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

Free download. Book file PDF easily for everyone and every device. You can download and read online Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension file PDF Book only if you are registered here. And also you can download or read online all Book PDF file that related with Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension book. Happy reading Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension Bookeveryone. Download file Free Book PDF Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension at Complete PDF Library. This Book have some digital formats such us :paperbook, ebook, kindle, epub, fb2 and another formats. Here is The CompletePDF Book Library. It's free to register here to get Book file PDF Optical Fluorescence Microscopy: From the Spectral to the Nano Dimension Pocket Guide.

High resolution TERS map.

Dr. Paolo Bianchini - Google Scholar Citations

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.

Optical Tweezers - Fluorescence Microscopy: Workflow

Graphene flakes 30x30 um. Ni foil 20x20 um. MoO3 30x30 um. Optical viewing system image with approached AFM probe. Raman map conductive polymer nanowires. Photoluminescence CdS. Raman and PL spectrum of CdS nanowire. Sample courtesy: prof. Carpick, Penn State University. G band intensity. Raman spectra. AFM topography.

Applications

Yanul, S. Magonov, P. Recording of the webinar presented by Dr. Novel optical scheme consists of three independent channels of sample excitation: from top, side and bottom directions. Each channel is developed as independent module Sample excitation directions are easy to exchange between each other. Light collection could be done by excitation optical channel or by different one Open design provides great opportunities in system customization. AFM registration laser system independent on Raman objective and this allows fast and easy exchange of objectives which are centered onto the same point on the surface Automated AFM laser, probe and photodiode alignment minimize customer routine operations User-friendly change of AFM registration system wavelength provides compatibility with any excitation or collection wavelength Stand-alone optical periscope allows to combine AFMRaman system with virtually all Raman spectrometers available on the market upon customer request Spectrometer could be equipped with number of etectors: PMT, APD, CCD.

Both Rayleigh optical image and Raman map could be obtained simultaneously. Video demonstration of Spectra II usability.

Education in Microscopy and Digital Imaging

TERS nano-Raman mapping. Remarkable lifetime without considerable enhancement degradation. Lee et al. September , p. Kotlyar, S. Stafeev, A. Nalimov, M.

Duplicate citations

Kotlyar, L. Express, vol.

Imaging the chemical activity of single nanoparticles with optical microscopy

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.


  • Select Your Region and Language.
  • Introduction to Fluorescence Microscopy | MicroscopyU!
  • Goldoni: Volume Two: 2 (Absolute Classics).
  • Global Health and Global Health Ethics!
  • Citations per year.

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.


  1. The Feather Friends (A Fun Rhyming Childrens Picture Book for ages 2-6).
  2. Spectral Imaging and Linear Unmixing?
  3. ZEISS Microscopy Online Campus | Introduction to Spectral Imaging?
  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.


    • Download Optical Fluorescence Microscopy From The Spectral To The Nano Dimension 2011.
    • Under One Roof Again: All Grown Up and (Re)learning to Live Together Happily.
    • NTEGRA Spectra II.
    • The Cryptogram of Rennes-le-Chateau: A Guide to an Enigmatic Village.
    • The Renal Survival Cookbook?
    • Rock Climbing: The Ultimate Guide (Greenwood Guides to Extreme Sports).
    • Ancient Society or Researches in the Lines of Human Progress from Savagery through Barbarism to Civilization.

    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.