The 2 major technologies currently available at the Bioimaging Platform are light microscopy and electron microscopy.

Two-photon confocal microscope

Zeiss 710 NLO Intracellular physiology can be dependably studied in intact organisms only. By multiphoton microscopy it is possible to explore fundamental mechanisms in functional tissue. A perfect choice for neurobiologists, developmental biologists, immunologists and plant biologists: the 2-photon microscope captures high-resolution images of fluorescent structures in living animals and thick tissue specimen. Our new 2-photon microscope (Zeiss 710 NLO) has a high signal-to-noise ratio enabling brilliant imaging even in deep-lying tissue layers. Pulsed, ultrafast infrared lasers excite fluorescent dyes at the focus only - the only place where photon density is high enough to produce fluorescence. With low absorption and next to no scattering loss, the red excitation light penetrates much deeper into tissue. With some tissues it excites fluorochromes at depths up to 1 mm. Thanks to the small excitation volume, images of subcellular resolution are obtained. Being only excited at the focal plane, the dye won’t bleach above and below it: ensuring unmatched preservation of your fluorescence-labeled specimen. Specifications • Femtosecond biphoton laser (690-1040 nm) plus single-photon excitation lasers (405, 458, 476, 488, 514, 543 and 633 nm). The LSM710 can also be used as a traditional confocal microscope with its 3 descanned detectors. • Non-descanned detectors with 3 combinations of emission filters (blue⁄green, green⁄red, CFP⁄YFP). Fluorescence light excited with the multiphoton laser originates from the focus only. Thus, no confocal pinhole is needed, and the signal can be projected directly from the specimen onto the detector, gaining sensitivity. • GaAsP non-descanned detector. High efficiency detection (up to twice the level of that of standard non-descanned detectors) and low dark noise: the ideal tool for visualizing very fine details in tissue that may be extremely light-scattering. • The system is equipped with a very high NA, long distance, 20x dip-in objective, a 40x dip-in and a 63x oil objective.

Spinning disc confocal microscope

3i Marianas For imaging of rapid events like calcium signaling, vesicle transport or chromosome dynamics, traditional point scanning confocal microscopes are often not fast enough. With the spinning disc technology it is possible to do confocal optical sectioning at video rate enabling the visualization of very fast processes in living cells at high resolution. Our new spinning disc confocal system (Intelligent Imaging Innovations Marianas SDC) is mounted on an inverted microscope (Leica DMIRE2). It is equipped with two lasers having 488 nm (for excitation of green-emitting fluorochromes) or 560 nm (for excitation of red-emitting fluorochromes) excitation wavelengths. For imaging a highly performing 63x glycerin objective is available.

Classical confocal microscopes

Leica TCS SP2 AOBS The Leica SP2 AOBS has 8 excitation lines (405, 458, 476, 488, 514, 543, 594 and 633 nm) and 4 tuneable detection channels. The prism technology allowing the free selection of detection windows makes the system optimal for separating closely juxtaposed emission signals and adaptable for newly developed fluorochromes. The microscope stand is inverted making it thus possible to look at live cells growing in 35 mm glass bottom Petri dishes. The user interface is easy to use and has applications wizards for doing FRAP (fluorescence recovery after photobleaching) and FRET (fluorescence resonance energy transfer) experiments. For live cell imaging we can control the temperature of the microscope and run it with 5% CO2.

Zeiss 510 Meta The second confocal microscope, the Zeiss 510 Meta, has 7 excitation laser lines (405, 458, 476, 488, 514, 543 and 633 nm) and 2 detection channels (photomultipliers). In addition it has the special Meta-detector, a polychromatic multichannel detector that is able to instantly acquire fluorescent emission spectra. The spectral information allows then the separation of complex fluorescent signals through a process called emission fingerprinting. The microscope stand is upright and the system is equipped with three dip-in objectives (20, 40, 63). These objectives make it possible to directly view cell cultures and other live organisms from the top. Temperature and CO2 content can be controlled.

Widefield microscopes

Leica AF6000 LX The Leica AF6000 LX is an integrated system for advanced widefield fluorescence imaging and analysis. This ultra-fast system offers the ultimate in hardware and software integration to study the processes of life. Imaging fast cell dynamics or 4D experiments over several days can easily be performed. Carefully selected components ensure the necessary stability for long term experiments keeping the cells in optimal condition. The system is equipped with two cameras. 1. Coolsnap HQ from Roper Scientific. Pixel size is 6.4 µm in a 1024 by 1024 CCD chip. This camera is fast, has a good sensitivity and allows high resolution due to the small pixel size. 2. CascadeB from Roper Scientific. Pixel size is 16 µm in a 512 x 512 CCD chip. This backthinned CCD offers highest sensitivity (over 90% quantum efficiency) plus a very high read-out time. Due to the larger pixel size resolution is inferior compared to the Coolsnap camera. Filters available on the system are: A4 (UV-filter), BGR (blue-green-red triple filter), GFP, CFP, YFP, Y5 and I3 (FITC). In addition there is a special FRET filter for the measurement of sensitized emission available. Objectives are the following: 10x (dry), 20x (multi-immersion), 40x (dry, long distance), 63x (glycerine immersion, corrected to work at 37°C) and 100x (oil immersion). The microscope stand sits in a thermo-box where the temperature can be regulated from 22°C to 37°C. For mammalian cells, humidification and CO2 are equally available.

Zeiss Axio Imager Z1m The Axio Imager Z1m from Zeiss is a fully motorized upright microscope. The system is intended for an easy and fast approach to fluorescence microscopy of live and fixed samples. It is equipped with a sensitive monochrome camera (Zeiss, Axiocam MRm) and has filter cubes for UV (Dapi, Hoechst), CFP, FITC, YFP, TRITC and Cy5 (far red). It is possible to take image stacks in multiple colors and to do time laps experiments. The user interface is very simple and the entire system is straightforward and easy to use. Users especially like the possibility to use dip-in objectives they can utilize for the analysis of fluorescent expression patterns of cells growing in petri or multi-well dishes.

Bioluminescence workstation

Olympus LV200 For cell biology the use of luminescence bears the advantages of stronger sensitivity and highest signal to noise ratio compared to fluorescence. With conventional luminometers it was so far only possible to measure the average luminescence intensity of a sample, but particularly for the analysis of cultured cells or tissues it is of great interest for many researchers to resolve cellular structures in detail. At the Bioimaging Platform we have now installed a system that is able to just do this. The LV200 has been developed by Olympus and could be best described as an imaging luminometer. Based on a new optical concept and special components the LV200 allows not only to characterize the specimen by the luminescence intensity but to image cellular samples and to quantify the luminescence structures at cellular and even sub-cellular resolution. The imaging luminometer is therefore ideally suited for the long-term observation of luminescence based reporter gene assays where phototoxicity of the fluorescence excitation is an issue.

Live cell imaging

Nikon BioStation IM The Nikon BioStation IM incorporates a microscope, an incubator and a sensitive cooled CCD camera in a compact body. This all-in-one package provides a stable environment for live cells and advanced solutions for simple long-term time-lapse data acquisition. Moreover, focus drift caused by thermal change and mechanical instability has been minimized, and fail-proof data acquisition is possible even during lengthy timelapse imaging. The system is user-friendly and easy to use. Equipment and configuration • Inverted microscope • Incubator (5% CO2, 37°C) • Objectives: dry: 40x (20x, 40x, 80x by switching magnification tube lens), NA 0.8 • Semrock filter blocs: - GFP: EX 450-490/DM 495/BA 500-545 - YFP: EX 480-415/DM 520/BA 525-560 - TxRed: EX 540-580/DM595/BA 600-650 • Illumination: Intensilight precentered fiber mercury lamp • Red LED illumination is used for phase contrast imaging (to reduce cell damage) Applications • Designed for live cell imaging • 2D, 3D (Z stacks), multipositions, timelaps • Acquisition in constant condition • Phase contrast • Fluorescence • Perfusion during acquisition • 1x 35 mm glass bottom dish, single or multichamber (4 different conditions) or 1x slide

Imaging software

• Imaris® by Bitplane provides capabilities for working with three-dimensional images.
• AutoDeblur® from AutoQuant uses deconvolution algorithms to reassign out-of-focus light in image stacks to where it belongs.

Transmission electron microscope

FEI Tecnai™ G2 Sphera The Tecnai™ G2 Sphera from FEI has been installed in 2003. It runs at 200 kV with a LaB6 cathode. It is equipped for tomography and has a cryo-stage. The system has a 2000 by 2000 pixel high resolution digital camera. The contrast of this microscope is generally much lower than that obtained on the older Philips EM 410, which is now no longer available at the Platform.

Scanning electron microscope

JEOL JSM-6510LV The JEOL JSM-6510LV low vacuum scanning electron microscope (SEM) is a high-performance SEM for fast characterization and imaging of fine structures (high resolution of 3.0 nm at 30 kV). It enables observation of specimens up to 150 mm in diameter. The selectable “low vacuum” mode allows for observation of specimens that cannot be viewed at high vacuum due to excessive water content (like many biological samples) or because they have a non-conductive surface. In addition to routine imaging at hundred time greater resolution than optical microscopes, and with a focal depth over ten times greater, the SEM allows for detailed measurements, including 3D measurements from stereo images. Dual live image display of the secondary electron image and a backscattered composition image allow the user to contrast and compare specific details. Other highlights • Mechanically eucentric stage • Fast, unattended data acquisition (with stage automation) • Smart settings for common samples (create/store/recall) • Streamlined design • Compact footprint • Enhanced SE imaging • Superior low kV imaging • Multiple live image display (including picture in picture) • Signal mixing • Live, full screen image • Video capability (.avi files)

Specifications

Resolution	• High vacuum mode: 3.0 nm (30 kV) • Low vacuum mode: 4.0 nm (30 kV)
Accelerating voltage	0.5 to 30 kV
Magnification	5x to 300,000x (printed as a 128 mm x 96 mm micrograph)
Objective lens apertures	Three position, controllable in X⁄Y directions
Maximum specimen size	125 mm ∅ full coverage (152.4 mm ∅ loadable)
Specimen stage	• Eucentric goniometer • X = 80 mm, Y = 40 mm, Z = 5-48 mm • R = 360° (endless) • Tilt -10⁄+90° • Option: computer controlled 2, 3 or 5 axis motor drive

Utra-cryo-microtome


Leica UCT

This microtome is equiped with cryo-unit FCS for low temperature sectioning of biological samples.



High pressure freezer


Leica EM PACT

This frezer is designed for high pressure ultra-fast freezing of biological samples for electron microscopy.



Freeze substitution system


Leica EM AFS

This is a system designed for freeze substitution and low temperature embedding after cryofixation and for the PLT (Progressive Lowering of Temperature) technique.