Color Image Acquisition using a Monochrome Camera and Standard Fluorescence Filter Cubes
Gregory F. Weber, A. Sue Menko · BioTechniques · 2005
BioTechniquesVol. 38, No. 1 BenchmarksOpen AccessColor image acquisition using a monochrome camera and standard fluorescence filter cubesGregory F. Weber & A. Sue MenkoGregory F. WeberThomas Jefferson University, Philadelphia, PA, USA & A. Sue Menko*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: E-mail Address: [email protected] Jefferson University, Philadelphia, PA, USAPublished Online:30 May 2018https://doi.org/10.2144/05381BM06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail 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 charge-coupled 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 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 eosin-stained transverse sections of human jejunum were viewed using a halogen white light source. In order to acquire the digital color image, we took three pictures, each with a different filter cube in place: red, green, and blue. The auto-expose feature in Metamorph was used to obtain proper color balance. The resulting three images were then combined using the color combine feature in the Metamorph software. This feature creates a 24-bit image from three 8-bit images by assigning each image to a different color channel. Alternatively, the layer blending options feature of Adobe® Photoshop® software could be used to assign the monochrome images to the appropriate 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 image and compares favorably with the image acquired with the color camera. We have further tested our technique with various histological samples, including sections of human ear pinna that were stained with aldehyde fuchsin and then counterstained with ponceau de xylidine, acid fuchsin, and fast green (Figure 1, C and D). We found that our method produced color images that were comparable to those acquired on a color camera for all staining techniques that we have tested.Figure 1. Three histological samples stained with different dyes were used to assess the color attributes of images obtained through our described method.The test samples included: (A and B) hematoxylin and eosin (H&E)-stained transverse sections of human jejunum; (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 was Verhoff and van Gieson-stained. Images were acquired using either a monochrome camera with fluorescent filter sets (panels A, C, and E) or a color camera (panels B, D, and F).Table 1. Specifications of the Fluorescent Filter Cubes Used to Obtain Color ImagesAlthough the negative aspects of our described process are minimal, we feel that any users of this method should be aware of the potential limitations. Using the fluorescent filter sets, certain wavelengths will not be acquired because the dichroic mirrors and emission filters 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.AcknowledgmentsWe 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.Competing Interests StatementThe authors declare no competing interests.References1. Mahon, B. 2003. The Man Who Changed Everything: The Life of James Clerk Maxwell. John Wiley & Sons, Ltd., Chichester, West Sussex, UK.Google Scholar2. Inoue, S. and K. Spring. 1997. Video Microscopy, The Fundamentals, 2nd. ed. Plenum Publishing, New York.Crossref, Google Scholar3. Matsumoto, B., K. Linberg, and R. Crandall. 2004. Colorful fluorescence stands out in black and white. 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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.Competing Interests StatementThe authors declare no competing interests.PDF download