Process to achieve advanced knowledge of functional neuroanatomy and systems neuroscience. Studies involve detailed explorations of the anatomical structures and pathways that underlie sensation and perception in each of the sensory modalities.
Confocal upright microscope (Zeiss LSM 880) for mounted dry tissue sections and wet specimens
The confocal laser scanning microscope is an optical imaging technique used to visualize extremely small structures, down to the cellular and sub-cellular size. Its use permits the acquisition of clearer and more detailed pictures when compared with a general widefield microscope, plus the possibility of creating three-dimensional representations of the tissue visualized.
In our facilities it is possible to use two models of upright Zeiss LSM 880 fluorescence confocal microscope: an AxioImager.Z1 used for mounted dry tissue sections and an AxioExaminer.Z1 for wet specimens. The microscopes are equipped with a range of high quality objectives (10-63x magnification, using air, oil and water as immersion mediums). A varied set of lasers is used as light source, comprising most of the visible spectrum (405, 458, 488, 514, 561, 594 and 633 nm lines). Signal detection is achieved through a spectral detector (34 PMTs, 32 of them being GaAsP detectors). The microscopes are controlled by Zen Black edition software.
Automated slide scanners
Two Zeiss AxioScan.Z1 automated slide scanners are available for large-scale widefield digitizing of histological material. Each system fits up to one hundred 26×77 mm microscope slides, it is also possible to load 52×77 mm slides. In addition to a white lightsource for transmitted light brightfield imaging, the systems are equipped with Zeiss Colibri2 LED sources of different wavelengths for epifluorescence imaging (365, 470. 555, and 625 nm). The two imaging modes employ respectively a 24-bit Hitachi RGB camera and a 16-bit Hamamatsu BW camera. The slide scanners are equipped with high quality air objectives ranging from 5-40X magnification. The systems are controlled by Zen Blue software which also includes options for image processing and analyses such as fluorescence intensity profile.
Standalone upright widefield microscopes
The facility provides one standalone Zeiss AxioImager.M1 upright microscope for checking histological material and taking single, non-tiled images. The stage is manually controlled and can fit 26×77 mm microscope slides. The microscope is equipped with transmitted light brightfield as well as epifluorescent Zeiss Colibri7 LED lightsources (wavelengths 385-630 nm), in addition to air objectives ranging from 1.25-40x. A Zeiss AxioCam MRc camera is fitted on the microscope and is controlled by Zen Blue software.
For detailed neuroanatomical analyses we provide two upright Zeiss AxioImager microscopes, models M1 and M2. Both microscopes are equipped for transmitted light brightfield as well as epifluorescent imaging and have high quality air and oil objectives ranging from 1.25-100x. The microscopes are also fitted with MicroBrightField CX9000 cameras. The stages are motorized and can fit 26×77 mm microscope slides. The microscopes can also be controlled by MicroBrightfield software installed on the accompanying computers. The StereoInvestigator software is used for unbiased stereology, whereas Neurolucida gives the opportunity to reconstruct neurons or tracing other anatomical structures in 3D. The software may also be used for taking tiled images of smaller areas but not the whole slide.
Processing and analysis of imaged material
Three work stations are available for processing and analysis of imaged material. Software installed includes Zeiss Zen Blue Desk (two licenses) for processing .czi images from the AxioScan.Z1 systems, SVI Huygens software for deconvolution of images (one license), and MBF NeuroLucida360 software for 3D neuron/tissue reconstruction and analysis (one license).