Seven-color Fluorescence Imaging of Tissue Samples Based on Fourier Spectroscopy and Singular Value Decomposition

Abstract

Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images.

Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor Quantum Dots

“…In addition, due to the large spectral overlaps between the emissions of dye fluorophores, multicolor imaging requires complex mathematical analysis of the data. 16 Secondly, the emission intensity of organic dyes depends on their environment, and photobleaching imposes a stringent upper limit on the number of photons detectable per molecule. Third, the fast fluorescence emission of the dyes (<5 nsec) coincides with a high degree to short-lived autofluorescence background from many naturally occurring species reducing therefore the signal to noise ratio.…”

Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory

“…For example, biotin-and digoxigenin-conjugated antibodies can be used simultaneously for immune labelling (Tsurui et al, 2000). In addition, tyramide signal amplification, a highly sensitive method allowing the dilution of the first antibodies far beyond the detection limit of fluorescently labelled secondary antibodies, has also been used for this purpose (Brouns et al, 2002).…”

“…To solve this problem, several approaches have recently been proposed (Buchwalow et al, 2005). These methods include: (1) the use of directly labelled primary antibodies (Tsurui et al, 2000); (2) a two-step protocol using two or three primary monoclonal or polyclonal antibodies (Lewis Carl et al, 1993;Negoescu et al, 1994;Shindler and Roth, 1996;Brouns et al, 2002 ); (3) the use of different haptenated primary antibodies (Tsurui et al, 2000;Brouns et al, 2002); (4) the use of specific anti-Ig isotype/subclass secondary antibodies (Tidman et al, 1981;Suzuki et al, 2000;Buchwalow et al, 2005); (5) the use of immune complexes of primary antibody and the Fab fragment of the secondary antibody for staining (Ino, 2004); (6) the use of two or more biotin-conjugated primary antibodies (Ferri et al, 1999). The immune labelling of cell or tissue samples with either of these methods is however not without its limitations.…”

“…However, because of the limitations of the dyes employed in confocal microscopy, only a few studies with four or more colour immune stains have been reported (Buchwalow et al, 2005). A seven-colour imaging technique using mainly directly labelled primary antibodies has been reported for the simultaneous detection of up to seven antigens on thymus tissue (Tsurui et al, 2000). With respect to multi-colour staining at the single-cell level, a four-colour labelling method has been described for the identification of mesenchymal stem cells (Schieker et al, 2004).…”

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

“…Spectral imaging with the ability to unmix multiplexed images is only marginally applied for fluorescence microscopy (Tsurui et al 2000;Siboni et al 2001;Greenbaum et al 2002;Weier et al 2005). Papers describing spectral imaging of bright field tissue samples are rare (Ornberg et al 1999;Ornberg 2001;Levenson and Mansfield 2006).…”