Clearing Up the Signal: Spectral Imaging and Linear Unmixing in Fluorescence Microscopy

Zimmermann, Timo; Marrison, Joanne; Hogg, Karen; O’Toole, Peter

doi:10.1007/978-1-60761-847-8_5

Cited by 96 publications

(90 citation statements)

References 36 publications

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“…There are many sophisticated tools for spectral unmixing, which include the use of optimization algorithms; however, we focus on a simple mathematical approach to spectral unmixing that is adequate for many applications. A more exhaustive introduction and dissertation on spectral unmixing may be found in [362–366]. …”

Section: Image Renderingmentioning

confidence: 99%

A pragmatic guide to multiphoton microscope design

et al. 2015

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Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope.

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Section: Image Renderingmentioning

confidence: 99%

A pragmatic guide to multiphoton microscope design

et al. 2015

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“…Multicolor intravital TPM can benefit from the use of spectral chips, composed of an array of PMTs and a prism or diffraction grating to separate the fluorescence photons (Im et al 2010; Shi et al 2012, 2015; Zimmermann et al 2014). Spectral segmentation of the wavelengths arising for the different fluorophores will be thereby both more accurate and easier to achieve.…”

Section: Introductionmentioning

confidence: 99%

“…, spectral deconvolution), the contribution of each of the fluorophores used in the experiment in each of the NDD must be measured. Then, using dedicated algorithms based on maximum-likelihood unmixing (Davis and Shen 2007), each image from each NDD can be spectrally unmixed and then analyzed (Thaler and Vogel 2006; Brenner et al 2013; Zimmermann et al 2014). As an example of the crucial importance of spectral unmixing, Ducros et al have used it to detect small spectral variations of odor-evoked FRET transients up to 250 μm in the olfactory bulb of living mice (Ducros et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Ricard

Arroyo

et al. 2018

Brain Struct Funct

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Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

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“…Imaging using conventional bandpass based microscopy is common, where spectral separation is limited to four to five colors that span the ultraviolet (UV) to near-infrared (NIR) spectra. Unique fluorophore detection in a single sample can be improved using spectral imaging and linear unmixing based fluorescence microscopy to simultaneously identify overlapping emission spectra, increasing multicolor imaging capability 1,2 . Fluorescence linear unmixing studies have demonstrated up to five color imaging with overlapping subcellular entities using two excitation wavelengths and emission distributed over 150 nm of spectral space 3 .…”

Section: Introductionmentioning

confidence: 99%

Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

Bittel

Davis

Wang

et al. 2018

Sci Rep

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Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.

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Clearing Up the Signal: Spectral Imaging and Linear Unmixing in Fluorescence Microscopy

Cited by 96 publications

References 36 publications

A pragmatic guide to multiphoton microscope design

A pragmatic guide to multiphoton microscope design

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

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