Zeiss LSM 980 laser scanning confocal with Airyscan 2

 

The AMI laser scanning confocal fluorescence microscope is capable of performing a large variety of applications. The confocal microscope can be used by visiting researchers to image thick specimens such as tissue sections or cell aggregates (and many others). The benefit of confocal microscopy can also be combined with enhanced resolution "Airyscan" to enable access to sub-cellular information within both fixed and live samples. The AMI confocal also has an array detector that can be used to capture fluorescence emission spectra information and it is compatible with a large variety of fluorescent probes. In addition to this, the confocal microscope can be incubated to allow for live-cell microscopy experiments to study temporal phenomena.

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Laser scanning confocal microscopy

The pinhole within the AMI confocal microscope prevents out-of-focus light from causing "blurry" images. This means that the images captured are more detailed and contain light only from the plane of interest. This makes confocal microscopy an excellent approach for capturing 3-dimensional images of both fixed and slow moving live samples.

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"Airyscan 2" microscopy

An adaption of confocal microscopy called "Airyscan 2" eliminates the need for a pinhole while still being able to optically section thick samples. This approach has various advantages such as the need for less photo-exposure to capture high-quality images. As a results, images can be acquired faster than standard confocal microscopes.

 

Using computational reconstructions, the resolution limit in Airyscan images can be reduced by roughly two-fold. This means that enhanced resolution can be captured relatively deep within live or fixed samples with the benefit of a laser scanned image. 

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Spectroscopy

By employing a diffraction grating and a series of adjacent point detectors within the AMI confocal, the emitted light can be divided spectrally. Visiting researchers can use this to perform spectral unmixing for samples with a large number of fluorescent markers or samples with high background autofluorescence.

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Fluorescence (cross-)correlation

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are techniques that can provide information on the dynamics of (fluorescent) molecules within live samples. These two techniques help researchers gain insight into the dynamics of specific molecules within living samples, such as diffusion coefficients and molecular concentrations.

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FCS is a single-molecule technique that measures the fluorescence emitted by a single molecule or a small number of molecules within a very small position of the sample. It can be used to study the kinetics of chemical reactions, the diffusion and transport of molecules, as well as the interactions between molecules. With FCCS, the fluorescence emitted by two different populations of (fluorescent) molecules in a sample is measured. This can provide researchers with quantitative information on the interactions between the two molecules of interest.

 

Instrument specifications

• Inverted microscope configuration

• Auto focus for time-lapse acquisitions of live samples in aqueous media

• Contrast methods:

  • bright-field transmitted light

  • differential interference contrast (DIC)

  • fluorescence

• Temperature- and CO₂-controlled incubation

• Objectives:​

  • 63× 1.4 NA oil-immersion DIC

  • 40× 1.2 NA water-immersion with correction collar

  • 40× 1.3 NA oil-immersion DIC

  • 20× 0.8 NA

  • 10× 0.3 NA

• Laser excitation:

  • 405nm ■

  • 445nm ■

  • 488nm 10mW ■

  • 514nm ■

  • 561nm 10mW ■

  • 639nm 7.5mW ■

  • 730nm 10mW ■

• Axiocam 712 mono sCMOS monochrome camera

• Near-infrared GaAs, MA, and 32 GaAsP point detectors

  • Array/spectral detection ■ ■ ​■ ■ ■ ■ ■ ■ 

• Automated stage control with piezo X-, Y-, and Z-control

 

Instrument capabilities

• Resolution: ~200nm (~100nm with Airyscan SR)

• Speed: down to 500ms for 512×512 pixel size image and 5s for a 15×15×5μm volume

• Depth: up to 100μm

• Field of view: dependent on zoom, magnification, and pixel number

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Instrument uses

• Photon counting

• FCS

• FCCS

• Image scanning microscopy super-resolution (Airyscan SR)

• 8Y imaging

• Live-cell imaging

• Automated stage

• Tiling (for large fields of view)

• Intracellular dynmics

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