IGC

WARNING

This is to remind you the basic rules of fluorescence lamps and gas lasers:

  • Never turn OFF the fluorescent lamp if it's been on for less than 30 min.
  • Never turn OFF the lasers if they've been on for less than an hour (1 Hour).
  • Never turn on the lamp if it's been off for less than 30 min (check to see if it's still warm).
  • Never turn on the lasers if they've been off for less than hour (1 hour).
  • Never turn off the lamp or lasers if someone else is using the microscope within the next 4 hours.
  • The Mercury lamp should always be first-on and last-off.
  • Not respecting these instructions may lead to instrument malfunction and complete loss of function! Both lasers and lamps are extremely expensive.

Roper TIRF | Reservations

  • Roper TIRF
  • Location: UIC Room 4 (0B05), Bartolomeu Dias wing

    Manufacturer: Roper

    Model: Nikon

    Year Installed: 2011

    Camera: Photometrics 512 EMCCD

    Operating: Full temperature atmosphere control (37°C + Humidity + CO2)

    Keywords: spinning disk, tirf, frap, inverted microscope, live imaging, fast imaging

Spinning disk confocal microscopy is rapidly emerging as the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems are capable of providing high acquisition speeds with acceptable contrast and minimal photobleaching at the low light levels available with this technique.

In this system, a spinning disk with multiple small holes is installed between the light source and specimen to excite point-like sources on specimen. The same hole serves as a detecting pinhole to remove out-of-focus light.

Spinning disk mechanism

In addition to the Spinning Disk confocality, this microscope is prepared with a TIRF module. TIRF (Total Internal Reflection Fluorescence) is a microscopy technique where only a thin region/band of the specimen is illuminated. If you interested in this technique, feel free to speak with the UIC personnel.

TIRF illumination


Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).

Maintenance log:

  • 07-04-2016 - Added a second power bar to the microscope controls, to prevent problems with some equipment beeing constantly on.
  • 12-03-2015 - Laser launch configuration changed and a new Optical switcher installed by the Roper tech. Change should prevent further laser misalignments. Camera firmware updated.
  • 09-03-2015 - Laser lauch tray replaced by a marble table, which is heaver and sturdier. Less vibration shoud be expected.
  • 15-01-2015 - Lasers re-aligned and power increase for all the modules, Spinning Disk, FRAP and TIRF.
  • 26-07-2014 - Lasers re-aligned. Output at the objective increased.
  • 24-04-2014 - Computer backup made.
  • 24-04-2014 - Log-in confirmation system installed in the microscope computer.
  • 17-04-2014 - Field ilumination was corrected for flatness.
  • 17-04-2014 - Lasers re-aligned and power increased.
  • 20-03-2014 - Lasers were realigned and fiber recoupled to increase the laser power output at the backfocal plane of the objective.
  • 18-03-2014 - Room structure changed and microscope moved to the back of the room.

Booking Calendar details

Configuration
Value
Booking type
Regular reservation
Responsible
Unidade Imagiologia
Minimum booking time (in min)
30
Schedule tolerance (in days)
7
Detele tolerance (in hours)
1
Booking restriction status
Disabled

Laser Unit

Laser
Excitation lines
Maximum power
405
405 nm
100 mW
Cobolt 491
491 nm
100 mW
Cobolt 561
561 nm
100 mW
642
642 nm
165 mW

Available objectives

Mouse hover Magnification to see the objective.
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Pixel size (µm)
Pixel size * 1.5x (µm)
Nyquist Sampling @525nm in Z (µm)
Link
SpD
TIRF
SpD
TIRF
10x
PLAN FLUOR
0.30
-
16
0.700
1.33
1.6
0.89
1.07
1.05
40x
PLAN Fluor
1.30
OIL
0.2
0.162
0.33
0.4
0.22
0.26
0.24
50x
PLAN Achromat
0.9
OIL
0.35
0.233
0.26
0.32
0.18
0.21
0.35
100x
PLAN APO
1.49
OIL
0.13
0.141
0.13
0.16
0.09
0.11
0.21

- Spinning Disk

Remember to choose the proper calibration if doing montages/size measurements.

(1) NA - Numerical Aperture. For more information, follow this link.

(2) These values were calculated using the Resolution formula adapted to confocals as seen in the Olympus micro website: R = 0.4*λ/NA

The pixel size in microns of your picture is saved automatically. Use Bio-formats to recognize it with ImageJ/Fiji (already installed in the Fiji package).


The filter wheels are not up to date yet. Under construction! Check with the UIC personnel for more info!

Spinning Disk Emission Filterwheel

Name
Transmission
Description
MetaMorph position
DAPI
447/60
DAPI, CFP or Hoechst filter
1
GFP/FITC
525/45
GFP or FITC filter
2
Dual RFP
523-612/25
RFP or TRITC filter
3
Dual mCherry
523-612/25
mCherry or RFP filter
4
Cy5
685/40
Cy5/Far red filter
5
Quad
400/521/607/700
Quadruple filter
6
Trans
Open
Transmited light filter
7

TIRF Emission Filterwheel

Name
Transmission
Description
MetaMorph position
DAPI
460/50
DAPI, CFP or Hoechst filter
1
GFP/FITC
525/50
GFP or FITC filter
2
Dual RFP
595/50
RFP or TRITC filter
3
Dual mCherry
-
mCherry or RFP filter
4
Cy5
685/40
Cy5/Far red filter
5
Quad
400/521/607/700
Quadruple filter
6
Trans
Open
Transmited light filter
7

Dichroic Mirror Yokogawa CSU-X

Name
Transmission
Description
Manufacturer
Link
Quad
405/488/568/647
Quadruple filter
Semrock

Spectra, lines & filters:

Setup

Turn on procedures

  1. Turn on the computer.
  2. Turn on the fluorescence lamp, should you need it.

    Fluorescence lamp

  3. Turn on the Master Control Switch 1 (below the computer).

    on/off button 1

  4. Turn on the Master Control Switch 2 (behind the spinning disk, left side of the microscope).

    on/off button 2

  5. Turn on the lasers in the Laser Remote control.

    Laser Remote

  6. Start Metamorph. If you are using the Spinning Disk module, start the "Metamorph CSU only" icon in the desktop. If you are using the TIRF module, use the "Metamorph TIRF" shortcut.

When you are finished and there is someone immediately after you, leave the entire system on (in particular, don't turn off the fluorescence lamp even if there is a large time difference between you and the next person). If you're the last or only person of the day, see the turn off procedures below.


Turn off procedures

  1. Close the software and save all your files

    • Save all the files in the server using the shortcuts provided in the desktop.
  2. Turn off the Master Control Switch.
  3. Turn off the microscope and the Spinning disk unit.
  4. Turn off the Fluorescence lamp.
  5. Clean any immersion objectives you used.
  6. TAKE OUT EVERYTHING YOU BROUGHT! (pipettes, slides, etc...)

Common problems

If you can't focus, make sure you haven't clicked the escape button. After you click escape, the focus doesn't move unless you click refocus.

If the camera doesn't see anything when using Metamorph, remember to select the L100 microscope port.

If you can't see anything, check the laser output switch and check if it is open.

No light reaches the objective. Check if the lasers are on.

Acquisition

Acquiring images in a spinning disk confocal microscope is a complex procedure, first time users are required to contact the UIC staff for training. If you need assistance don’t forget to press “with assistance” at reservation time.

  • Grid Colection / Multi-well / Mosaic

    It is possible to acquire grids and tiles of big samples through MetaMorph. For a more detailed protocol, please check the following link or click in the image to the right.

  • Tile Scan

  • Perfect Focus System (PFS)

    Nikon has a hardware autofocus system called Perfect Focus System (PFS) which allows continuous and constant sample focus using the coverslip position as the reference, for a faster and less damaging focusing system compared to normal software ones. For more detailed information, follow the link or click the image to the right side.

  • Nikon PFS


Extra Info