Epi-Fluorescence Microscopy

Webb, Donna J.; Brown, Claire M.

doi:10.1007/978-1-62703-056-4_2

Cited by 73 publications

(42 citation statements)

References 59 publications

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“…Although it is usually assumed that the ratio of intensities of a fluorophore detected in two distinct wavelength ranges by two detectors is constant, intensity dependence of overspill parameters has recently been reported . Intensity dependent detector gain and spread of the signal to neighboring pixels have been blamed for this phenomenon . Here, we intend to show that such apparent intensity dependence of ratio parameters calculated on a pixel‐by‐pixel basis also arises as a result of the statistical nature of photon detection.…”

Section: Resultsmentioning

confidence: 86%

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy

et al. 2014

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Ratiometric determination of the efficiency of fluorescence or F€ orster resonance energy transfer (FRET) is one of the most widespread methods for the characterization of protein clustering and conformation. Low photon numbers, often present in pixel-by-pixel determination of FRET efficiency in digital microscopy, result in large uncertainties in the derived FRET parameter. Here, we propose a method based on maximum likelihood estimation (MLE) of FRET efficiency using photon counting detectors to overcome this limitation. Intensities measured in the donor, FRET, and acceptor channels were all assumed to follow Poisson statistics as a result of detector shot noise. The joint probability of photon numbers detected in the donor, FRET, and acceptor channels was derived using an equation describing the relationship between the three measured intensities. The FRET efficiency generating the measured photon numbers with the largest likelihood was determined iteratively providing a single FRET value for all pixels in the calculation. Since as few as 100 pixels are sufficient to provide a maximum likelihood estimate for FRET, biological variability in FRET values can be revealed by performing the analysis for regions of interests in an image. Since the algorithm provides the probability of a combination of donor, FRET, and acceptor intensities observed in each individual pixel given a certain FRET efficiency, outlier pixels with low probabilities could be excluded from the analysis. Simulations carried out with low photon numbers in the presence and absence of outlier pixels revealed that the proposed approach can reliably and reproducibly estimate FRET efficiency. In addition, systematic evaluation of the simulation results showed that the distribution of pixel-by-pixel FRET efficiencies is skewed, and the mean of these FRET values is a biased and unreliable estimate of the FRET efficiency. In the absence of outlier pixels, FRET calculated from summed donor, FRET, and acceptor intensities proved to be as reliable as MLE. We conclude that MLE of FRET outperforms calculations using summed and pixel-bypixel intensities in biologically relevant situations involving low photon numbers and outlier pixels.

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Section: Resultsmentioning

confidence: 86%

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy

et al. 2014

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“…The majority of fluorescence microscopes are “epifluorescence” microscopes, where the fluorescence is produced by reflected rather than transmitted light. The excitation and detection of fluorescence are performed through the objective, which occurs on the same path of light . A typical fluorescence microscope comprises a light source (typically, a laser), an excitation filter, a dichroic filter (a beamsplitter), and an emission filter.…”

Section: Biomedical Applicationsmentioning

confidence: 99%

Multifunctional Photonic Nanomaterials for Diagnostic, Therapeutic, and Theranostic Applications

Kim¹,

et al. 2018

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The last decade has seen dramatic progress in the principle, design, and fabrication of photonic nanomaterials with various optical properties and functionalities. Light-emitting and light-responsive nanomaterials, such as semiconductor quantum dots, plasmonic metal nanoparticles, organic carbon, and polymeric nanomaterials, offer promising approaches to low-cost and effective diagnostic, therapeutic, and theranostic applications. Reasonable endeavors have begun to translate some of the promising photonic nanomaterials to the clinic. Here, current research on the state-of-the-art and emerging photonic nanomaterials for diverse biomedical applications is reviewed, and the remaining challenges and future perspectives are discussed.

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“…They can be delivered with an excellent spatial resolution: a light beam can be focalized down to the diffraction limit, which permits to confine photoactivation to a few femtoliters—three orders of magnitude smaller than a cell—when relying on multiphoton excitation. Similarly, the associated temporal resolution can be very good, down to the femtosecond range when using a pulsed laser . Varying the illumination intensity can also easily be achieved with acousto‐optic modulators, shutters, or more simply LED sources (giving access to

t_{P}^{\min}

values above 0.1 μs).…”

Section: Why and How To Use The Kinetics Of Reactions For Selective mentioning

confidence: 99%

Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging

et al. 2016

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Living cells are chemical mixtures of exceptional interest and significance, whose investigation requires the development of powerful analytical tools fulfilling the demanding constraints resulting from their singular features. In particular, multiplexed observation of a large number of molecular targets with high spatiotemporal resolution appears highly desirable. One attractive road to address this analytical challenge relies on engaging the targets in reactions and exploiting the rich kinetic signature of the resulting reactive module, which originates from its topology and its rate constants. This review explores the various facets of this promising strategy. We first emphasize the singularity of the content of a living cell as a chemical mixture and suggest that its multiplexed observation is significant and timely. Then, we show that exploiting the kinetics of analytical processes is relevant to selectively detect a given analyte: upon perturbing the system, the kinetic window associated to response read-out has to be matched with that of the targeted reactive module. Eventually, we introduce the state-of-the-art of cell imaging exploiting protocols based on reaction kinetics and draw some promising perspectives.

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Epi-Fluorescence Microscopy

Cited by 73 publications

References 59 publications

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy

Multifunctional Photonic Nanomaterials for Diagnostic, Therapeutic, and Theranostic Applications

Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging

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