Spectral overlap in specimens labeled with synthetic fluorophores and fluorescent proteins can often lead to analysis artifacts when interpreting images. The technique of spectral imaging, which involves gathering incremental emission lambda stacks, coupled to linear unmixing can significantly aid in the interpretation of images and in FRET measurements. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spectral imaging.
Zimmermann, T., Rietdorf, J. and Pepperkok, R.
Spectral imaging and its applications in live cell microscopy. FEBS Letters 546: 87-92 (2003). The authors provide a concise and well-written review of spectral imaging techniques as applied to fluorophores with highly overlapping emission spectra in living cells. Discussed are the recent progress on the technical implementation of the methods, the limitations, and applications to the imaging of biological specimens.
Dickinson, M. E., Simbuerger, E., Zimmermann, B., Waters, C. W. and Fraser, S. E.
Multiphoton excitation spectra in biological samples. Journal of Biomedical Optics 8: 329-338 (2003). A review on the application of spectral imaging and linear unmixing to multiphoton microscopy. The authors demonstrate how excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in a similar manner as the emission stacks acquired in laser scanning confocal microscopy.
Hiraoka, Y., Shimi, T. and Haraguchi, T.
Multispectral imaging fluorescence microscopy for living cells. Cell Structure and Function 27: 367-374 (2002). Discussed in this review article are the basic aspects of microscope configurational variations for spectral imaging and the accompanying detection strategies. The authors include several examples of spectral imaging and linear unmixing with living cells expressing fluorescent protein fusions and describe other biological applications, such as FRET.
Lansford, R., Bearman, G. and Fraser, S. E.
Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy. Journal of Biomedical Optics 6: 311-318 (2001). This research report describes spectral imaging and linear unmixing techniques using multiphoton microscopy for three fluorescent proteins (EGFP, ECFP, and EYFP) that are difficult to otherwise resolve spectrally. Included is a discussion of spectral imaging basics, establishing a spectral library, and the analysis of unmixing algorithms.
Lerner, J. M. and Zucker, R. M.
Calibration and validation of confocal spectral imaging systems. Cytometry A 62: 8-34 (2004). The authors describe calibration of laser scanning confocal microscopy systems using a multi-ion discharge lamp (MIDL) that contains mercury, argon, and inorganic fluorophores emitting distinct, stable spectra. Reference spectra from the MIDL data can be used to predict spectral resolution, contrast, and aliasing parameters for spectral imaging instruments.
Zimmermann, T.
Spectral imaging and linear unmixing in light microscopy. Advances in Biochemical Engineering/Biotechnology 95: 245-265 (2005). An excellent review article that discusses the microscope techniques available for spectral imaging and deciphers the theory of linear unmixing. Discussed are the limitations of the methodology and approaches for image optimization, as well as biological applications.
Neher, R. A. and Neher, E.
Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. Journal of Microscopy 213: 46-62 (2003). The authors present a theoretical analysis on the separation of signal contribution from multiple fluorophores in a single sample. Topics addressed are signal-to-noise, spectral detection channel width, photobleaching effects, and the inclusion of fluorescence lifetime information.
Garini, Y., Young, I. T. and McNamara, G.
Spectral imaging: principles and applications. Cytometry A 69: 735-747 (2006). This review focuses on the basic principles of spectral imaging and linear unmixing and describes several representative applications. Included are a summary of variations in spectral imaging system configuration, techniques used to generate lambda stacks, and implementation of the algorithms.
Levenson, R. M. and Mansfield, J. R.
Multispectral imaging in biology and medicine: Slices of life. Cytometry A 69: 748-758 (2006). A comprehensive discussion spectral imaging that focuses on the techniques and specimen range for both fixed samples and in vivo imaging. Among the topics discussed are brightfield microscopy, fluorescence microscopy, image acquisition, hardware, software, and several specific applications.
Berg, R. H.
Evaluation of spectral imaging for plant cell analysis. Journal of Microscopy 214: 174-181 (2004). The author discusses fluorescence imaging at high spectral resolution with linear unmixing as a method to computationally distinguish spectrally similar fluorophores and to remove autofluorescence. Comparisons are made between several commercially available laser scanning confocal microscopes equipped with spectral detectors.