Fluorescence Microscopy

A research technique in optical microscopy that relies on excitation of fluorescent molecules with a specific wavelength region to produce an image generated by the secondary fluorescence emission at longer wavelengths. Modern fluorescence microscopes are equipped with reflected light illuminators that incorporate neutral density filters and a specialized combination of interference filters to segregate incident illumination from the detected fluorescence emission.
The advantage of fluorescence for microscopy is that you can often attach fluorescent dye molecules to specific parts of your sample, so that only those parts are the ones seen in the microscope. You can also use more than one type of dye. By changing the excitation light, you can cause one type of dye to fluoresce, and then another, to distinguish two different parts of your sample. Examples of this are different cell types within a tissue, or different targets within a cell.

Facility Equipment

  1. ZEISS Axioplan 2 epifluorescent/light microscope with high-resolution Axiocam greyscale digital camera.
  2. Nikon Axiophot epifluorescent/light microscope with Spot digital colour camera.
  3. The Spot digital colour camera and imaging system can be setup on the ZEISS if required.

ZEISS Axioplan 2 Epifluorescent Light Microscope

Transmitted Light (Kohler illumination)       
Phase Contrast: viewing unstained cells
Epi-Fluorescence
DIC: 3D effect (Nomarski filtering). Uses polarized light.
HR Greyscale Digital Imaging (AxioCam MRm)
Image acquisition: colour assignments in Axiovision acquisition software
Scalings: all objectives are calibrated within the Axiovision acquisition software

Objectives:   

10x Phase 1 dry
20x Phase 2  dry
40x Phase 2   dry
63x Phase 3 oil immersion
100x Phase 3 oil immersion
1.25x requires change of condenser

Nikon Axiophot Epifluorescent Light Microscope

nikon.jpg

Transmitted Light (Kohler illumination)
Phase Contrast: viewing unstained cells
Epi-Fluorescence
Dark-field illumination
Digital Colour Imaging: Spot Jn
Image Acquisition: True colour imaging
   
Objectives:

 

4x Phase L dry
10x Phase 1  dry
20x Phase 2 dry
40x   dry
100x   Oil immersion


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

ZEISS Filter sets for epifluorescence:
Filter Set 1.    Ex = UV 365/12; Em > 397 blue
DAPI = 4',6-Diamidino-2-Phenylindole
        (DAPI + DNA: Exmax = 359, Emmax = 461)
Filter Set 10.    Ex = 450- 490 blue; Em = 515-565  green
        FITC = Fluorescein Isothiocyanate (Exmax = 490, Emmax = 525)
Filter Set 15.    Ex = 546/12 green; Em >590 red
        TRITC = Tetramethyl Rhodamine Iso-Thiocyanate.
        (Exmax = 540, Emmax = 580 )
        Filter set can also be used for Cyanine 3 (Exmax = 550, Emmax = 570)    

NIKON Filter Sets:    standard red, green, blue filter sets.

Contacts

Microscopes are located in room 541 of the Molecular Biosciences Building
Contact: Dr Steven Mason, Room 433, Molecular Biosciences Building
Email: steven.mason@uq.edu.au
Phone: 33654872

Facility Charges

Available on application.

Requirements for using the Facility

All users must undertake a training session and be registered to use the microscopes.

Care of Microscopes and Slides

The microscopes must be treated with care at all times. Do not force sliders or remove any parts including the objectives. If any problems occur, contact the facility supervisor immediately.
All immersion oil must be removed from objectives after use.
Any coverslip/slide mounting media must be set and dry before viewing the slides.

 

 

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