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.

Leica SP5 Live Upright | Reservations

  • SP5 Upright
  • Confocal microscopy permits one to optically section a fluorescent sample (such as a cell that has been stained with contrasting fluorescent dyes) with superior resolution by using a pinhole to reject light that originates outside of the chosen area. By collecting a series of such images through the depth of a sample, the user may assemble a highly accurate three-dimensional reconstruction of the entire sample. The Leica SP5 confocal microscope is equipped with a spectral head that employs a prism, movable slits, mirrors, and computer control to permit the operator to choose which bands of light at specific wavelengths will be focused simultaneously onto each of three detectors. This system also allows emission spectra (with 5-nm resolution) to be collected from a diffraction-limited-size spot.

    This particular SP5 is equiped with Hybrid detectors (HyD) which provide increased sensitivity and fast resonant scanners for live confocal imaging.

    Location : Bartolomeu Dias wing, UIC room 2

    Manufacturer : Leica

    Model : SP5

    Year Installed: 2012

    Optics : Confocal fluorescence and DIC

    Detectors : 2X HyD + 1 PMT + 1 TLD (Transmitted light - brightfield)

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, using HyD detectors in Standart Mode/Photon Counting Mode.

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:

  • 04-04-2015 - Lasers realigned. Slight power increase in both 488 and 561 lasers.
  • 23-09-2015 - Lasers realigned. 488 power increased by 2x.
  • 31-03-2015 - Data Hard Drive has hardware malfunction, causing windows to fail loading. Replaced temporarily by a smaller one.
  • 11-02-2015 - Power Supply and Ar laser replaced by new units. Fully working with 1,09 mW at 20% power using 10x obj.
  • 20-01-2015 - Power Supply of Ar laser replaced by an old one (old SP5 Inv replaced PSU) but the Ar still does not start.
  • 14-01-2015 - Argon laser Power supply fuse broken. No Argon laser available.
  • 08-01-2015 - Mercury lamp broke and was substituted bu a new one.
  • 04-12-2014 - Faulty HyD 3 detector replaced by a new one.
  • 24-09-2014 - Argon laser fiber was misaligned and losing 90% laser power. Realigned and power restored to values similar to May 2014.
  • 18-08-2014 - Computer HDD damaged and failing to load Windows. Replaced by a new one and Windows restored with a backup image.
  • 07-08-2014 - Leica LAS AF software updated to a newer version.
  • 04-06-2014 - System hard drive backed up.
  • 10-04-2014 - Incubation chamber installed in the microscope to control temperature.

Laser Unit

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

Available objectives

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @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
HCX PL APO CS
0.70
-
0.59
0.300
0.106
0.900
0.450
40x
HCX PL APO CS
1.3
OIL
0.22
0.162
0.057
0.485
0.242
63x
HCX PL APO CS
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) 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. Start the Leica Confocal Software - “LAS AF”.

NOTE: Clicking the checkboxes on the software is what turns them 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 anyone after you, 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 Colection / TileScan

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

  • Tile Scan


Extra Info