CHAPTER 1 - SYSTEM OPERATIONZEISS Hardware Aspects LSM 88014 000000-2071-464 10/2014 V_012.5 Performance Features of the LSM 880Optical and Mechanical AspectsThe highly integrated system design makes for the shortest possible optical paths, top-grade opticalprecision and high stability. The Airyscan module can be mounted to the LSM 880 scan head of anyconfiguration and with upright or inververted microscopes. The BiG.2 module is either directly attachedto the scan head and can be used in combination with upright and inverse microscope stands.The spectral range available extends from the UV to the IR region.For the VIS (visible-light) Laser Module, the user can select from up to six lasers with wavelengths of 633,594, 561, 543, 514, 488, 458 and 405 nm. The UV Laser Module provides wavelengths of 355 nm. Ti:SaLaser provide pulsed laser light from 680 to 1300 nm for Multiphoton imaging (NLO). UV and visiblelasers are coupled to the scan head via polarization-preserving single-mode optical fibers. Ti:Sa lasers arecoupled to the sytem via free beam coupling kits. One variable beam collimator each for the UV, NLO andvisible ranges provides optimum adaptation of the respective laser wavelength to the objective used and,thus, optimum correction for Z aberrations.Acousto-optical tunable filters (AOTF) adjust the necessary brightness for up to 8 laser lines withinmicroseconds. Ti:Sa lasers are attenuated via AOM within less than a microsecond.The internal image acquisition channels usable for reflection or fluorescence, and an additionaltransmitted-light channel are ideal for the investigation of multiple fluorescence specimens. Airyscan andBiG.2 are attached directly to the scan head. Both are equipped with Gallenium Arsenide Phosphidedetectors (GaAsP). The diameter of the pinhole and the XY positions can be optimized, and the desiredemission can be selected. This adjustment also includes positioning along Z. For the simultaneousregistration of multiple fluorescence signals, identical optical sections can be obtained in each confocalchannel.The microscope's transmitted-light channel is equipped with a photomultiplier, too. It is thereforepossible to superimpose a multiple fluorescence image on a brightfield, differential interference or phaseimage.Airyscan is equipped with a filter wheel consisting of 8 positions of which 6 are used for specific doubleband pass emission filters. BiG.2 dichroics and emission filters are housed in push & click filter cubeswhich are exchanged manually.The high-NA C-APOCHROMAT objectives specially developed for the LSM technique reach the physicallimit in resolving power, and can be used throughout the 380...900 nm spectral range with the samehigh quality, producing brilliant images. Note, for fluorescence correlation spectroscopy theC-APOCHROMAT objectives are highly recommended.A two-mirror scanner system, controlled by real time electronics, offers several advantages. The largedeflection angle of the scanning mirrors allows a wide area to be scanned. With a 1.25× objective, theobject area scanned is 11 × 11 mm². The scanning field size can be freely selected between 4 × 1 and8192 × 8192 pixels.It is possible to rotate the XY scanning field through 360° and carry out XY scans without having torotate the specimen itself under laser radiation load.For FCS operation all detectors able to count photons can be used for FCS. These are GaAsP PMTs of thearray detector, BiG.2 and the cooled multialkali PMT in the red spectral range. Multialkali PMTs of thethree channel configuration without GaAsP PMTs or NDDs cannot be used for FCS.Selection of the specimen detail of interest for zooming is fast and convenient, and the zoomed image isautomatically centered. This saves the job of specimen centration with the microscope stage.Bi-directional scanning doubles the scanning rate to a maximum of 13 frames/sec (at 512 × 512 pixels).Two fluorescent dyes can be viewed and documented in a quasi-simultaneous mode when using twodifferent laser wavelengths for bi-directional scanning. This will prevent cross talk between detectionchannels.
