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.

Zeiss META | Reservations

  • Zeiss META
  • The LSM 510 META system is a point scanning laser confocal microscope with spectral analysis suitable for live cell imaging and photobleaching experiments. The system is currently equipped with a large size incubator with high-volume for warm air incubation and CO2 control, ensuring optimal conditions for live imaging. Due to its excellent resolution, high power lasers and extended filter sets it is one of the most used microscope in the institute.

    Location : Not defined

    Manufacturer : Zeiss

    Model: LSM 510 META

    Year Installed: 2007

    Keywords: confocal, inverted microscope, scanning confocal

    Some of it's features:

    • Z-sectioning
    • Spectral analysis
    • Co-localization of proteins
    • Fluorescent Resonant Energy Transfer (FRET)
    • Fluorescent Intensity Measurement

Suggestion for description in "Materials and Methods":

Confocal Z-series stacks were acquired on a Zeiss LSM 510 META, using a 63x 1.4NA Oil immersion objective, the 488nm and 543nm 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:

  • 05-04-2016 - Motherboard and Power Supply unit replaced by newer ones. Faulty Motherboard was causing booting problems and computer crashes.
  • 07-03-2016 - Problematy HDD was reaplaced due to corrupted boot files. Data backup available.
  • 23-09-2014 - PMT high voltage module was malfunctioning and causing Ch2 to turn off. Replaced by the Zeiss technician and working normally once again.
  • 22-09-2014 - Fluorescent lamp broke, replaced by a new one.

Booking Calendar details

Configuration
Value
Booking type
Inactive
Responsible
Unidade Imagiologia
Minimum booking time (in min)
30
Schedule tolerance (in days)
7
Detele tolerance (in hours)
0
Booking restriction status
Disabled

Laser Unit

Laser
Excitation lines
Argon
458, 476, 488, 496 & 514 nm
HeNe 543
543 nm
HeNe 633
633 nm

Available objectives

Place mouse over Magnification to see the objective.
Magnification
Objective type
NA*
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
PLAN NEOFLUAR
0.30
-
5.2
0.700
0.247
2.100
1.050
20x
PLAN APO
0.75
-
0.55
0.280
0.099
0.840
0.420
40x
ECPLAN-NEOFLUAR
1.3
OIL
0.21
0.162
0.057
0.485
0.242
63x
PLAN APO
1.4
OIL
0.19
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).

Available filters

Type
Wavelength
PMT
LP
685nm
1
BP
390-465nm
1
BP
435-485nm
1
BP
480-520nm
1
BP
500-530nm
1
BP
500-550nm
1
LP
505nm
2
LP
560nm
2
BP
500-620nm
2
BP
500-550nm
2
BP
535-590nm
2
BP
565-615nm
2
BP
650-710nm
2

Spectra, lines & filters:

Setup

Turn on procedures

  1. Log-in using you Agendo credentials.
  2. Turn on the fluorescence lamp power source.
  3. Turn on the “SYSTEM PC” and “COMPONENTS” switch on the control box.

    Control box

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 the next user isn't coming for a few hours, just turn off the lasers on the software. If you're the last or only person of the day, see below.


Turn off procedures

  1. If there is someone booked after you, simply close the LSM software (leave the lasers ON) and log-off from windows.
  2. If no one is going to use the microscope after you, turn off the lasers on the software and the fluorescence lamp power source.
  3. Shut down the software and log off the computer.
  4. If you turned the lasers off wait 5 minutes for laser cooldown and then turn off the “SYSTEM PC” and “COMPONENTS” switches on the control box.
  5. Clean any imersion objectives you used.
  6. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipets, slides, etc...

Acquisition

Acquiring images in a confocal is a complex procedure, first time users are encouraged 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.

Don’t forget that we regularly run theoretical courses, which should give you the basis for understanding what you are doing. If you don’t think it is enough, just talk with us for further discussion.


Extra Info