Confocal Microscopy
Laser scanning confocal microscopy is the default technique in the CVR for detection and localisation of viral and host fluorescent molecules in cells and thin tissues. The sensitivity and resolution of modern systems allow for fine detailing of viral infection and host molecular behaviour in 3 dimensions. The CVR is served by two confocal microscopes, the Zeiss LSM 880 and the Zeiss LSM 710 and both are heavily used instruments by groups across the CVR. Open source and commercial software is implemented to enable 2D image analysis (HALO software, Indica Labs) and 3D image analysis (Imaris software, Oxford Instruments). The LSM 880 sits within an incubated chamber, this addition of full environmental control providing the further dimension of time-lapse live-cell microscopy.
Note for local users: The local user pages provide details of equipment curators and locations.
Zeiss LSM 710 Meta Confocal Microscope
Purchased in 2012, the Zeiss LSM 710 meta utilises Zen 2011(black) software. The microscope’s spectral detector, which can be used to unmix up to 10 (spectrally similar) fluorophores simultaneously and remove autofluorescence, is also ideal for FRET and FRAP. Both scan speed and detector sensitivity are significantly enhanced compared to the CVR’s older LSM 510 meta system. Extra features include the functionality to create image montages, accurately identify colocalisation and enhance low signal in samples with a large range of signal intensities using the software’s High Dynamic Range module.
The microscope is fitted with the following hardware:
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Objective lenses |
Laser lines |
Detectors |
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10x (dry) – DIC ready |
405nm (eg DAPI) |
2 x PMTs |
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20x (dry) – DIC ready |
458nm (eg CFP) |
1 x spectral detector |
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40x (oil) – DIC ready |
488nm (eg GFP) |
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63x (oil) – DIC ready |
514nm (eg YFP) |
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100x (oil) – NOT DIC ready |
561nm (eg Alexa 568) |
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633nm (eg Alexa 633) |
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Zeiss LSM 880 Confocal Microscope with Airyscan FAST
Purchased in 2015, the Zeiss LSM 880 utilises Zen 2 (black) software. The system is fitted with a choice of autofocus options and an incubator incorporating full CO2 and temperature control allowing for extended live-cell time-lapse imaging. Other fitted hardware:
|
Objective lenses |
Laser lines |
Detectors |
|
10x (dry) – DIC ready |
405nm (eg DAPI) |
2 x PMTs |
|
20x (dry) – DIC ready |
458nm (eg CFP) |
1 x GaAsP |
|
40x (oil) – DIC ready |
488nm (eg GFP) |
1 x Airyscan |
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63x (oil) – DIC ready |
514nm (eg YFP) |
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|
|
561nm (eg Alexa 568) |
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594nm (eg Alexa 594) |
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633nm (eg Alexa 633) |
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The quantum efficiency of the GaAsP and Airyscan detectors provides significant improvement in signal:noise. In comparison to the CVR’s LSM 710, which has PMT detectors. The LSM 880 also offers improved scan speed.
Incorporation of an Airyscan detector allows for super-resolution imaging, providing an improvement of up to 2x higher resolution (in x,y and z dimensions) compared to ‘standard’ confocal microscopy.
The addition of the Airyscan FAST module in 2021 has increased the speed of Airyscan acquisition 4-fold and provides an expanded range of image resolutions. The associated workstation also allows for synchronised Airyscan processing which further speeds up the image acquisition process.
