Super-resolution and Conventional Fluorescence Microscopy



   Service Description

Conventional widefield fluorescence microscopy is still the best choice for many applications. The CCD and CMOS-based sensors used for conventional micros-copy are typically much more sensitive than the photomultiplier tubes used in confocal microscopes and flow cytometers. As the camera captures the whole field of view at the same time, it also allows for faster imaging in many cases. Examples where conventional microscopy may be advantageous include the visualization of individual molecules, receptors, or small organisms such as bacteria and yeast.
Total Internal Reflection Fluorescence (or TIRF) is a powerful technique that combines the sensitivity of conventional fluorescence with selective illumination to improve the contrast of features very close to the sample coverslip. TIRF is often used for studies related to membrane dynamics, receptor-ligand interactions, and vesicular transport.
The intrinsic diffraction of light has historically made it difficult to use fluorescence to distinguish structures closer than 200nm apart. Super-resolution microscopy refers to techniques that selectively activate fluorescent molecules to map their position with up to 10 times more accuracy than conventional fluorescent microscopy. The Nikon N-Storm system is capable of localizing molecules with a resolution of up to 20nm and is compatible with all most current localization protocols and fluorophores (including Alexafluor 647 and Atto488).

   Equipment

  • Nikon Eclipse TiTIRF / N-STORM
  • Nikon Eclipse Ti basic Fluorescence Microscope
  • Nikon / EppendorfMicroinjection System
  • Nikon /Hamilton ThornePhotoablation System
  • Leica M165 Fluorescent Stereo-microscope

    • Andor iXon3 897 EMCCD camera
    • 100x Plan Apo Oil immersion Objective 149 NA
    • 4 Excitation lasers405nm, 488nm (200mW), 561nm and 647nm (300mW)
    • TIRF optical system
    • Multipass TIRF and STORM filters for highspeed multicolor imaging
    • Removable cylindrical lens for 3D STORM
    • Nikon PFS (Perfect Focus System) for improved stability

    • Inverted Nikon Ti Eclipse fully motorized microscope
    • Hamamatsu Orca R2 monochrome 13 MP CCD camera
    • Objective lenses: 60x (14 NA), 40x, 10x and 4x
    • Filter sets for DAPI, GFP, TRITC
    • Configured for both NISElements and the opensource Micromanager suite for advanced longterm imaging experiments
    • Differential interference contrast (DIC) for improved contrast in brightfield illumination

    • Inverted Nikon Ti Eclipse Microscope
    • Eppendorfmicromanipulation and injection system
    • Colour fluorescence camera with filters for multicolor imaging

    • Inverted Nikon Ti Eclipse
    • Hamilton Thorne XYClonephotoablation module

    • Highend 1 x Plan Apo objective lens with 165:1 optical zoom
    • Resolution of up to 11 microns
    • Fluorescence (Hoechst/DAPI, GFP/FITC, and TRITC)
    • 8 MP Colour and 13 MP Monochromatic camera options
    • Epi, Trans, and Oblique white light illumination options

   Applications

  • Nanoscale imaging of fixed samples
  • Dynamic imaging of cytoskeletal structures, focal adhesion formation, as well as endocytosis and vesicle dynamics in live cells
  • Single-molecule studies and localization microscopy modalities including N-STORM, Direct STORM, and PALM
  • Fluorescent analysis of histological samples
  • Microinjection of DNA, RNA, morpholinos, vital dyes, or pharmacological agents into cultured cells, oocytes, or embryos
  • Non-contact ablation of targeted membranes or structures as a powerful tool for stem cell, cloning, and developmental biology areas
  • High-quality macro-to-micro imaging of both biological and non-biological samples

   Services Offered

  • Super-resolution (SR) advice and consulting: we can help you evaluate and choose the best localization microscopy strategies for your samples
  • We can supply super-resolution specific reagents, including buffers and fluorochromes, and custom-labeled antibodies
  • STORM image processing: we can convert localization imaging data into SR images using both proprietary and open source software packages
  • TIRF imaging of membrane and/or cytoskeletal dynamics in live adherent cells using commercial fluorescent labels such as DiR, FM4-64, and Cell-Light or user-supplied reagents
  • Long-term (>4 days) imaging of bacterial colonies and bacterial interactions using DIC, temperature control, autofocus, and multi-field imaging
  • Routine multi-color fluorescent analysis of histological samples where confocal or other more advanced methods are not necessary
  • Imaging of bone, muscle, or connective tissues in histological samples using circularly-polarised light


Technical staff


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Calle Severo Ochoa, 35
Parque Tecnolgico de Andaluca (PTA) Campanillas, Mlaga 29590

(+34) 951 440 260
Fax: (+34) 951 440 263