Leica DMI6000b

Inverted motorized fluorescence microscope for High-Throughput Screening

BOOK THIS DEVICE


PRESENTATIONDMI6000b

The Leica DMI6000 B is a fully automated inverted fluorescence microscope. It features automated contrast and illumination manager, motorized Z focus, automatic brightness and diaphragm adjustment, autofocus, automated multiposition imaging and other automated functions. This system is suitable for high throughput projects and automated imaging in multiwell imaging media. It can accomodate an SP8 confocal module (not installed). This tutorial shows the location and function of some of the major components of the microscope.


Room 8.1.79

Computer name: leicadmi6000b / 10.101.93.191 (why?)


PositionMagnificationNumerical ApertureImmersion MediumCorrectionWD (mm)Glass (mm)Color CodeObservations
15x0.15AirPL S-APO13.7-DIC
210x0.40AirHC PL APO2.20.17
320x0.75Water, glycerol, oilHC PL APO0.68-Correction collar
440x1.10WaterHCX PL APO0.630.14-0.18Correction collar

No phase contrast; DIC ONLY ON 5x


PositionExcitation (nm)Emission (nm)FluorophoresModel
1340-380450-490DAPI, hoechstA
2425-445455-485CFPcustom CFP
3490-510520-550GFP, Alexa488, cy2, FITC, YFPYFP
4515-560>590mCherry, cy3, TRITC, Alexa568N2.1
5630-660>670Alexa 633, Alexa647, cy5custom Cy5
6---DIC ANA + BF


Transmitted light

Halogen lamp

Fluorescence

EL6000 

Mercury metal halide bulb

Lifetime: 2000 hours

Integrated fast shutter


Hamamatsu Orca-Flash4.0

Monochrome CMOS camera 4.0 MP (2048 x 2048 pixels)

Pixel size 6.5 μm × 6.5 μm

Bit depth 16 bit

Peltier cooling


Water immersion micro dispenser

Bartels Mikrotechnik Extended Micropump Controller

Allows continuous water distribution for automated imaging of multiwell plates. Available for all water immersion objectives (20x and 40x).

Universal supports for for multiwell plates and microscopy slides

Immersion oil (RI 1.5180)


 

  • Turn on microscope controller box (1), fluorescence source if needed (2), Hamamatsu camera (3), and immersion pump (4). Do not switch fluorescence source off for 30 minutes after switching on, and DO NOT switch on if the metal casing is still warm and it was switched on less than 45 minutes before.
  • Allow motorized stage to finish full range motion for calibration. Start LAS X software, and click “OK” on the configuration window.

 

 

  • Add desired fluorescence channels using button 1, and match each one to the appropriate filtercube (2). Change to preferred lookup table color in each one by clicking in the colored window. If using brightfield, select TL-BF instead of FLUO from the drop-down box (3)
  • Cycle through viewing lookup tables (button 4) and pick the “saturation” one. Grey levels will appear as reddish-brown, black pixels will show in green and white pixels will show in blue. This will help you choose proper exposure for each channel.
  • Select a low magnification objective (5) – 10x is a good starting point. Click one of the channels (preferably one with good signal, usually nuclei stained with DAPI/Hoechst), press the “Eyepiece” selector button on the microscope console, and then the “Shutter” button. Look through the eyepieces, move through your slide with the X-Y-Z joystick, and bring your sample into focus.
  • Press “Live” button (6), and adjust proper exposure for each channel (7). Avoid white/saturated pixels which show up in blue in the viewing window.
  • When all channels are set up with the proper exposure, press “Start” button (8) for acquisition. If you change objective, readjust exposure.
  • Your acquisitions will be gathered as *.lif files, in the “Open projects” tab (9). Save it in your image folder. You may create “collections” inside *.lif files by right-clicking on them. A typical structure may be [Project: Experiment name or Date] \ [Collection: slide name or number] \ [Image: name of experimental condition]
  • When you’re done with work, check if there are users scheduled after you. If not, close program, switch off machinery in reverse order, and leave a notepad.exe note on the computer desktop informing the time you switched off the fluorescence.

This microscope allows stage experiments (mosaic, mark&find) and automated fine focus (box 10). Contact Luís for further help with these settings.

Live imaging (with temperature and CO2 control) is also available, as well as High Content Data acquisition. Contact Hugo or Luís for assistance in setting up this kind of experiments.


Leica LAS X

Including Leica High Content Screening Software (MatrixScreener)


LINKS / USER MANUALS


BioISI members: Free

Non-BioISI members: 10 €/h


EQUIPMENT RESPONSIBLE

HugoQ67DIEPD 10922654_10153982169038222_2387869307937053110_o
Hugo Botelho

BioISI

FunGP

hmbotelho@fc.ul.pt

Room 8.1.76

Ext: 28176

Luís Marques

BioISI

Microscopy technician

lfmarques@fc.ul.pt

Room 8.1.79

Ext: 28179