The technique

A popular mode of optical microscopy in which a focussed laser beam is scanned laterally along the x and y axes of a specimen in a raster pattern. The emitted fluorescence is sensed by a photomultiplier tube and displayed in pixels on a computer monitor. The pixel display dimensions are determined by the sampling rate of the electronics and the dimensions of the raster. Single photons that are emitted away from the focal plane are blocked by a pinhole aperture located in a plane confocal with the specimen. This technique enables the specimen to be optically sectioned along the z axis.

Applications

Confocal laser-scanning fluorescence microscopy can be used to determine the localisation of proteins or e.g. mRNA in live or fixed cells and tissues. This means that one can also follow transport of protein- or RNA-containing granules in cells.

Facility Equipment

ZEISS LSM 510 META Confocal Microscope

Capabilities:

  • Multitrack imaging
  • Optical sectioning and 3D image analysis
  • Time series and photo-bleaching
  • Spectral analysis and emission fingerprinting
  • Co-localisation studies
    LSM510META_web.jpg




    Confocal Pathways

Facilities available

The ZEISS LSM 510 META confocal microscope is primarily available for SCMB-based users. All users must undertake an induction/training session. Experienced users may book and use the microscope at any time. New users will be supervised within the facility core hours of 9.00 am to 3.00 pm week days:  assistance, training and supervision is available during this period.
The maintenance period is from 8.00 am to 8.30 am daily, and from 8.30 am to 10 am on Mondays. No bookings are allowed before 8.30 am on week days.
 
Access to the facility after 3.00 pm weekdays and on weekends and public holidays is available to experienced users only. An access key will be required and is available by authorisation only.

The facility is located in Room 160 of MBS. There is secure, swipe-card access to the facility and its surrounds. As the facility is certified as PC2, all users will need to sign the PC2 log to verify that they have completed PC2 training. PC2 regulations must be adhered to at all times, this includes wearing lab coats and not consuming food or drink in the facility. The facility should also be locked when unoccupied.

Contact

Dr Steven Mason, Room 433, Molecular Biosciences Building.
Email: steven.mason@uq.edu.au
Phone: 33654872

Charges for use 

External users by agreement with the Facility Manager. 

UQ users have priority of access.

External Client Availability and Conditions

The confocal microscope is available for external users, subject to internal demands. For conditions of use, contact the Facility Manager. All external users must undertake an induction/training session.  New users will be provided with extensive training.

ZEISS Filter sets for epifluorescence

Microscopes are fitted with far red, red, green, blue filter sets (commonly used for Cy5, TRITC, FITC and DAPI fluorophors). Colours are based on Emission wavelengths.
Red = 610-750; Green = 500-570, Blue = 450-500

Filter Set 1.          Ex = UV 365/12; Em > 397 blue
Filter Set 9.          Ex = 450- 490 blue; Em >520 green
Filter Set 15.       Ex = 546/12 green; Em >590 red
Filter Set 50.       Ex = 640/30 red; Em = 690/502 red


Objectives:   10x/0.45  dry  ∞/0.17  Plan-APOCHROMAT
                        20x/0.75  dry  ∞/0.17  Plan-APOCHROMAT 
                        40x/1.3   oil  DIC   ∞/0.17  Plan-NEOFLUAR
                        63x/1.4   oil  DIC   ∞/0.17  Plan-APOCHROMAT
                       100x/1.4  oil  DIC   ∞/0.17  Plan-APOCHROMAT

 

Lasers and Laser Lines Available

Laser Unit Wavelengths nm

Fluorophors/dyes

EM EM max
Diode 405 DAPI >420 461
  405 Hoechst >420 447

Argon/2

458 Fluoro-Gold >475 536
  458 ECFP >475 501
  458 Lucifer Yellow >475 536
Argon/2 477 EGFP >477 507
  477 FM 1-43 >477 598
Argon/2 488 Alexa Fluor 488 >505 520
  488 Cy2 >505 508
  488 Fluo-3 >505 520
  488 FITC >505 520
Argon/2 514 EYFP >530 535
  514 Oregon Green >530 535
  514 SYTOX Green >530 536
  514 FM 4-46 >530 640
HeNe1 543 Cy3 >560 565
  543 Alexa Fluor 546 >560 572
  543 TRITC >560 576
HeNe2 633 Alexa Fluor 633 >650 654
  633

Cy5

>650 666

Important Notices

  • Authorised/registered users are not permitted to remove microscope objectives or to attempt any maintenance.
  • Non-authoried users are not allowed access to the microscope. 
  • Users are not permitted to setup and save new configurations of lasers and filters. The preprogrammed configurations should cover most applications.
  • There is only one on/off switch for the entire system. Do not touch any other on/off switches on the microscope, computer or power supplies.
  • When you have completed your imaging, switch off the lasers and wait until they have shut down before exiting the LMS software.
  • Copy your images to CD, DVD, USB stick or network drive before you leave. Images left on the miscroscope operating system may be deleted at any time without warning.

 

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