Dorigny resources MICROSCOPES - BIOPHORE. Confocal Microscope Zeiss LSM 700

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1 Dorigny resources Contributed by Yannick KREMPP Friday, 13 July 2007 Last Updated Monday, 30 October 2017 MICROSCOPES - BIOPHORE Confocal Microscope Zeiss LSM 700 This Zeiss LSM700 confocal is mounted on an inverted Zeiss Axio Observer Z1 using a motorized XY stage controller. We have 4 solid state laser lines (445, 488, 555 and 639nm) available on this system. Currently four objectives are on the system: a Plan Apo 10x Ph1 objective with a 0.45 NA, a Plan Apo 25x Oil immersion DIC objective with 0.8 NA, a Plan Neofluar 40x Oil immersion DIC objective with a 1.3 NA and an Apochromat 63x Water immersion DIC objective with a 1.2 NA. Two photomultiplier detectors are available, each coupled with 8 emission filters for spectral flexibility on the detection of the fluorescence signal. An additional transmitted light detector may also be selected to allow the users to acquire a transmitted light image of the samples in confocal mode. The software running the LSM700 is the ZEN ("Zeiss Efficient Navigation") software. That software allows a really easy and straightforward use of the confocal. Using the so-called "Smart Setup" function, the software will drive you in a few clicks through the light path configuration to set up for your own experiments. Additionally, the focus stabilizer ("Definite Focus") implemented on this confocal microscope enables maintaining the focus stable in the z axis for in long term (typically time-lapse) acquisitions.

2 Confocal Microscope Leica SP 8 FLIM This confocal microscope comes with a pulsed supercontinuum (White Laser Line) tunable laser in the visible range from 470nm to 670nm. Tune freely your own BP filter settings for detection that are matching for your fluorescent detection. The fine tunable WLL (every wavelength with 1nm accuracy can be selected from the nm range) will allow to perform any type of excitation within this range and also measuring the excitation spectrum of your fluorescent dyes. Complete excitation emission spectra can be recorded. A FLIM (Fluorescence Lifetime Imaging) module is also attached to this system. So with that you can measure precisely fluorescence lifetime of your dyes and perform Single Molecule Detection (SMD). This feature is fully integrated to the confocal system (developed by PicoQuant company using Symphotime software). This SMD FLIM can also be used to make FRET experiments. The microscope stand is an inverted one fully motorized. The system has a motorized xy stage controller. The motorized z focus can use the microscope motorization or the table top piezo z for fine and fast z movements. On the detection side of the fluorescence of your samples its fully filter free you can design and fine tune your emission spectrum window freely. Two sensitive HyD + 1 HyD-SMD/FLIM detectors (Hybrid Detectors) are installed with one standard PMT detector. The HyDs are special detection devices that will allow you to time shift the detection of the light coming from the sample using the so called gating feature (adjusting in time gate switches off the data collection during the WLL laser pulse) this will help in many cases to get read of autofluorescence/biological background of the sample used. The HyDs are also generating no electronical noise and can be used for photon counting experiments.

3 A transmitted light PMT is added to the system for the confocal brightfield recordings. As complementary lasers on the Leica SP8 we have an Argon ML laser generating the 458, 488 and 514nm lines. A 440nm solid state laser is also present for the FLIM measures. On the objectives side the system is equipped with a: 10x HC PL FLUOTAR NA 0.30 Dry, 20x HC PL NA 0.75 IMM, 40x HC PL IRAPO NA 1.10 WATER, 63x HC PL APO NA 1.20 WATER. In summary this system will enable you to perform all the traditional confocal multi-dimensional experiments as consecutive optical slices (z-stacks), xy tiles stitched automatically together, time lapses, fluorescent multi channels acquisitions, spectral characterized experiments and all these can be combined. Also precise FRAP/FRET or FLIM experiments can be performed. This brand new system is now installed at the CIF Dorigny campus (Biophore, Room 4416).

4 MICROSCOPES - GENOPODE Confocal Microscope Zeiss LSM 510 Meta INVERTED Available Laser lines : Diode 30mW 405 nm Argon Multiline 30mW 458/477/488/514 nm HeNe 1mW 543 nm HeNe 2mW 594 nm HeNe 5mW 633 nm Equiped with transmited detector for DIC and Phase Contrast. Meta detector for emission spectra of fluorochromes. Attached software : LSM Software Rel. 3.5

5 INVERTED Microscope Zeiss AXIO Vert 200 M XY motorized Stage Filter cubes Set 01 DAPI Ex BP 365/12, FT 395, Em LP 397 Set 09 FITC Ex BP , FT 510, Em LP 515 Set 15 Rhod Ex BP 546/12, FT 580, Em LP 590 Objectives EC Plan-NEOFLUAR 10x/0,30 EC Plan-NEOFLUAR 20x/0,50 EC Plan-NEOFLUAR 40x/1,30 Oil DIC Plan-APOCHROMAT 63x/1,40 Oil DIC Plan-APOCHROMAT 100x/1,40 Oil DIC Camera : AxioCam MRm Axiovision Software Rel. 4.4 Illumination: HAL 100, HBO 100

6 Zeiss LSM 710 NLO (Non Linear Optics) Laser lines Available visible laser lines 458, 488, 514, 561 and 633 nm for conventional laser scanning confocal microscopy. The available laser for the 2 Photon acquisitions is a Ti:Sapphire Chameleon Ultra II infrared laser, tunable from 690 nm to 1040 nm with an maximum output power of 4W at 800 nm. Microscope An upright Zeiss AxioExaminer Z1. Objectives available on the system, currently 3 objectives are available on the system:

7 Achroplan 10x W objective with a 0.3 NA Plan-Apochromat 20x W objective with a 1.0 NA Apochromat 63x Water-Oil immersion objective with a 1.2 NA Attached Software ZEN 2009 (Zeiss Efficient Navigation 2009) Detection modes The light may be detected in the confocal scanhead (internal or 'descanned' detection) Confocal detection on external 'non-descanned' detectors (NDD) Confocal detection on GaAsP non-descanned detector (GaAsP NDD)

8 System Capabilities In depth confocal microscopy for thick samples 3D optical sectioning. Spectral infrared excitation fingerprinting. Spectral emission fingerprinting. FRAP, FRET, Time lapse, fluorescent dyes uncaging Confocal Microscope Zeiss LSM 510 Meta UPRIGHT Cellular Imaging Facility

9 Available Laser lines : Diode 30mW 405 nm Argon Multiline 30mW 458/477/488/514 nm HeNe 1mW 543 nm HeNe 2mW 594 nm HeNe 5mW 633 nm Equiped with transmited detector for DIC and Phase Contrast. Meta detector for emission spectra of fluorochromes. Attached software : LSM Software Rel. 3.5 UPRIGHT Microscope AXIO Imager Z.1 Fully motorized Filter cubes Set 49 DAPI Ex G 365, FT 395, EM BP 445/50 Set 38 HE egfp EX BP 470/40, FT 495, EM BP 525/50 Set 43 HE Cy3 Ex BP 550/25, FT 570, EM BP 605/70 Set GFP Chroma Objectives EC Plan-NEOFLUAR 10x/0,30 ACHROPLAN 10x/0,30 W Ph1 ACHROPLAN 20x/0,50 W Ph2 ACHROPLAN 40x/0.80 W EC Plan-NEOFLUAR 40x/1,30 Oil DIC ACHROPLAN 63x/0,95 W Plan-APOCHROMAT 63x/1,40 Oil DIC

10 Camera : AxioCam MRm Axiovision Software Rel. 4.4 Illumination: HAL 100, HBO 100 Stereomicroscope Leica MZ16FA Illumination Standard + Fluorescence Fluorescence Leica EL6000 (lasts up to 2000 hours) Magnification 1x up to 180x Filters DAPI, GFP, CY3, Transmitted light Attached software : Leica Application Suite (LAS)

11 Cameras : Leica DFC350FX Monochrome Leica DFC320R2 Color Motorized filter wheel, focus and zoom. Laser Microdissection and laser tweezers Microscope : Inverted OLYMPUS IX71 Illumination: HBO, HAL Filters cubes for Dapi, FITC, TRITC: narrow-band DAPI Ex BP , BS DM400, Em BA wide-band FITC Ex BP , BS DM505, Em BA wide-band TRITC Ex BP , BS DM570, Em BA Objectives: Plan Fluorite 4X/0.13 PHL wd 17.00mm

12 Plan Fluorite 10X/0.30 PH1 wd 1.60mm Plan Fluorite 20X/0.45 PH1 wd mm.60mm Plan Fluorite 40X/0.60 PH2 wd 2.70mm 4.00mm Plan Fluorite 60X/0.70 PH2 wd 1.50mm 2.20mm Plan Super Apochromat 100X/1.40 oil imm wd 0.12mm Modules : mmi CellCut Laser Microdissection System Computer controlled xy-table mmi CellManipulator Laser tweezer Multi Cap System Multi-Slide System Attached Camera : 3 Chip RGB CCD Sony camera 20 Interactive Pen-Screen Display Laser for cutting and isolation of tissue samples : Solid state UV-A laser, 350 nm Trapping Laser CellManipulation : YAG IR-laser, 3 W, 1064 nm Attached Software : mmi UV Cut Software Cellexplorer image analysis software package CellManipulator Software Package

13 TIME LAPSE / ION-imaging, Multidimentional acquisition station Illumination : Visichrome monochromator Halogen lamp Microscope stand : Inverted microscope Zeiss Axiovert 200 M with XY motorized stage controller Full automatic Objective changer Automatic Z-focus Automatic filter change Installed filter cubes : Single Filter sets for DAPI, FITC, Rhodamine Triple Filter set for DAPI, FITC, Rhodamine Objectives : Plan-Neofluar 20x/0.50 Ph2 Plan-Neofluar 40x/1.30 Oil DIC Plan-Apochromat 63x/1.40 Oil DIC Plan-Apochromat 100X/1.40 Oil DIC Camera : Roper Scientific CoolSnap HQ

14 Objective Heating Ring system Thermostatized Heating tabletop incubation chamber CO2 controller Attached Softwares : MetaMorph 6.3 (Universal Imaging) MetaFluor 6.3 (Universal Imaging) WORKSTATIONS - GENOPODE Stations short description : Dell Precision 670 2x Intel Xeon 3.00 GHz (800MHz FSB / 2 MB L2 cache), processor Hyper-threading 2.0 GB (2x1024MB DIMM) 400MHz DDR2 ECC SDRAM 128Mo PCIe x16 (DVI/VGA) nvidia Quadro FX " LCD TFT flat wide aspect screen Installed softwares :

15 Metamorph Image acquisition and analysis. NIH Image / Scion Image / Visilog / Image J General purpose image analysis software with programming language. Bitplane IMARIS suite 4.1 Consisting of Imaris, ImarisMeasurement Pro, ImarisTrack and ImarisColoc. Large range of specific image file readers for 3D microscopes. SLicer, Section, Gallery, Easy 3D shadow Projection, real time volume rendering. Interactive 3D distance measurement, volume and voxel intensity statistics in 3D and 4D, manual object segmentation with contours. Leading 3D and 4D colocalization. Bitplane ImarisXT - programing interface Bitplane Filament Tracer 4.1, to segment and visualize filamentous structures AutoAligner 2.0, for automatic and manual registration of serial 2D images SVI HUYGENS Essential Rel D Deconvolution software Huygens Essential is an image processing software package tailored for deconvolution of microscopic images. Huygens Essential is able to deconvolve a wide variety of images ranging from 2D widefield (WF) images to 4D multi-channel two-photon confocal images. To facilitate comparison of raw and deconvolved data, or results from different deconvolution runs the Essential is equipped with a dual 4D slicer tool. Powerful and fast renderers produce different representations of your data, not needing any special graphic card. Xenogen Living Image Xenogen 3D analysis package Imaging software from the Whole animal imaging system, IVIS 3D from Xenogen. Leica Confocal Software

16 Zeiss LSM 510 software Zeiss Axiovision ROOMS ROOM System CIFD_1A Confocal Zeiss LSM 510 Meta CIFD_2A Time lapse / Ion imaging CIFD_3A Laser Microdissection CIFD_4A Leica Macro Fluo CIFD_4B Whole Animal Imaging CIFD_5A Culture Lab CIFD_6A Workstations

17 Phone numbers : Confocal Leica SP2 AOBS (Biophore) +41 (0) CIFD_6A +41 (0) CIFD Office Technical Manager +41 (0)