Olympus IX50

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PRESENTATION

IX50

Olympus IX50 manual fluorescence microscope with mercury fluorescence illuminator, Nomarski/DIC Prism, cool CCD for fast low-light imaging, motorized filter wheels and micromanipulation / microinjection


Room: 2.1.15

IP: 10.101.134.37 (why?)


Check “What are the costs” at the Users section


PosMagNAIMCorr.WD (mm)Glass (mm)Color CodeObs
110x0.40Air / WaterUPlanApo3.10--
220x0.20AirLCPlanFL7.55 - 6.75--
340x0.85AirUPlanApo0.20--
4100x1.25OilPlanC N---


PositionExcitation (nm)Emission (nm)FluorophoresModel
1BP 330-385LP >420DAPI, hoechst
2BP 470-490LP >515GFP, Alexa488, cy2, FITC
3BP 530-550LP >590RFP, YFP, Alexa546, Alexa568, cy3, TRITC


Transmitted light

Halogen lamp

Fluorescence

Olympus U-RFL-T Mercury lamp


PCO Sensicam-QE 12bit


Ludl filter wheels for 6 excitation and 6 emission filters

Narishige micromanipulator


 

BASIC PROCEDURE FOR MULTICHANNEL GRAYSCALE IMAGES

  • Turn on PCO SensiCam camera (button 1).
  • Turn on transmitted light source box (button 2)
  • If doing fluorescence imaging, turn on fluorescence source box (button 3). Do not switch it 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.
  • Turn the Eyepiece/Camera knob to eyepieces, rotate the Fluorescence shutter to open, and select desired filtercube with the manual filterwheel. Select desired objective and bring your sample into focus.
  • Turn on the acquisition PC (4) and start uManager program

 

  • Turn the Eyepiece/Camera knob to camera position.
  • Select both desired filtercube and objective in the drop-down boxes in box (1). THESE ARE SOFTWARE SETTINGS AND DO NOT CONTROL THE MICROSCOPE. But they do allow you to set different exposures for each channel for multidimensional acquisition, and snap spatially calibrated images for each objective.
  • Pick the second lookup table in box (2). It will show black pixels as BLUE and white pixels as RED. This will help you with exposure settings.
  • Press “Live” button (3) to see your image.
  • Increase exposure (4) in each filter combination (change to the adequate “Filtercube” in (1) each time) until you see your signal, but very little to no saturation (white/red pixels). If the image “freezes”, insert exposure value again.
  • When you have picked correct exposure settings for all your channels, go to menu Tools > Scripts, and click and run the manualAcq2.bsh script – The software will now ask you for the specific filterwheel position for each acquisition.
  • Press Multi-D. Acq. Button (5) and check “Channels”. Add desired channels, pick a suitable lookup table color for each, and insert previously set Exposure values.
  • Press “Acquire” to start multi-dimensional acquisition. Turn manual filterwheel as requested by the software, and minimize table vibrations as the software is acquiring images.
  • Go to ImageJ File > Save As > save as TIFF. You will be saving a composite image with “x” 12/16-bit greyscale channels. Save it on your image folder. Copy your images from Voxel 6.
  • Turn off system in reverse order.

This system is equipped with DIC and two motorized filterwheels which can be enabled for fine fluorescent spectra adjustment. Contact Luís for help setting this up.

The system is also equipped with Narishige micro-manipulators/injectors. Contact Prof. Rui Malhó for instructions on setting up these kinds of experiments.


uManager for semi-automated multi-dimensional acquisition and control of filter wheels.

(Deprecated) Image Pro-Plus with Scope-Pro (control of filter wheels) and Sharpstack (deconvolution module)


LINKS / USER MANUALS


PEOPLE RESPONSIBLE FOR THE EQUIPMENT

Rui Malhó Telmo 10922654_10153982169038222_2387869307937053110_o
Rui Malhó

FCUL / BioISI

Head of Microscopy Facility

r.malho@fc.ul.pt

Room 2.1.44
Ext: 22144

Telmo Nunes

FCUL

Microscopy technician

mevarrimento@fc.ul.pt

Room 2.1.15
Ext: 22181

Luís Marques

FCUL

Microscopy technician

lfmarques@fc.ul.pt

Room 8.1.79 / 2.1.15
Ext: 28179 / 22181