Manual Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension

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Sample: BCB thin molecular layer on Au substrate right. AFM Probes shop. Ask online. Working principle. Contact Us. Applications Graphene, carbon nanotubes and other carbon materials Semiconductor devices Nanotubes, nanowires, quantum dots and other nanoscale materials Polymers. Optical device characterization: semiconductor lasers, optical fibers, waveguides, plasmonic devices Investigation of cellular tissue, DNA, viruses and other biological objects Chemical reaction control.

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Kotlyar, L. Express, vol.

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Slekiene, L. Ramanauskaite, and V. Lipids, vol. Li et al. Yun et al. Kharintsev, A. Fishman, S. Saikin, and S. DOI: Plasmonic lens focused longitudinal field excitation for tip-enhanced Raman spectroscopy. The resulting spectral information can be used to pinpoint the location of specific fluorophores and dyes with high spatial precision, and is also potentially capable of producing information about interactions between two or more probes. Under routine circumstances, and where experimental protocols permit, traditional confocal and widefield imaging techniques can be successfully applied through the careful selection of fluorescent probes and associated filter sets, as well as by implementing multitracking scan strategies with the proper controls to produce reasonable separation of fluorophore signals.

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Unfortunately, the increased use of multiple fluorescent protein colors with their associated high degree of spectral overlap to monitor intracellular interactions often limits the choice of experimental parameters. Furthermore, in live-cell imaging using a single fluorophore, natural autofluorescence can significantly interfere with detection in channels where the most popular green-emitting fluorescent probes such as enhanced green fluorescent protein are visualized.

This background noise problem can also be seriously compounded by extraneous fluorescence that is introduced through the use of fixatives or DNA transfection reagents. In situations where fluorescent probe spectra significantly overlap or autofluorescence is excessively high, spectral imaging coupled with post-acquisition image analysis using linear unmixing algorithms can be utilized to untangle mixed fluorescence signals and clearly resolve the spatial contribution of each fluorophore.

Similar in concept to the optical section or z -stack obtained from thicker specimens using high numerical aperture objectives in laser scanning confocal or deconvolution microscopy, the lambda stack is a three-dimensional dataset that consists of an image collection using the same specimen field acquired at different wavelength bands, each spanning a limited spectral region ranging from 2 to 20 nanometers. In contrast, typical imaging scenarios in all forms of optical microscopy involve acquiring a single image or a successive group of images in time-lapse experiments over the entire wavelength response band of the detector.

By plotting pixel intensity versus wavelength on a linear graph see Figure 5 b , the emission spectral profile of the particular fluorophore spatially located at pixel i can readily be determined. It should be noted that the accuracy and resolution of an emission spectrum obtained using this technique is a function of the number of lambda stack images gathered at distinct wavelength bands, the spectral width in nanometers of each wavelength band shorter bandwidths produce higher resolution , the physical quality of the specimen under investigation, and the photon sensitivity quantum efficiency of the detector.

A real-world example of a lambda stack acquired on a laser scanning confocal microscope in living cells using three fluorescent proteins having overlapping spectra is presented in Figure 6.

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  4. The fluorescent protein markers used in this experiment are enhanced green fluorescent protein EGFP from jellyfish; emission maximum at nanometers , enhanced yellow fluorescent protein EYFP from jellyfish; emission maximum at nanometers , and the monomeric version of Kusabira Orange mKO , emission maximum at nanometers , a high-performance probe developed from a naturally-occurring coral protein. In this case, the individual lambda stack images were scanned in nanometer wavebands ranging from to nanometers Figure 6 a to generate a total of 16 spectral sections for the fluorescent protein mixture.

    The first image of the lambda stack reveals the spectral signature of the specimen in the emission range of to nanometers, while the second image contains emission data from to nanometers see Figure 6 b. Note that virtually all of the fluorescence emission in the first two lambda sections arises from the short-wavelength tail of EGFP alone with only a very minor contribution from EYFP in the longer wavelength section to nanometers.

    In the next two lambda sections to nanometers and to nanometers , the contribution from EYFP steadily increases as the emission from EGFP reaches a plateau.

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    In the three lambda sections between and nanometers, The EGFP signal begins to decrease as the contribution from EYFP emission reaches a maximum at approximately nanometers. Likewise, the emission contribution from mKO becomes more significant in the band between and nanometers. In review, the wavelength bands at the extremes of the lambda stack between to nanometers and between to nanometers feature emission contributions that are dominated by the shortest and longest wavelength-emitting proteins, EGFP and mKO, respectively.

    Those wavelength bands in the center of the lambda stack to nanometers contain fluorescence emission that represents some contribution from all three fluorescent proteins. As will be discussed below, the distribution of the mixed emission signal across the wavelength bands of the lambda stack can be linearly unmixed using reference emission spectral profiles from each probe to clearly separate the contribution of the individual fluorescent proteins.

    The principal instrumental consideration in optical microscopy for spectral imaging includes the ability to accurately segregate fluorescence emission or source light not absorbed by the specimen into its component wavelengths using a dispersive element or similar technique. A number of different methodologies have been implemented to generate lambda stacks in widefield and confocal microscopy using several detector designs.

    The most useful instruments have proven to be laser scanning confocal microscopes equipped with spectral detectors that rely on prisms or diffraction gratings to disperse fluorescence emission, which is then directed to one or more photomultipliers. Advanced confocal instruments contain a prism or diffraction grating to disperse the emission beam into its component spectrum, which is then passed to either a multi-anode photomultiplier that can simultaneously detect up to 32 individual channels of spectral information, or to slits that pass selected wavelengths to one detector and reflect shorter and longer wavelengths to additional slits.

    Widefield instruments utilize interference filters, acousto-optical tunable filters AOTFs , liquid crystal tunable filters LCTFs , interferometry, prisms, prisms coupled to reflectors, or gratings to generate lambda stacks for image analysis. Among the many techniques that have been used to generate lambda stacks, the so-called wavelength-scan methods represent perhaps the simplest approach.

    In practice, a series of narrow bandpass interference filters usually 5 to 20 nanometers wide is used to gather a stack of images of the viewfield with each filter. Alternatively, a combination of shortpass and longpass filters having particularly sharp cut-off wavelengths can be used instead of bandpass filters.