Design and application of a confocal microscope for spectrally resolved anisotropy imaging

Esposito, Alessandro; Bader, Arjen N.; Schlachter, Simon C.; Heuvel, D.J. van den; Schierle, Gabriele S Kaminski; Venkitaraman, Ashok R.; Kaminski, Clemens F.; Gerritsen, Hans C.

doi:10.1364/oe.19.002546

Cited by 23 publications

(19 citation statements)

References 32 publications

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“…Intuitively, multi-parametric detection [8], [9] is an obvious strategy to achieve this goal. Indeed, various techniques developed in the past decades, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, various techniques developed in the past decades, e.g. , spectrally resolved lifetime imaging [8], time-resolved anisotropy imaging [10] and spectrally-resolved anisotropy imaging [9], already provide detailed information concerning certain parameters. Detection technologies are mature to integrate all these modalities into a single instrument.…”

Section: Introductionmentioning

confidence: 99%

“…For conciseness, this technique will be referred to as Hyper Dimensional Imaging Microscopy or HDIM [9]. Furthermore, as phasor transformation [11] has become widely used in the analysis of biophysical imaging data, we propose a generalization of this technique to multi-dimensional datasets.…”

Section: Introductionmentioning

confidence: 99%

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Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

2013

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Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems.

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“…Intuitively, multi-parametric detection [8], [9] is an obvious strategy to achieve this goal. Indeed, various techniques developed in the past decades, e.g.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

2013

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“…Detection of polarization and spectral information can be achieved with the use of optics [41], but the timing properties of light require pico/nano-second sensitive pixels [11]. The integration of smart pixels and dedicated optics can provide an extremely versatile optical detector that can be reconfigured on-pixel to detect the features of biological interest.…”

Section: A Vision For the Futurementioning

confidence: 99%

Beyond Range: Innovating Fluorescence Microscopy

Esposito

2012

Remote Sensing

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Time-of-Flight (ToF) technologies are developed mainly for range estimations in industrial applications or consumer products. Recently, it was realized that ToF sensors could also be used for the detection of fluorescence and of the minute changes in the nanosecond-lived electronic states of fluorescent molecules. This capability can be exploited to report on the biochemical processes occurring within living organisms. ToF technologies, therefore, provide new opportunities in molecular and cell biology, diagnostics, and drug discovery. In this short communication, the convergence of the engineering and biomedical communities onto ToF technologies and its potential impact on basic, applied and translational sciences are discussed.Keywords: Time-of-Flight (ToF); fluorescence lifetime; microscopy Converging CommunitiesIt was the summer of 2005 when in the laboratory of Fred Wouters we started the first autonomous runs of our new in-house developed high throughput fluorescence lifetime imaging (FLIM) system. However successful that development was, one key element in allowing the biomedical community to benefit from FLIM systems, to enable the screening technologies we were pioneering [1] and to get closer to the routine application of FLIM, was still missing: replacing the expensive, comparatively fragile, and not very user-friendly, multi-channel plates used in FLIM with better technologies. The basic idea was to find a silicon-based detector capable of demodulating fluorescence signals on a bi-dimensional array of pixels. Frequent searches of primary literature returned only a few, though interesting, papers including: Nishikata et al. [2] on the development of a dedicated lock-in imager in CCD technology and, Mitchell et al. [3,4] on the adaptation of conventional CCD cameras to achieve demodulation. Unfortunately, ad hoc technologies for lock-in imaging appeared to be operating at OPEN ACCESS

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“…A different approach, which is simpler to implement and interpret, and which, similarly to NBA, only requires a single fluorophore moiety for labeling, is fluorescence anisotropy imaging microscopy (FAIM) [18]. FAIM measures energy transfer, homo-FRET, occurring between identical and proximate fluorophores.…”

Section: Introductionmentioning

confidence: 99%

Analyzing Receptor Assemblies in the Cell Membrane Using Fluorescence Anisotropy Imaging with TIRF Microscopy

et al. 2014

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Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.

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Design and application of a confocal microscope for spectrally resolved anisotropy imaging

Cited by 23 publications

References 32 publications

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

Beyond Range: Innovating Fluorescence Microscopy

Analyzing Receptor Assemblies in the Cell Membrane Using Fluorescence Anisotropy Imaging with TIRF Microscopy

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