Advanced fluorescence service

The Advanced Fluorescence service is equipped with cutting-edge equipment suitable for an extensive range of applications. Its facilities are designed to support research activities at Bionand and other institutions such as universities, hospitals, research institutes and companies in Malaga, Andalucía and beyond.

For investigators at Bionand these applications can range from the initial characterization of the fluorescent properties of novel compounds up to the evaluation of their medical applications using tissue culture and animal models.

For external researchers the range of possible applications goes far beyond nanomedicine including the characterization of patient tissues, preclinial studies using rodent models, small scale drug screening and studies using of model organisms such as Zebrafish or Drosophila Melanogaster.

As part of the objective of making the advanced technologies available in Bionand as accessible as possible we offer full technical support and training services to both internal and external users.

The service is operated in close collaboration with the other advanced facilities of the Nanoimaging Unit and the other units of Bionand ensuring researchers can easily combine fluorescence-based techniques with other complementary approaches such as electron microscopy or nuclear magnetic resonance.

Below we give a brief introduction to different techniques available from the Advanced fluorescence service. More detailed information can be found on the individual equipment pages and by contacting Dr. John Pearson, the Bionand Advanced Fluorescence Specialist.

Confocal Microscopy

    Laser-scanning confocal microscopy is a very popular technique that uses a combination of laser illumination and a "pinhole" mask to ensure that only fluorescence from the focal plane reaches the detector. This avoids the characteristic blur typical of conventional fluorescence microscopy and allows images to captured as detailed optical slices and then used to build up rich 3D models. Confocal microscopy is one of the most versatile techniques available in a optical microscopy. The Advanced Fluorescence Service offers two extremely well specified Leica SP5 confocal microscopes. Their features include HyD hybrid detectors for the best possible sensitivity, as well as full environmental control for live cells and high-speed resonant scanning.

  • Leica SP5 HyD
  • Leica SP5 MP HyD
  • HyD detector advantages (external site)

Multiphoton Microscopy

    Multi-photon (or two-photon microscopy) takes advantages of the near simultaneous absorption of two or more photons which act to excite a fluorescent molecule with the combined energy of both photons. In practice this means that low energy infrared light can be used to see fluorescent molecules that are normally excited by high energy ultraviolet and blue light. Using this method we can visualize fluorescent molecules at much greater depths than conventional microscopy. Infrared light is also much less damaging to live tissues than UV or blue excitation, making it ideal for visualizing model organisms or tissue explants. The Leica SP5 MP HyD combines both confocal and multi-photon modes in a single machine and can easily switch between different modalities. It is also fitted with the brand new, super-sensitive external HyD hybrid detectors dedicated for multi-photon microscopy.

  • Leica SP5 MP HyD
  • Two-photon microscopy basics (external site)
  • HyD detector advantages (external site)

Super-resolution microscopy

    Fluorescence microscopy offers a wide array of tools for visualizing microscopic events. 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. With the aim of promoting the use nanoscale imaging in biomedicine, Bionand has established one of Spain´s first N-STORM super-resolution platforms dedicated to practical applications. The Nikon N-Storm system is capable of localizing molecules with a resolution of up to 20nm.

  • Nikon Eclipse Ti N-Storm
  • N-Storm Superresolution explained (external site)

Flow cytometry / Cell sorter

    Flow cytometry is a highly effective method for analyzing thousands cells or other particles in a matter of minutes. Flow cytometers detect the fluorescence, size and complexity of particles that pass through laser beams as a stream. In this way they can quickly generating statistically valid data by count very large numbers of particles - something that would be extremely difficult with traditional microscopy approaches. Flow cytometers are very well established in hospitals for routine diagnosis and now widely used for different types of research application from marine biology to stem cell research. A Cell Sorter is a specialized flow cytometer which is able to physically separate different classes of particles based on their fluorescence or other physical characteristics.

  • Beckman Coulter Galios flow cytometer
  • Beckman Coulter MoFlo cell sorter
  • Cytometry basics (external site)

Fluroesence microscopy

    For many applications conventional widefield fluorescence microscopy is still the best choice. The CCD detected used for conventional microscopy are often more sensitive that the photomultiplier tubes used in confocal microscopes and flow cytometers. As the camera capture the whole field of view at the same time - it also allows for faster imaging in many cases. Examples where conventional microscopy may be more appropriate 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 which combines the sensitivity of conventional fluorescence without out of focus light. User have access to a dedicated conventional fluorescence system equipped for time-lapse microscopy and a laser-based multi-color TIRF system (also used as the basis for the N-STORM system)

  • Nikon Eclipse Ti basic
  • Nikon Eclipse Ti TIRF
  • TIRF explained (external site)

High Content Screening

    High content screening (HCS) attempts to bridge the gap between microscopy and cytometry, providing both large quantities of statically useful and phenotypically rich data. High content screening combines automated microscopy with customizable algorithms able to detect and quantify many different types of phenotypic information in both fixed and living cultured cells. Part of the Bionand Cell Culture Unit, the Perkin Elmer Operetta high content screening combines the quality of a spinning disc confocal microscope with Harmony HCS software, which contains many common algorithms such as nuclei and cells detection, as well as the ability to "learn" new phenotypes. Bionand will also offer access to the advanced server-based Columbus HCS software.

  • Perkin Elmer Operetta HCS confocal
  • Perkin Elmer Columbus HCS software (external page)