Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Zucker, Robert M.; Rigby, Paul; Clements, Ian P.; Salmon, W. D.; Chua, Michael

doi:10.1002/cyto.a.20371

Cited by 33 publications

(21 citation statements)

References 36 publications

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“…Light was delivered to the sample with a 60 × Plan Apo 1.4 numerical aperture (NA) objective; the system also uses diode lasers of 404 nm, 488 nm, 561 nm, and 633 nm. Prior to each experiment, the confocal microscope was tested for field illumination alignment, optical efficiency, colocalization, and axial resolution (Lerner and Zucker 2004; Zucker 2006a, 2006b; Zucker and Lerner 2005; Zucker et al 2007); and the lens was inspected and cleaned before use. Cells were exposed sequentially to ZnSO 4 (catalog no.…”

Section: Methodsmentioning

confidence: 99%

An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

Cheng

Tong

Miller

et al. 2010

Environ Health Perspect

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BackgroundThe mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn2+ does not generate reactive oxygen species (ROS) through redox cycling.ObjectiveTo characterize the role of oxidative stress in zinc-induced toxicity.MethodsWe used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells.ResultsZinc treatment in the presence of the Zn2+ ionophore pyrithione of A431 skin carcinoma cells preloaded with the H2O2-specific indicator PG1 resulted in a significant increase in H2O2 production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H2O2 formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts.ConclusionTaken together, these findings show a disruption of mitochondrial integrity, H2O2 formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

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Section: Methodsmentioning

confidence: 99%

An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

Cheng

Tong

Miller

et al. 2010

Environ Health Perspect

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“…Fluorescence imaging is a powerful technique that can identify fluorescent labels (fluorophores) in a variety of biological applications, including in vivo imaging, 1,2 fluorescent protein-and quantum dot-tracking, [3][4][5][6] and clinical imaging. [7][8][9][10] Traditional fluorescence microscopy approaches can detect one-to-several spectral bands using bandpass filters with center wavelengths (CWLs) corresponding to peak emission wavelengths of specific fluorophores.…”

Section: Hyperspectral Imaging With Tunable Filtersmentioning

confidence: 99%

Thin-film tunable filters for hyperspectral fluorescence microscopy

et al. 2013

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Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging-the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes.

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“…Parallel spectral imaging, on the other hand, samples fluorescence simultaneously onto several detectors allowing fast image acquisitions, a prerequisite to follow fluorescence transients. To date the latter has only been used in the confocal mode [22]–[25] and thereby suffers limitations when imaging in depth in brain tissue [26], [27].…”

Section: Introductionmentioning

confidence: 99%

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

et al. 2009

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BackgroundThe generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.Methodology/Principal FindingsUsing fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.Conclusions/SignificanceOur method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.

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Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Cited by 33 publications

References 36 publications

An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

Thin-film tunable filters for hyperspectral fluorescence microscopy

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

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