Microscopes

 

How confocal microscopes work
Confocal microscopes provide "optical slice" images (z-sections) of fluorescent specimens, with out-of-focus light removed, providing improved resolution through the depth of the image (z-dimension).  The capture of a series of such z-sections (z-stack) generates a 3D data set from a specimen.

 

Resolution achievable with confocal microscopes
The maximum achievable resolution and the depth within a specimen from which high resolution optical slice images can be obtained depend on the optics of the microscope, refractive indices through the sample and the depth of penetration of the labelling.
All our confocal microscopes are equipped with the highest quality lenses. With good sample preparation and minimum refractive index mismatch, resolution of up to 200 x 200nm in x & y and 500nm in z can be achieved.  Resolution in z can be further improved with post image acquisition deconvolution (discuss prior to capturing images if you intend to do this).  Imaging to a depth of up to ~50µm can be achievable in specimens that present minimal refractive index mismatch and good label/antibody penetration, but ideally the part of the specimen to be imaged should be as close to the coverglass as possible, preferably mounted or grown on the coverslip, then sealed onto a slide.

 

Imaging capabilities

  • Our confocal microscopes are suitable for fluorescence imaging of any fluorophore requiring excitation between near UV and red (choice of excitation between 405nm to 633nm) and any emission between 400 and 800nm.
  • All three confocal microscopes feature adjustable scan speed, pixel format and optical zoom, and have very high quality, high numerical aperture (and very expensive) objective lenses.
  • Transmitted light imaging, with or without contrast optics is also available on all three confocals.

Live cell imaging modes available

Both the Leica SP5 and the Olympus FV1000 havelive cell imaging capabilitiesincluding:
  • pH and ion imaging
  • FRET
  • FRAP
  • FLIP
  • FCS
  • RICS

A similar Leica SP5 at Adelaide Microscopy (www.adelaide.edu.au/microscopy) can be used for FLIM.

Users

Upon successfully completed training, users are given access to the facility and the respective instruments for which they have been trained. All equipment use must be booked and equipment charges, known as user fees, apply.
User fees are kept to a minimum and funds from user fees go back into the Facility to cover repairs, parts replacement and equipment upgrades.
Confocal microscope user fee charges are invoiced to supervisors bi-annually.
Please refer to the current table of User Fees (PDF 13KB) for more information.

Booking Equipment

All usage of microscopes and associated equipment must be booked by using the online Calcium Booking Calendar .
The calendars may be viewed by everyone. However, the ability to book specific equipment will only be enabled after successfully completed training with Flinders Microscopy Management Personnel.

Useful Resources and Links

Confocal imaging principles and virtual instrument / simulator tutorials: Software to analyse microscopic images

Tutorials on the basics of fluorescence, excitation and emission – Life Technologies

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Leica SP5

Features
  • suitable for fluorescence imaging of any fluorophore requiring excitation between near UV and red (choice of excitation between 405nm to 633nm) and any emission between 400 and 800nm
  • five (5) fully tuneable detectors (can set desired emission range for a given fluorophore, rather than being locked into available filter cube emission bandwidths)
  • additional laser lines for CFP & YFP imaging
  • adjustable scan speed, image size, pixel bit depth, zoom and rotation
  • very high quality, high numerical aperture (and very expensive) objective lenses
  • additional single photon sensitive filtercube-based detectors (avalanche photo-diode detectors) and resonant scanner for where extremely high sensitivity or high speed scanning is required
  • coupled to a fluorescence correlation spectroscopy (FCS) system which enables direct measurement of the diffusion of fluorescently-labelled particles.
  • suitable for short term live cell experiments
Specifications
  • Eight laser lines; 405, 458, 476, 488, 496, 514, 561 & 633nm
  • Five fully adjustable PMT detectors (ie can be set to capture any given range of emission from  400 to 800nm),
  • transmitted light detector with DIC option
  • Two filter cube based avalanche photo diode (single photon sensitivity) detectors which can be used with green and far-red fluorophores for imaging or FCS
  • 10x & 20x multi-immersion fluid lenses, 40x, 63x & 100x oil immersion lenses and a 63x water immersion lens
  • Resonant scanner (8kHz) for very high speed image acquisition
  • Uses Leica Specific Software (LAS AF)

 

Olympus FV1000

Features
  • suitable for fluorescence imaging of any fluorophore requiring excitation between near UV and red (choice of excitation between 405nm to 633nm) and selected emission between 400 and 800nm
  • adjustable scan speed, image size, pixel bit depth, zoom and rotation
  • very high quality, high numerical aperture (and very expensive) objective lenses
  • silicon-immersion lenses
  • based on Olympus inverted IX81 scope
  • housed within in a fully enclosed gas and temperature controlled chamber
  • on a gas-floated anti-vibration table – for when stage stability is critical (eg. for FRET-acceptor bleaching and FRAP measurements)
  • autofocus options
  • confocal of choice for medium to long term live cell experiments

  Specifications

  • Four laser lines; 405, 473, 599, & 635nm
  • Three PMT detectors for emission capture through fixed filter cubes suitable for most blue, green, red and far red fluorophores
  • additional transmitted light detector
  • 10x, 20x & 40x dry lenses, 30x & 60x silicon-oil immersion lenses
  • Uses Olympus Specific Software

     

Biorad MRC 1024

Features
  • suitable for fluorescence imaging of any fluorophore requiring excitation between blue and red (choice of excitation between 488nm to 647nm) and any emission between 500 and 800nm
  • adjustable scan speed, image size, pixel bit depth, zoom and rotation
  • very high quality, high numerical aperture (and very expensive) objective lenses
  • Based on Olympus AX70 scope
Specifications
  • Three laser lines; 488, 568 and 647nm
  • Three PMT detectors for emission capture through fixed filter cubes suitable for most green, red and far red fluorophores
  • 10x, 20x, 40x, 60x and 100x oil lenses
  • 4x and 40x long working distance dry lenses
Note
  • This microscope has been modified for a specific user’s application. Please consult with Flinders Microscopy Management regarding the suitability of using this instrument for other applications.

 

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