSSION With all components powered up, the Image program is started and capturing initiated with the menu option "Specials/Start capturing." The focus and the iris of the camera are adjusted manually to yield a sharp picture of the desired brightness on the monitor. Image then offers many features to enhance contrast, adjust the gray scale, produce inverted images, etc. However, we found the default settings to yield the best results. Image offers editing (similar to MacPaint™, Apple Computer) of the images, allowing labeling, enlarging and editing in many ways before an image is sent to the printer or electronically stored as a tagged information fIle format (TIFF) or pictorial (PICT) file for later use. Image provides many tools and macros for data analysis, such as automated particle analysis or measurement of the area, average gray value, and center and angle or orientation of a user-defined region of interest. Density calibration can be done against density standards using user-specified units and eight different curve fitting methods. A macro programming language (similar to PASCAL) incorporated into Image allows automation of complex routine tasks. Image directly supports, or is compatible with, flatbed scanners, film recorders, graphic tablets, PostScript laser printers and photo typesetters. Figure 3 shows a comparison of the hard-copy images of the same DNA gel, recorded with either conventional Polaroid instant photography with a type 664 film or the video system described here. The same sensitivity is obtained by both methods, which allows the detection of 500 ng of DNA in a band. The response of a video camera to light intensity is much more linear than that of photographic film, as long as
the pixels are not saturated. If a color monitor is used, saturated pixels can be visualized in color on the screen. It is thus possible to accurately quantify band. intensities onthe computer screen. Quantitative imaging by video photography has previously been discussed in detail (1,4). The video imaging system described here was originally developed to provide a low-cost alternative to Polaroid instant photography. However, the versatility of the available software expands the range of applications far beyond the preparation of hard copies of DNA gels and should be useful to many laboratories. The setup offers two additional advantages: first, through the high signal strength, the light box arrangement described here does not require the use of a darkroom, and second, the use of surface illumination obviates the need to remove gels from UV-absorbing trays. Finally, the sensitivity of existing video recording equipment can be greatly increased by using the UV illumination described here and replacing the interference fIlter with a standard red filter. ACKNOWLEDGMENTS We thank Ulrich Marti for help with the operation of the Macintosh computer and Catherine Solioz and Thomas Weber for expert technical assistance. This work was supported by Grant 32-37527.93 from the Swiss National Foundation. Polaroid is a registered trademark of the Polaroid Corporation, Cambridge, MA, USA. REFERENCES I.Freeman, S.E.. L.L. Larcom and B.D. Thompson. 1990. Electrophoretic separation of nucleic acids: evaluation by video and photographic densitometry. Electrophoresis 11:425-431. 2.Humphreys, G.O., G.A. Willshaw and E.s. Anderson. 1975. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383:457-463. 3.Maniatis, T., E.F. Fritsch and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,NY. 4.Southerland,J.C.. B. Lin, D.C. Mooteieone,J. Mugavero, B.M. Southerland and J. Trunk. 1987. Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA. Anal. Biocllem. 163:446-457. Address correspondence to: M . Solioz Department of Clinical Pharmacology Murtenstrasse 35 3007 Bem Switzerland Intemet: solioz@ ikp.unibe.ch
