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Leica SP8 Manual

Leica SP8 Manual

Upright confocal microscope
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Leica SP8 Upright confocal microscope
• System description:
• DMi6, Upright microscope: BF, Fluorescence (blue, green, red)
• BF TL Detector (transmitted light image)
• Scanning stage with z-Galvo and Navigator (overviews, selection of regions of
interest...)
• Scanner with Scan-field Rotation
• 2 PMT detectors (photo multipliers)
• 1 Hybrid Detector normal (more sensitive than PMT and no detector noise nor
background)
• 1 Hybrid Detector SMD (fast, for FLIM, FALCON)
• Laser lines: 405, White Light Laser (470-670nm, up to 8 laser lines simultaneously)
• FALCON (FAst Lifetme CONtrast) including Pulse Picker for WLL
• LIGHTNING (Deconvolution) for Superresolution and super sensitive imaging
• Phasor plot for direct examination of lifetime data
Objectives:
• Objective
HC PL FLUOTAR 5x/0.15
• Objective
HC FLUOTAR L 16x/0.60
use with 0.0 - 0.17 mm cover glass) + BABB cleared samples
oil)- (506533)
• Objective
HC FLUOTAR L 25x/0.95
• Objective
PL APO 20x/0.75 IMM
• System Start Up
Turn on heating Unit if needed
• Switch on 'PC-microscope' button (1). This gives power to the computer and
microscope controller
• Switch on 'scanner power' (2)- This enables the scanning head
• Switch on 'Laser power' (3)
• Turn the 'Laser ignition key' (4) in position 'on'- enables Laser shutters (-> Only an
interlock)
• Switch on fluorescent lamp(5) for visual examination
• TL/LED manual switch is at the back of the mic (if you don't see the TL light in the
eyepiece, it might be the switch)
14, 07, 2020
Dry , WD=12mm (overview) (506224)
IMM CORR VISIR, WD=2.5mm
Water-VISIR,
WD=2.5 mm, wide angle (41°)
CORR VISIR,
WD 0.67 mm
(Correction collar for
(Oil, water, glycerol, silicon
(Oil, glycerol)- (506343)
(506374)

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Summary of Contents for Leica SP8

  • Page 1 Leica SP8 Upright confocal microscope 14, 07, 2020 • System description: • DMi6, Upright microscope: BF, Fluorescence (blue, green, red) • BF TL Detector (transmitted light image) • Scanning stage with z-Galvo and Navigator (overviews, selection of regions of interest…) •...
  • Page 2 • For imaging: start LASX software. Select configuration Machine.xlhw or machine with zgalvo and DM6microscope (You should insert zgalvo stage and fix in place using 2 knobs at the back-> If you are not using it, place it face down in the heating chamber!) •...
  • Page 3 In User Config -> Your data can be organized into Folder structure or Project based structure. In the folder based structure files are saved as TIFF while projects are saved as Lif/Lof. (Due to a software bug do not use Folder base file saving) Additional info @: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserH elp/UserHelpEnglish/Default.htm?Highlight=user configuration#DLG/Configuration/DLG_configuration_user_configuration.htm?Highlight=us er configuration...
  • Page 4: Before You Start

    • A simple way to plan your confocal imaging is using the dye assistant. Open the menu and enter your fluorophores. Choose PMT. Then choose the imaging methods (simultaneous and/or sequential scanning according to your needs considering cross talk and imaging speed. Press "Apply" ). Additional info @: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserH elp/UserHelpEnglish/Default.htm#INS/INS_initial_image_acquisition.htm%3FTocPath%3DSt ep-by-step%2520Instructions%7CImage%2520Acquisition%7C_____5...
  • Page 5 Fig.3 • The system is turning on laser lines “or ask for it if you didn’t turn them on from config” (tunes to the excitation maxima of selected dyes), activates detectors with appropriate bandwidth a, sets the gain and shows the emission spectra of selected dyes.
  • Page 6 Fig.4 Set your confocal image: !!! Note: In addition to the software (Fig4), gain, offset, scan field rotation, pinhole, zoom, and z-position settings may be changed via the control knobs in front of you Position your sample: Change the zoom or rotate your image -> Changing scan speed (>700 Hz ) will change the minimum zoom.
  • Page 7 Applying frame or line averaging, reduces the imaging speed. Check the pixel dwell time • Gated detection (Useful for removing Reflection or background auto fluorescence) Note: Only possible using HyD detectors!!! • Used to reduce auto fluorescence or reflection with shorter lifetimes than actual fluorescence •...
  • Page 8 This way you can reduce the crosstalk and increase the speed of imaging. • You can save "sequential files" in your folder and load them again. Additional info @: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN /UserHelp/UserHelpEnglish/Default.htm?Highlight=lineseq#INS/INS_sequential_scan .htm?Highlight=line seq • Setting up a Z-stack •...
  • Page 9 Press ‘begin’ in the Z-stack menu. Move focus up with ‘Z position’, select the most superficial slice, press ‘End’. Set up Z-step size according to your needs or activate system optimized for Nyquist settings according to the objective and pinhole settings used Additional info @: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserHelp/U serHelpEnglish/Default.htm#DLG/Acquire/DLG_acquire_z_stack.htm...
  • Page 10 • To start time series, press ‘start’ • In the Navigator, selecting multiple marks with z-stacks and time will not show the correct estimated time. • Tile Scan See the manual for Navigator module on the desktop Additional info @: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserH elp/UserHelpEnglish/Default.htm#INS/INS_stage_overview_create_overview.htm...
  • Page 11 • go to “Open projects” menu • Save your data directly to DATAINT (E) on the Additional info@: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserH elp/UserHelpEnglish/Default.htm#OVW/Acquire/OVW_acquire_projects.htm • Please save the data in the correct group foloder the data will automatically updated to the scratch bioimaging: Data upload is triggered by Log off/Log on, data will be uploaded to the following...
  • Page 12 \\scratch3.ist.local\scratch-bioimaging\{Your group Name}\_ImageDrop\ For manual upload, map the above path. login your usermane should be inserted as : IST\username • PLEASE NOTE: All data on our E drive are deleted after 30 days automatically!!! • Acquiring confocal images using the Lightning – Deconvolution environment Fig.9 •...
  • Page 13 Fig.10 • Select your deconvolution Strategy: Adaptive or Global !!! Note: In the Adaptive setting Background and bright pixels are treated differently which might lead to non-linearity in your results (may create problems in quantitative measurements). The Global method is treating every pixel in the same way!!! •...
  • Page 14 • Raw data and deconvolved image will be saved as separate files in Open project Additional info@: file:///C:/Program%20Files/Leica%20Microsystems%20CMS%20GmbH/LAS%20X/BIN/UserH elp/UserHelpEnglish/Default.htm#DLG/Wizards/DLG_lightning_lightning_grade.htm • Deconvolution processing after imaging in standard mode • Images taken in the regular confocal environment may be deconvolved afterwards •...
  • Page 15: Laser Safety Instructions

    Laser Safety Instructions • During operation of class 3B and class 4 lasers red warning lights have to be switched on manually • Red warning light at the door of the room, containing laser-based equipment, prohibits the entrance • Optical path of the laser beam at all setups has to stay intact and should never be disassembled by a user.