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Spectral Imaging and Linear Unmixing
Spectral imaging and linear unmixing is becoming an important staple in the microscopist's toolbox, particularly when applied to the elimination of autofluorescence and for FRET investigations. Instruments equipped for spectral imaging are becoming increasingly popular and many confocal microscopes now offer this capability. Widefield fluorescence and brightfield microscopy are also being used more frequently for resolving complex fluorophore and absorbing dye mixtures, a trend that should continue into the future.
Introduction to Spectral Imaging and Linear Unmixing - Spectral imaging combined with linear unmixing is a highly useful technique that can be used in combination with other advanced imaging modalities to untangle fluorescence spectral overlap artifacts in cells and tissues labeled with synthetic fluorophores that would be otherwise difficult to separate.
FRET Microscopy with Spectral Imaging - In FRET applications, spectral imaging relies on excitation of the donor alone, followed by acquisition of the entire emission spectrum of both the donor and acceptor fluorescence. Spectral imaging is very useful in the examination of fluorescent protein FRET biosensors.
Practical Considerations for Spectral Imaging - Among the most important considerations for spectral imaging and linear unmixing is to note that the success of this technique depends upon a number of factors that are within the control of the microscopist. This section outlines critical aspects and artifacts with spectral imaging and linear unmixing.
Interactive Flash Tutorials
Additive Properties of Emission Spectra - This interactive tutorial explores how multiple spectra can be added to produce a composite emission spectrum similar to those encountered in spectral imaging of specimens labeled with multiple fluorophores.
Spectral Imaging with Linear Unmixing - Explore how mixed fluorophores having highly overlapping emission spectra can be separated into individual components using spectral imaging and linear unmixing techniques. This tutorial contains several examples with fluorophores emitting in the green and red spectral regions.
Emission Fingerprinting with Lambda Stacks - Use this tutorial to examine how lambda stacks can be used to extract information about individual spectral profiles in specimens labeled with highly overlapping fluorophores.
LSM 700 Light Pathways - The LSM 700 laser scanning confocal microscope from Carl Zeiss is designed for efficient separation of signals by efficient splitting of the emission using the variable secondary dichroic (VSD) beamsplitter to prevent crosstalk and enable spectral imaging as well as linear unmixing of highly overlapping fluorophores.
Spectral Imaging FRET with Biosensors - Spectral Imaging FRET with Biosensors - Spectral imaging of FRET biosensors using fluorescent proteins is an emerging technique for the analysis of events in cell biology. This tutorial explores the performance of a cameleon calcium biosensor and a caspase apoptosis indicator in spectral imaging.
Fluorescent Protein FRET Biosensors - Spectral imaging has been very useful for the examination of fluorescent protein biosensors to determine the presence or absence of FRET in response to a biological stimulus.
3-Channel QUASAR Detection Unit - The ZEISS QUASAR photomultiplier detection technology is based on a filter-free system that guides the desired wavelength range to the target detector using adjustable optical wedges and slider light stops.
34-Channel QUASAR Detection Unit - Employing a special 32-channel photomultiplier, the ZEISS multichannel QUASAR detection unit is ideal for enhancng lambda stack acquisition speed for live-cell imaging experiments.
References and Resources
Spectral Imaging and Linear Unmixing - The technique of spectral imaging coupled to linear unmixing can significantly aid in the interpretation of images. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spectral imaging.
FRET with Spectral Imaging and Linear Unmixing - FRET with Spectral Imaging and Linear Unmixing - Spectral imaging FRET enables gathering of the entire fluorescence spectrum for deconvolution of distinct shapes of the spectra rather than simply monitoring emission intensity in a limited bandwidth region using a filter.
Contributing Authors
Mary E. Dickinson - Department of Molecular Physiology and Biophysics, One Baylor Plaza BCM 335, Baylor College of Medicine, Houston, Texas, 77030.
Rene Hessling - Division of Microscopy, Carl Zeiss, Ltd., P.O. Box 78 Woodfield Road, Welwyn Garden City, Herts, AL7 1LU, United Kingdom.
David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 702 Light Hall, Nashville, Tennessee, 37232.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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