Leica SPE

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PRESENTATION

Leica SPE

The Leica TCS SPE is a high-resolution spectral confocal system, based on a Leica DMI4000B microscope.


Location: 2.1.15

Computer name: 


TBA


PosMagNAIMCorr.WDGlassColor CodePixel sizeStep size
14x0.1AirHCX 4x W> 1mm0.17 mm (#1.5)>100 um
210x0.3AirHC PL APO CS1500 um0.17 mm (#1.5)1.615 um/px~8.0 um
320x0.7AirHC PL APO CS590 um0.17 mm (#1.5)0.750 um/px~2.0 um
4option
540x1.15OilACS APO240 um0.17 mm (#1.5)0.380 um/px~1.0 um
663x1.4 - 0.6OilHCX PL APO100 um0.17 mm (#1.5)0.242 um/px~0.6 um
Option2.5x0.07Air>2 mm0.17 mm (#1.5)
Option40x0.7AirHCX PL APO CS200 um0.17 mm (#1.5)0.380 um/px~2.0 um
Option63x1.15WaterHCX 63x W150 um0.15 - 0.19 mm0.242 um/px~1.0 um
Option40x1.25 - 0.75OilHCX PL APO CS100 um0.17 mm (#1.5)0.380 um/px~1.0 um

Pos Position

Mag Magnification

NA Numerical Aperture

IM Immersion Medium

Corr. Correction Type

WD Working Distance

Glass Coverglass

Pixel size @ 1024×1024; 1x zoom

Step Confocal section thickness (1 AU)

DIC prism available for HCX 63x W


PositionExcitation (nm)Emission (nm)FluorophoresModel
1450-490>515GFP, Alexa488, cy2, FITCI3
2515-560>590mCherry, YFP, cy3, Alexa546, Alexa568, TRITCN2.1
3330-385>420DAPI, HoechstA

For eyepieces only!


Transmitted light

Halogen lamp

Fluorescence

Mercury metal halide bulb

Lifetime: 2000 hours

Integrated fast shutter

Laser lines

488 nm, 532 nm and 635 nm

ATTENTION: this system does not have a UV laser so DAPI staining cannot be imaged in confocal mode. Use red DNA dyes instead (eg ToPro3)


1 PMT spectral (400-700nm) 1 transmitted light detector;

(non-normalised spectra)

X Y Z T Lambda acquisition


Immersion oil (RI 1.5180)


Turning system ON:

  1. Before mounting sample in microscope:
  2. Turn on the microscope controller box Turn on the “PC”
  3. Start LAS AF software Click “continue” (if errors occur default parameters should be “Machine” and “DMI4000”)
  4. Turn on Fluorescence light power source (check first if the lamp is not hot), otherwise leave it off and continue

Using the system:

  1. Create new experiment and load acquisition settings (you can also open a previous experiment and choose “properties” and then “apply settings”)
  2. Change from confocal mode to visualization mode to view through eyepieces (TL/IL on left side of microscope and “eye” button on top left of front panel)
  3. Using microscope to inspect sample and center image in field: Push button TL/IL to switch from brightfield and fluorescence (display will show BF or FLUO). In fluorescence mode press shutter to open/close light (7). Switching fluorescence filters: – RFP, Alexa568,543, Cy3, red fluorophores – GFP, Alexa488, Fluorescein/FITC, green. – DAPI and other blue fluorophores (not to be used in confocal!) – For DIC (HCX 63x W only!): Switch to DIC mode (button on left side of scope: LCD reads DIC, not POL or BF). Adjust compensator on turret, left side of scope below lens 6. Turn “laser” key on and wait ~1min for laser “warm-up” and start collecting images. Attention: if acquiring long time-lapse images avoid leaving fluorescence lamp on, and if interrupting the session for more than 30 min turn off lasers/fluorescence

Turning system OFF:

  1. Turn off fluorescence illuminator as soon as not needed
  2. Turn off the “LASER” key on the PC unit (red light goes off)
  3. Make sure you saved all images in “Experiments panel”
  4. Remove Sample from the microscope and gently wipe-off oil from lens (DO NOT RUB!)
  5. Exit “LAS AF” software and go to the “download” PC do copy your images. User/pass are “microscopia”
  6. Turn off microscope controller box; NEVER COVER THE MICROSCOPE WITHOUT TURNING OFF CONTROLLER BOX…IF BRIGHTFIELD LIGHT IS LEFT ON AND PLASTIC COVER IS PUT ON THE MICROSCOPE YOU CAN START A FIRE!!!!
  7. If you think the lens is dirty please inform one of the lab directors and we will clean it. DO NOT REMOVE AND CLEAN LENSES YOURSELF UNLESS YOU WERE TRAINED AND AUTHORIZED BY ONE OF THE LAB DIRECTORS TO DO IT

Make sure you log usage in the “user record” book, mentioning any problems


To open the images you can either use the Leica LAS-AF software (windows only!) or ImageJ with the latest LOCI plugin installed


LINKS / USER MANUALS


PEOPLE RESPONSIBLE FOR THE EQUIPMENT

Telmo gabriela_rodrigues

Telmo Nunes

FCUL

Microscopy technician

mevarrimento@fc.ul.pt

Room 2.1.15

Ext: 22181

Gabriela Rodrigues

FCUL / CE3C

Assistant professor

mgrodrigues@fc.ul.pt

Room 2.3.10

Ext: 22310