IGC

AdvancedImagingUIC

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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.

Leica SP5 Inverted | Reservations

  • SP5

  • The Leica SP5 confocal system is mounted on a Leica DM6000 inverted microscope, equiped with an Argon-ion laser (458,476,488,514nm lines) + 561nm and 633nm lasers, and with 3x spectral PMT detectors.

    Location : UIC Room 2 (0B03), Bartolomeu Dias Wing

    Manufacturer: Leica

    Model: SP5

    Year Installed: 2007

    Optics: Confocal fluorescence and DIC

    Detectors: 3x PMTs + 1TLD (Transmitted light - brightfield)

    Operating conditions : Temperature control

    Keywords: confocal, sp5, inverted microscope, scanning confocal, live imaging

Suggestion for description in "Materials and Methods":

Confocal Z-series stacks were acquired on a Leica SP5 confocal, using a 63x 1.3NA Oil immersion objective, the 488nm and 568nm laser lines, and spectral detection adjusted for the emission of the Alexa488 and Alexa 568 flurochromes.

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:

  • 11-04-2016 - Bad PSU component of the Mic control box was replaced by a new one. Will fix intermitent power when turning on.
  • 17-11-2014 - Modulation cable of AOBS1 had a bad connection due to broken pins. Broken pins fixed and solved.
  • 07-08-2014 - AOTF driver board software/firmware updated and working properly now.
  • 07-08-2014 - Transmitted light arm secured/fastened to the microscope body since it was slightly loose.
  • 06-08-2014 - Laser realigned at the objective back focal plane. Illumination should be homogeneous through the whole field of view.
  • 06-08-2014 - Leica control box was unresponsive sometimes and was fixed.
  • 06-08-2014 - Argon Laser (458/476/488/514) replaced by a new one.
  • 06-08-2014 - AOTF driver board replaced by a new working one.
  • 27-03-2014 - Stage was recalibrated by Gabriel Martins (calibration factor 0,16)

Booking Calendar details

Configuration
Value
Booking type
Pre-reservation with real-time tracking confirmation
Responsible
Unidade Imagiologia
Minimum booking time (in min)
30
Schedule tolerance (in days)
7
Detele tolerance (in hours)
1
Booking restriction status
Enabled
Restriction by
User
Maximum number of h/day
3
Maximum number of h/week
12

Installed lasers

Laser
Excitation lines
Argon
458, 476, 488, 496 & 514 nm
DPSS 561
561 nm
HeNe 633
633 nm

Installed objectives

Place mouse over Magnification to see the objective.
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution3 @525nm in XY (µm)
Nyquist Sampling @525nm in XY (µm)
Resolution @525nm in Z (µm)
Nyquist Sampling @525nm in Z (µm)
Link
10x
HC PL APO CS
0.40
-
2.2
0.525
0.186
1.575
0.788
20x
HC PL APO CS
0.70
-
0.59
0.300
0.106
0.900
0.450
20x
HC PL APO
0.70
IMM2
0.26(Water)-0.17(oil)
0.300
0.106
0.900
0.450
40x
HCX PLAN APO
0.85
-
0.24
0.247
0.087
0.741
0.371
40x
HCX PL APO CS
1.25-0.75
OIL
0.10
0.168
0.059
0.504
0.252
63x
HCX PL APO
1.40-0.60
OIL
0.10
0.150
0.053
0.450
0.225

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

(2) Requires manual adjustment of the correction collar; works with oil, glycerol or water.

(3) This value is the theoretical optical resolution limit depending on the NA. To achieve optimal lateral (XY) resolution and Nyquist Rate, please speak with the UIC personnel for optimal pixel size.

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).

Setup

Turn on procedures

  1. Turn the fluorescence lamp power on.

    Fluorescent Mercury lamp

  2. Turn on the “PC”, “SCANNER” and “FAN” switches.

    SP5 power switches

  3. Turn on LASER key.
  4. Turn on the computer.
  5. Log on to the computer using your Agendo credentials
  6. Start the Leica Confocal Software - “LAS AF”.

NOTE: Clicking the checkboxes on the software is what turns the lasers ON or OFF. The key controls the power to the lasers: turning it on DOESN't turn on the lasers unless you check them on the software, but turning it off WILL turn off the lasers.

Turn off procedures

If there is someone booked after you, close the software, save your files in the server and Log-off.

Leave everything else ON and clean any immersion lenses you used. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipets, slides, etc...

Proceed only if there is no one booked afterwards or for the next 4 hours after your session ends:

  1. Save your files and transfer them to the server.
  2. Log out and shut down the computer.
  3. Turn off the lasers key, the “PC” and “SCANNER” switches.
  4. Turn off the fluorescence lamp.
  5. Turn off the “FAN” switch after 10 minutes (to let the lasers cooldown).
  6. Clean any immersion lenses you used just to remove the oil overspills.
  7. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipets, slides, etc...

Acquisition

Acquiring images in a confocal is a complex procedure, first time users are required to contact the UIC staff to discuss the use of confocal microscopes and fluorescent imaging in general. If you need assistance don’t forget to press “with assistance” at reservation time.

Click here for the SP5 Manual.

  • Grid Collection/TileScan

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

  • Tile Scan

Extra Info