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Advanced light microscopy

Equipment

LBIC has three advanced light microscopes, all placed at Biomedicinskt Centrum (BMC), floor D11.

Setup of Nikon A1RHD microscope

Nikon Confocal A1RHD microscope

Fully-automated confocal imaging system equipped with both a high-resolution galvano scanner as well as a high-speed (15 fps) 1024 x 1024 resonant scanner capable of capturing high-quality confocal images of tissues, cells and molecular events at high speed and enhanced sensitivity. This system is also equipped with a spectral detector.

Key features:

  • Available laser lines: 405 nm, 488 nm, 561 nm, and 640 nm
  • Detectors: 2 GaAsP PMT (for 488 and 561 lines), 2 PMT (for 405 and 640 lines) and 1 GaAsP PMT for spectral detection
  • Piezo Z-drive (NanoDrive, MadCity labs) for fast Z-stacks
  • Software: NIS-Elements

Nikon Confocal A1+ microscope

Fully-automated confocal imaging system equipped with a high-resolution galvano scanner capable of capturing high-quality confocal images of tissues, cells and molecular events at enhanced sensitivity.

Key features:

  • Available laser lines: 405 nm, 488 nm, 561 nm, and 640 nm
  • Detectors: 2 GaAsP PMT (for 488 and 561 lines), 2 PMT (for 405 and 640 lines)
  • Software: NIS-Elements

To find out more about the confocal technique go to: 
http://www.microscopyu.com/articles/confocal/confocalintrobasics.html

Setup of Nikon STORM microscope

Nikon TIRF/STORM microscope

Total Internal Reflection Fluorescence (TIRF) microscopy gives superior signal to noise images compared to conventional wide-field fluorescence. The system is equipped with an Andor Ultra 897 EMCCD camera and an Andor Zyla 4.2 sCMOS camera (owned by Pontus Nordenfelts group), which enables imaging at very high speeds and sensitivity. The high speeds of the cameras allow for tracking of low-light dynamic events within a live cell.

The STochastic Optical Reconstruction Microscope (STORM) is a super resolution technique that uses technology licensed from Harvard University. N-STORM provides dramatically enhanced resolution that is 10 times that of conventional optical microscopes. The LBIC STORM is equipped with 2 high-powered lasers (561 and 647) that enable 2 color direct STORM. Compared to conventional wide-field and confocal imaging techniques STORM requires specific sample preparation and careful optimization as well as a data reconstruction process.

Key features:

  • Available laser lines: 405 nm, 488 nm, 561 nm (high-powered), and 647 nm (high powered)
  • Cameras: Andor iXon 897 EMCCD and Andor Zyla 4.2 sCMOS
  • Incubator: Stage top incubator with temperature control.
  • Software: NIS-Elements

To find out more about the TIRF technique go to 
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html

To find out more about the STORM technique go to: 
http://www.microscopyu.com/print/articles/superresolution/stormintro-print.html

Zeiss CD7 automated live cell microscope

Shared between LBIC and Filipe Pereira. Placed at Biomedicinskt Centrum (BMC), floor A12

M Squared Aurora Light Sheet Microscope

.Light sheet microscopy (LSM) differ from conventional light microscopy by having the illumination and detection optical pathways decoupled. In LSM, the sample is illuminated by a thin sheet of light (low- or sub-micrometer range) and the emitted light is detected orthogonally to the plane of illumination. Because of this, LSM imaging significantly reduces out-of-focus emitted light and an image of the illuminated plane can be quickly recorded using a CCD or CMOS camera. Optical sectioning and generation of 3D-images is there for possible using LSM by moving the sample through the light sheet or vice versa.

Furthermore, as only the optical plane of interest is illuminated in LSM, this technique offers reduced photo-bleaching and -toxicity compared to traditional light microscopy, where the whole detection path is illuminated. Due to the rapid imaging, high lateral and axial resolution, and reduced sample illumination associated with LSM, this technique is a great choice for imaging large biological samples (especially in combination with optical clearing techniques) and/or live imaging.

Key features: 

  • Immersion objectives (~16x magnification) compatible with aqueous clearing solutions (e.g. CUBIC, Clarity, and ScaleS) and hydrophobic solvents (e.g. ethyl cinnamate)
  • 7 laser lines: 405, 457, 488, 515, 561, 647, and 730 nm
  • 9 filters: compatible with, for example, DAPI, CFP, mTurq2, eGFP, AF488, BODIPY 493, eYFP, tdTomato, TagRFP, AF568, mScarlet, AF594, mCherry, AD647, miRFP, AF750, Cy7
  • Frame rate: 100 FPS at full FOV (reducing image size increases imaging speed)
  • Imaging mode: sequential imaging
  • Stage top incubation system able to control temperature (25-37°C), O2 (1-18%), CO2 (0-10%), and relative humidity
  • The system is additionally prepared for upgrades with an additional camera and a multiphoton laser

To find out more about light sheet microscopy go to:
https://www.microscopyu.com/techniques/light-sheet/light-sheet-fluorescence-microscopy (opens in a new window)