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Light microscopes

The CCI offers access to several different types of advanced fluorescence microscopes. Here is a brief overview of the different techniques:

Widefield fluorescence microscopes

See Carl Zeiss Axio Observer and Olympus ScanR/CellR

A widefield fluorescence microscope uses a lamp, e.g. a Mercury arc lamp, to illuminate and excite the specimen. This is a fast and economical way to obtain fluorescent images, which can be viewed directly with your eyes through the ocular or captured with a camera.

Thin specimens that do not require confocal imaging might be better analyzed using a conventional widefield microscope as it offers unsurpassed signal to noise.

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Laser scanning confocal microscopes

See Carl Zeiss LSM 510 META, LSM 700, LSM 710 and LSM 780.


Fig. 1 A widefield fluorescent image (left) vs a LSM image (right) of a mouse intestine (from Carl Zeiss).

The advantage of a confocal microscope is that it only collects the light reflected or emitted by a single plane of the specimen. This makes the images from thick specimens much sharper than with a conventional widefield fluorescent microscope (see Fig.1), and by collecting images from several focal planes, you can reconstruct a 3D representation of your fluorescent specimen. With a confocal laser scanning microscope (LSM) it is possibile to zoom in on small details, to perform multi-color imaging and to make time series.

The possibility to select certain regions of interest (ROI) in the confocal images makes it possible to measure dynamics and interactions with FRAP and FRET, see Techniques.

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Multiphoton microscope

See Carl Zeiss LSM 710 NLO

Fig. 2 The autofluorescence of a waterflea imaged with confocal (left) and MP (right) microscopy.

Multiphoton (MP) microscopy utilizes a non-linear excitation process, usually two-photon excitation, which occurs only at the focal point of the microscope. This gives inherent optical sectioning capabilities, without cutting off out-of-focus emission, and minimizes the photobleaching and photodamage that are the ultimate limiting factors in imaging live cells. The low energy/ long wavelenght infrared (IR) excitation light is less harmful to living species than the light range used for confocal microscopy. The IR light also undergoes less scattering, which results in less background and longer penetration depths, see Fig. 2. These advantages allows investigations on thick living tissue specimens that would not otherwise be possible with conventional imaging techniques. 

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High content screening microscope 

See Olympus ScanR/CellR

Fig. 3 Images aquired from a specimen from P. Johansson and O. Hammarsten, Clinical Chemistry, University of Gothenburg.

High Content Screening (HCS) microscopy is based on a highly automated imaging system combined with software for processing and analyzing large amounts of data from fixed or living cells.

The image analysis software can generate numeric data from the images for many different parameters, such as cell number and fluorescence intensities in labeled cell constituents. Unlike flow cytometry, which measures just the overall fluorescence of each cell, the strength of HCS image analysis is its ability to recognize and quantify cell morphology, localization, movement, structures and organization within the cells.

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Super-resolution microscope

See Carl Zeiss ELYRA PS.1

Fig. 4 Golgi apparatus in 3D as seen with widefield (left) and SIM (right). Image aquired at the ELYRA PS.1 by J. Fernandez-Rodriguez.

Superresolution microscopy is the common name of the different fluorescence-based microscopy techniques, which has a resolution beyond the diffraction limit of 200 nm in the lateral direction and 500 nm in the axial direction. CCI offers two such superresolution techniques: structured illumination microscopy (SIM), see Fig, which doubles the resolution in all directions, and the single molecule localization techniques (PALM/dSTORM), which have a resolution comparable to electron microscopy (down to 15 nm).

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Laser microdissection and pressure catapulting (LMDPC) 

See Carl Zeiss PALM MicroBeam

Fig. 5 The principle of the laser cutting and catapulting processes.

The Laser Microdissection and Pressure Catapulting (LMPC) technology offers non-contact sample handling without mechanical contact of specific tissue regions for downstream analysis of RNA, DNA and proteins without risk of contamination or infection. In addition, living cells can be catapulted for subsequent recultivation of specific clones of e.g. cells expressing fluorescent proteins.

Read more about the technique here

 

 

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Page Manager: CCI web manager|Last update: 10/11/2016
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