BENCHMARKS G418-resistant colonies (Figure 2). The murine B-16 cell line showed a 108% increase, and the 4T1 cell line had a 52% increase in stable colonies. It is important to note that heat treatment of 42°C for 10 min is mild in nature with treated cells, maintaining a viability of over 90% after heat treatment as measured by trypan blue staining (data not shown). In conclusion, we report an empirically observed phenomenon of enhanced stable plasmid integration in neoplastic cells by a brief (10 min) and mild heat treatment (42°C) following DMRIE-C lipid transfections. A similar phenomenon is seen in bacterial transformations where a brief (90 s) 42°C heat treatment increases transformation efficiencies (11). We have not studied the mechanisms that affect the increased integration rates, but it is clear from our data that heat is affecting the cell at two different levels. First, the increase in GFP-positive cells suggests an increase in the number of cells that had taken up the plasmid, possibly by affecting fluidity of the cell membrane (12). Secondly, the increased stable integration rate indicates that in more cells the DNA was able to cross the nuclear membrane and integrate into the chromosome, possibly due to a change in fluidity in the nuclear membrane or a change in chromatin structure (5), thus allowing the plasmid greater access to the chromatin. This technique could have potential in vitro applications for laboratories routinely using lipid-mediated transfections. The effect of heat on other types of lipidmediated transfections remains to be determined. ACKNOWLEDGMENTS
The authors would like to thank Marc Friedman for initial work on transfection characterization and Barb Carolus for help with flow cytometry. COMPETING INTERESTS STATEMENT
The authors declare no competing interests. 52 BioTechniques
REFERENCES 1.Felgner, P.L., T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, G.M. Ringold, and M. Danielsen. 1987. Lipofection: a highly efficient, lipid mediated DNA—transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413-7416. 2.Felgner, J.H., R. Kumar, C.N. Sridhar, C.J. Wheeler, Y.J. Tsai, R. Border, P. Ramsey, M. Martin, and P.L. Felgner. 1994. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269:2550-2561. 3.Zhdanov, R.I., N.G. Kutsenko, O.V. Podobed, O.A. Buneeva, T.A. Tsvetkova, S.O. Guriev, T.P. Lavrenova, G.A. Serebrennikova, et al. 1998. New cationic liposomes for transfection of eukaryotic cells. Dokl. Akad. Nauk. 362:557-560. 4.Liu, D, T. Ren, and X. Gao. 2003. Cationic transfection lipids. Curr. Med. Chem. 10:1307-1315. 5.Stevens, C.W., M. Zeng, and G.J. Cerniglia. 1996. Ionizing radiation greatly improves gene transfer efficiency in mammalian cells. Hum. Gene Ther. Sep. 7:1727-1734. 6.Stevens, C.W., G.J. Cerniglia, A.R. Giandomenico, and C.J. Koch. 1998. DNA damaging agents improve stable gene transfer efficiency in mammalian cells. Radiat. Oncol. Investig. 6:1-9. 7.Frost, E. and J. Williams. 1978. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue.
Virology 91:39-50. 8.Stow, N.D. and N.M. Wilkie. 1976. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33:447-458. 9.Luthman, H. and G. Magnusson. 1983. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11:1295-1308. 10.Goldstein, S., C.M. Fordis, and B.H. Howard. 1989. Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate. Nucleic Acids Res. 17:3959-3971. 11.Sambrook, J., E. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. CSH Laboratory Press, Cold Spring Habor, NY. 12.Vigh, L., B. Maresca, and J.L. Harwood. 1998. Does the membrane’s physical state control the expression of heat shock and other genes? Trends Biochem. Sci. 23:369-374.
Received 26 September accepted 25 August 2004.
2003;
Address correspondence to David T. Harris, Department of Microbiology and Immunology, Bldg. #90, Main Campus, University of Arizona, Tucson, AZ 85721, USA. e-mail:
[email protected]
Color image acquisition using a monochrome camera and standard fluorescence filter cubes Gregory F. Weber and A. Sue Menko Thomas Jefferson University, Philadelphia, PA, USA BioTechniques 38:52-56 (January 2005)
James Clerk Maxwell first demonstrated in the 1860s that all colors could be separated into three main components: red, green, and blue (1). He was able to produce color images by first taking a series of black and white photographs through red, green, and blue filters of an object illuminated by white light. The resulting images were then simultaneously projected through the color filters used in the acquisition process, and the combined image was seen in full color. These findings ultimately led to the development of trichromatic (RGB) color photography and imaging.
For a number of reasons, monochrome imaging has remained the preferred method of acquisition for fluorescence microscopy (2). Perhaps most important is the fact that monochrome cameras can achieve higher spatial resolution than color cameras resulting from simultaneous usage of all chargecoupled device (CCD) photodiodes for the acquisition of one image without color mosaics and the extrapolation of color information (3). Monochrome cameras also have increased sensitivity and acquisition speed, because transmitted light is not diminished by Vol. 38, No. 1 (2005)
BENCHMARKS filters necessary for color acquisition. When a color camera is used to acquire fluorescence images, the emitted light is filtered through the appropriate fluorescence dichroic mirrors and filters, following which it must pass through a second set of dichroics to achieve the color image. Therefore, color cameras limit the spectral range of the emitted light. And finally, monochrome cameras are generally less expensive than color cameras. Users of fluorescent microscopes have often had a need for taking pictures of tissue sections stained with traditional histological dyes, such as hematoxylin and eosin, to compliment their studies or to superimpose immunofluorescence with colorimetric immunolocalization. For many scientists, however, the costs and logistics of setting up digital color imaging equipment in addition to their monochrome acquisition equipment have outweighed the benefits. We have designed a simple way of converting a standard fluorescence microscope outfitted with a monochrome camera into a color acquisition platform for samples stained with histological dyes. Using Maxwell’s theory of color composition, we used the existing red, green, and blue fluorescence filter cubes in our microscope to produce high-quality color images from our digital monochrome camera. Most fluorescent microscopes are outfitted with filter sets for obtaining images of red (rhodamine or Texas Red®), green [fluorescein-5-isothiocyanate (FITC)], and blue [4′,6-diamidino-2-phenylindole (DAPI)] fluorochromes, which we have adapted in this technique for acquisition of color images. In establishing this new technique, we have used our Nikon® Eclipse 80i microscope (Optical Apparatus, Ardmore, PA, USA), which contained filter cubes with the specifications as detailed in Table 1. A Model C4742-95 monochrome digital camera (Hamamatsu, Bridgewater, NJ, USA) was attached to the rear port for image acquisition through Metamorph version 6.2 software package (Universal Imaging, Downingtown, PA, USA). For this study, hematoxylin and eosinstained transverse sections of human jejunum were viewed using a halogen 54 BioTechniques
Table 1. Specifications of the Fluorescent Filter Cubes Used to Obtain Color Images Color
Filter Cube
Fluorochrome
Excitation (λ)
Dichroic Mirror (λ)
Emission (λ)
UV2EC
DAPI
340–380
400 LP
435–485
Green
HQ41001
FITC
460–500
505 LP
510–560
Red
HQ41004
Texas Red
533–588
595 LP
608–683
Red
HQ41002
TRITC
510–560
565 LP
573–648
Blue
DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein-5-isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; LP, wavelength characteristics of a long pass filter.
white light source. In order to acquire including sections of human ear pinna the digital color image, we took three that were stained with aldehyde fuchsin pictures, each with a different filter and then counterstained with ponceau cube in place: red, green, and blue. The de xylidine, acid fuchsin, and fast green auto-expose feature in Metamorph was (Figure 1, C and D). We found that our used to obtain proper color balance. method produced color images that The resulting three images were then were comparable to those acquired on a combined using the color combine color camera for all staining techniques feature in the Metamorph software. that we have tested. This feature creates a 24-bit image Although the negative aspects of our from three 8-bit images by assigning described process are minimal, we feel each image to a different color channel. that any users of this method should Alternatively, the layer blending be aware of the potential limitations. options feature of Adobe® Photoshop® Using the fluorescent filter sets, certain software could be used to assign the wavelengths will not be acquired because monochrome images to the appropriate the dichroic mirrors and emission filters color channels. The picture taken with the blue (DAPI) filter set was assigned to the blue channel, the picture taken with the red (Texas Red) filter set was assigned to the red channel, and the picture taken through the green (FITC) filter set was assigned to the green channel. For comparison purposes, we acquired pictures of the same sample with an Optronics DEI-750 color camera (Optronics, Goleta, CA, USA). As Figure 1, A and B, illustrates, the image acquired through our new method is a near-perfect color Figure 1. Three histological samples stained with different dyes image and compares were used to assess the color attributes of images obtained through favorably with the our described method. The test samples included: (A and B) hemaimage acquired with toxylin and eosin (H&E)-stained transverse sections of human jejunum; the color camera. We (C and D) human ear pinna stained with aldehyde fuchsin, ponceau de xylidine, acid fuchsin, and fast green; and (E and F) a human vein that have further tested our was Verhoff and van Gieson-stained. Images were acquired using either technique with various a monochrome camera with fluorescent filter sets (panels A, C, and E) histological samples, or a color camera (panels B, D, and F). Vol. 38, No. 1 (2005)
BENCHMARKS in the filter cubes are designed to limit emission spectra to avoid overlapping fluorescent signals. With our filter sets however, we have found this to be small, only as high as 48 λ, and mostly in the yellow/orange range. Regardless, we were still able to acquire exceptional images of a Verhoff-van Gieson preparation of human vein sections (Figure 1, E and F). The specific wavelength parameters for the individual filter sets became most apparent when we compared images acquired through our two different red filter cubes. The monochrome image acquired through the Texas Red filter has greater contrast than what is achieved with the rhodamine filter. As a result, the Texas Red filter yields a picture with a slightly redder tint than the rhodamine filter set. However, we found the wavelength parameters had no more affect on color balance than any of the existing variabilities with color photography including film type, camera, colorimetric stains, and acquisition settings. We have described a method for color image acquisition through the use of standard fluorescent filters and a monochrome camera. This technique allows users with a fluorescence microscope to acquire high-quality digital color images from histological samples without a color camera. Until now, this has required the purchase of a costly color camera system or specialized filter sets for use with a monochrome camera. These filters function using similar principles to the method that we described here, which transforms the filter sets already available to most fluorescence microscope users into agents to provide color imaging of histologically stained samples. We are confident that this technique will be embraced by many scientists as an inexpensive method to obtain color imaging of their histological samples. ACKNOWLEDGMENTS
We would like to thank Dr. Nancy Philp for the use of her Optronics color camera. These studies were supported by National Institutes of Health (NIH) grants EY10577 and EY014258 to A.S.M. G.F.W received support from NIH Training grant ES007282. 56 BioTechniques
COMPETING INTERESTS STATEMENT
The authors declare no competing interests. REFERENCES 1.Mahon, B. 2003. The Man Who Changed Everything: The Life of James Clerk Maxwell. John Wiley & Sons, Ltd. Chichester, West Sussex, UK. 2.Inoue, S. and K. Spring. 1997. Video Microscopy, The Fundamentals, 2nd. ed. Plenum Publishing, New York. 3.Matsumoto, B., K. Linberg, and R. Cran-
dall. 2004. Colorful fluorescence stands out in black and white. Biophotonics International 11:36-39.
Received 21 July 2004; accepted 16 August 2004. Address correspondence to Sue Menko, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 571 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA. e-mail: sue.
[email protected]
Cleaning microsatellite PCR products with Sephadex™ in 96-well filtration plates enhances genotyping quality Delbert W. Hutchison1, Jared L. Strasburg2, and Christopher Shaffer2 1Whitman
College, Walla Walla, WA and 2Washington University, St. Louis, MO, USA
BioTechniques 38:56-58 (January 2005)
Automatic sequencing machines have greatly improved our ability to accumulate nucleotide sequence data. To enhance efficiency, sequencing reactions are routinely subjected to gel filtration through a porous particulate matrix prior to loading to remove salts, unincorporated nucleotides, and dye terminators that can interfere with electrophoretic dynamics and laser function. Such filtering is done either by column (1) or use of 96-well microplates for greater throughput (2). Microsatellite genotyping uses the same automatic technology to assess polymorphism. Interestingly, however, there is no corresponding general tendency to clean microsatellite PCR products prior to genotyping, even though filtration would likely improve genotyping efficiency. Some workers do use Sephadex™ columns (Amersham Biosciences, Piscataway, NJ, USA) to clean PCR products before genotyping, especially when using fluorescently labeled dNTPs (e.g.,
http://www.mnh.si.edu/GeneticsLab/ TechnicalPage/Protocols/Microsat/ MicroWorkshop.pdf). Unfortunately, this technique is not widely known, it does not apply to fluorescently labeled primers, and does not favor the highthroughput systems in use today. Our own efforts to run unfiltered multiplexed microsatellite PCR products on a BaseStation™ DNA Fragment Analyzer (MJ Research, Reno, NV, USA) are often frustrating. Many gels exhibit an inward bending of lanes, especially in the outer regions, due to excess salts interfering with electrophoresis. This makes it difficult to identify lanes, thus increasing the likelihood of improperly assigning fragments to neighboring individuals. Furthermore, the analysis software is often unable to accurately detect and/or size fragments due to interference from excess signal associated with unincorporated fluorescent dyes (Figure 1A). We therefore tested whether inclusion of a filtration step, analogous to what is Vol. 38, No. 1 (2005)
