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Infrastructure_

Light and laser microscopy

Capabilities in optical microscopy
Access an extensive range of light and laser microscopes across the campus to meet your research objectives.

At Charles Perkins Centre

Super Resolution Fluorescence Microscopy collects images of fluorescent molecules with resolution that is not limited by the diffraction limit of light. Stimulated Emission Depletion (STED) is built on a Confocal microscope and is a laser scanning technique.

STED can improve lateral resolution to a theoretical limit of 50nm, actual ~100nm. Axial resolution can also be improved with STED to a theoretical limit of 130nm, actual ~300nm. If optimal resolution in XY and Z is required the actual achievable resolution is approximately 150nm laterally and 500nm axiallly.

This microscope also features an Insight DeepSee laser for multiphoton and Second Harmonic Generation (SHG) imaging with excitation wavelenghts of 680-1300nm.

Black paneled box incubator around microscope for live cell imaging.

Lasers
  • UV: 405nm 
  • Argon: 458, 476, 488, 496, 514nm
  • White Light Laser (WLL): tunable 470nm - 670nm
  • Multi-photon 1: tunable 690nm - 1300nm
  • Multi-photon 2: 1040nm
  • STED 592nm
  • STED 775nm
Detectors
  • Internal, AOBS: tunable 400nm - 800nm
    • 3 standard PMTs
    • 2 Hybrid Detectors (high sensitivity PMTs)
  • External Non-Descanned (uses filter cubes)
    • Transmitted Light: 2 standard PMTs
    • Reflected Light: 2 standard PMTs & 2 Hybrid Detectors

This Leica SR GSD 3D is a widefield fluorescence and TIRF microscope which uses ground state depletion and single molecule return to localise molecules of interest. Using this technique coupled with optimal sample preparation and supported dyes, strucutures of interest can be resolved up to 20nm laterally and 50nm axially.

Lasers

Wavelength (power)

  • 405nm (30mW)
  • 488nm (300mW)
  • 532nm (500mW)
  • 642nm (500mW)

Objectives

  • HCX PLAPO 160x/1.43 Oil CORR GSD
  • HC PLAPO 10x/0.40
  • HCX PL FLUOTAR L 40x/0.60
    Note: Field of view with GSD objective 18x18μm or 51x51μm

Recommended Fluorophores

  • AlexaFluor 488, 532, 546, 555, 568, 647, 680, 700
    (Life Technologies)
  • Atto 488 520, 633, 647n, 655 (Sigma Aldrich)
  • Chromeo 488, 505 (Active Motif)
  • YFP, GFP

Confocal lasers: 403, 457, 488, 514, 560, 640nm

FLIM / FCS / FCCS lasers: 405, 488, 640nm

Detectors

DU4 uses Filter Blocks

  • 2 standard PMTs
  • 2 Galium Arsenide Phosphide (GaAsP; high sensitivity PMTs)

Spectral Detector

  • uses prism to split emission wavelengths to discrete bandwidths
  • 2 FLIM / FCS / FCCS GaAsPs

Total Internal Reflection microscopy is a Super Resolution technique.

Specifications
Lasers 405, 488, 561, 640nm
Lumencor SpectraX LED 395, 440, 470, 508, 555, 640nm
DMD for photoactivation and photouncaging
Cameras TIRF Monochrome - Andor iXon Ultra 888 (max 26 f/s at 1024x1024, 96f/s at 512x512)

Live cell fluorescence, DIC, large mosaics, multi-point, time series, perfect focus.

Light Source

  • Lasers: 405, 488, 561, 640nm
  • CoolLED pE-340fura LED illumination system for Fura2 Ratiometric Calcium Imaging
  • Lumencor SpectraX LED: 395, 440, 470, 508, 555, 640nm
  • Halogen lamp and optics for Brightfield, Phase contrast, DIC

Cameras

  • Brightfield Monochrome: DS-Qi2 (max 6 frames/second)
  • TIRF and SD Monochrome: Andor iXon Ultra 888 (max 26 f/s at
  • 1024x1024, 96f/s at 512x512)

Visualise, analyse, quantify and phenotype immune cells in situ.

The Mantra quantitiative pathology workstation, with inForm image analysis software, enables easy visualisation, quantification and phenotyping of cells in situ in tissue sections using multiplexed biomarkers. This integrated workstation features a spectral detector to collect spectral data for unmixing overlapping signals and removal of autofluorescence. inForm software features user-trained algorithm to enable automatic identification of specific tissue types based on tissue morphology and indentification of cells within the tissue for quantification.

Upright Confocal microscope with motorised stage.

Lasers: 405, 488, 561, 640nm

Detectors: 3 standard PMTs using Filter Blocks - DAPI/Cy5, FITC, TRITC

Upright microscope for mosaics and multipoint.

Light Source: Lumencor SpectraX LED - 395, 440, 470, 508, 555, 640nm

Cameras: Monochrome - DS-Qi2, Colour - DS-Fi2

Basic inverted widefield fluoresence microscope using a monochrome camera for fluorescence. It uses a white light source (mercury lamp) and a set of filter cubes.

Filter Cubes    Excitation Filter   Dichroic Mirror    Em Filter
A (Blue)            BP 360/40              LP 400                  LP 425
GRP (Green)    BP 470/80               LP 500                  BP 525/100
YFP (Yellow)    BP 500/40              LP 515                    BP 535/60
RFP (Red)        BP 546/24               LP 560                  BP 605/150
Y5 (Far Red)     BP 620/60             LP 660                   BP 700/76
Analyzer

A live cell imaging system for flasks, dishes and multiwell plates inside an incubator. Modalities include phase contrast, green, red fluorescence. Perfect for proliferation assays and scratch assays requiring many samples/wells and long time scales (e.g. Imaging every 2-12hrs for 7 days). The researcher can remove samples between acquisitions for drug treatments and to feed cells.

Fluorescence Stereomicroscope for brightfield or fluorescence with 0.63x and 2.0x objectives and 3 filter positions for blue, green and red fluorescence. Motorised zoom 16:1.

This automated slide scanner microscope can scan up to 100 slides at one time with brightfield or fluorescence mode. The system is deisgned specifically to digitise microscope slides for your research. It can also perform a Z-stack of up to 50um, to achieve a fully stitched 3D construction of the tissue of interest.

Filterwheel  Excitiation (nm)   Beam Splitter (nm)   Em (nm)
DAPI                G365                      FT 395                         445/50
GFP                 BP 470/40             FT 495                         525/50
DsRed             BP 550/25             FT 570                         605/70
Cy5                  BP 640/30            FT 660                         695/50
 

 

Live cell fluorescence and confocal laser scanning microscope with Airyscan detector for super resolution. Has a live cell incubator cage on the microscope to keep cells at appropriate temperature and CO2.


Super resolution: Lasers 405,488,561, 640nm and tuneable emission range 400-700nm with Airyscan detector for super resolution down to ~125nm XY and ~350nm Z.

Laser scanning and confocal microscopy: in addition to the Airyscan GaAsP PMT detector which can be used as a standard detector, there are two GaAsP PMT and a transmission detector with DIC optics. The system has tuneable emission wavelength selection between 400-700nm with clean up emission filters available.

Spectral Imaging and Unmixing: The system is capable of spectral imaging emissions from 400nm-700nm and unmixing for 5-6 channels. This works well for one blue, three green and red and one far red probe as well as autofluorescence.

Widefield fluorescence microscopy: This system has a metal halide white light source and monochrome camera for widefield fluoresence imaging.

At Madsen Building

Equipped with 405,488,561 and 640nm lasers and a transmitted ligt detector allwing for the majority of fluorophores to be imaged with a beautiful DIC (or brightfield) overlay. With the fastest Resonance Scanner on the market, it can capture up to 438fps at 512 x 32 or 30fps at 512 x 512. This, combined with a stagetop incubator results in excellent temporal resolution for imaging live cells.

Live cell imaging is made easy with Multiple Area Time Lapse acquisition and Micropplate navigator. TruSpectral technology prvovides fantastic spectral resolution that enables users to collect the emission profile of a visible fluorophore at any point in their sample. It can also be used for up to 16 channel sequential scanning. This gives rise to a spectral un-mixing function which allows separation of spectrally close fluorophores, particularly handy for users with autofluoesence interfering with the signal of their labelled target of interest.

This instrument covers a broad range of requirements in confocal and multiphoton imaging - with the full array of scan speeds at highest resolution. You can image your live cells then image the same “dynamic” event later at high resolution in the TEM. The microscope is equipped with a new Spectra-Physics Mai Tai DeepSee™ Ti:Sapphire femtosecond pulsed laser, specialised objectives and external non-descanned detectors. This means that we can image more than 300 microns deep into thick specimens without any signal drop-off. Single photon excitation at 458, 476, 488, 496, 514, 561 and 633nm. Multiphoton excitation variable 690-1060nm.

The system is equipped with a resonance and galvanometer scanner, so we can now image around three times faster at higher resolution than ever before (e.g. a 512 x 128 pixel array every 15ms). 

The system features Fluorescence Lifetime Imaging (FLIM) and forward and backward Second Harmonic Generation (SHG) capabilities.  It is equipped with a time-correlated single-photon-counting (TCSPC) board for (FLIM) which can measure the lifetime of a fluorophore faster and more accurately than before at various excitation wavelengths. Its photomultiplier tubes (PMTs) in the forward direction are calibrated to optimise forward and backward SHG imaging.

This is a conventional inverted fluorescence microscope equipped with motorised stage with definite focus. The Shuttle & Find from Zeiss is a correlative microscopy interface for light and electron microscopes. It can be used with the Zeiss Sigma HD SEM.

General-use light and fluorescence microscope for life sciences, material science and medical applications. Standard filter sets (FITC, GFP, Rhodamine, DAPI) for fluorescence. Bright field, phase and Nomarski optics.

Leica DFC400 camera with 1.4 megapixel for very fast image captures.

Example image:
Prepared fluid mount of Spirogyra showing Nomarski optics and fluorescence using three standard filter sets (FITC, Rhodamine, DAPI).

Inverted optical microscope for materials science. Ample working space to easily place large and heavy samples (up to 30kg). DMC4500 camera, reflected light only, DIC, Polarizer, manual objectives and stage, Leica LAS-X software.

At Brain and Mind Centre

This custom built system is the world's first commercially available multiphoton system to contain 3 tuneable lasers. Its special lasers can peer deep into the tissue of live animals, providing researchers with a view of biological or pathological processes as they occur. It uses 3 nano-second pulsed lasers which can illuminate up to 8 fluorophores simultaneously, providing exceptionally fast imaging.

This state of the art slide scanner allows researchers to observe labelled tissues on slides, using 5 channel fluorescence, dark field, Differential Interference Contrast (DIC) and transmitted light modalities. Researchers can either acquire immunoflourescently labelled tissues, or typical staining procedures including DAB and H & E stained tissue.

The system has a robotic arm used in conjunction with 2 x 50 slide racks allowing up to 100 slides to be put under the microscope objective one slide at a time. Then the system autofocuses onto the tissue on the slides, recognises the edges of the tissue and scans the entire tissue, stitching the image in real time, before moving onto the next slide. It also has the capability of performing a Z-stack of up to 50um, to achieve a fully stitched 3D construction of the tissue of interest.

This instrument also has a standalone analysis computer with software allowing for detailed image viewing and analysis of the massive files which are produced from the acquisition system.

Equipped with 405,457,488,514,640nm lasers and a transmitted light detector allowing for the majority of fluorophores to be imaged with a beautiful DIC (or brightfield) overlay. This microscope has both a stagetop and cage incubator resulting in excellent stability for imaging living cell dynamics 24,48 even 72 hrs. Live cell imaging is made easy with Multiple Area Time Lapse acquisition. Set up the microscope to image live cells from each well in a multiwell plate overnight.

We have 2 upright confocal microscopes with motorised stage ideal for imaging fluorescent slides. The motorised stage can be used to collect large stitched mosaics combined with z-stack function for very large volume images.

This slide scanner has the capacity to scan 400 slides in one run, saving the files directly onto a special server. The system only has a 20X objecitve (with capacity for doubling to 40X) and only performs brightfield scanning. Performs slide scanner with a fully automated system and is very simple to operate.

Basic upright widefield fluorescence microscope for mosaics using a combined monochrome/colour camera for fluorescence and histology. It uses a white light source (mercury lamp) and a set of filter cubes.

Filter Cubes      Ex                       Dichroic         Em
Ana
DAP                    BP 350/50          LP 400           BP460/50
L5                       BP 480/40          LP 505            BP 585/40
RHO                   BP 546/10           LP 560           BP 546/10
Y5                       BP 620/60          LP 660           BP 700/75

 

Basic upright microscope for mosaics using a colour camera for histology slides.

Lightsheet microscope for 3D imaging of large tissue samples or whoe organs. Ideal for cleared tissue. Zoom body configuration allows 1.26x to 12.6x magnification. Can image up to 1cm x 1cm samples. Refractive index matching 1.33-1.56. Equipped with whote light laser so excitation and emission use filters. Adjustable lightsheet for higher resolution or broader filed of view, six light sheets for homogeneous illumination.

Excitation Filters        Emission Filters
430/24                           480/40
470/40                           525/50
500/20                           535/30
545/30                           590/50
580/25                           625/30
640/30                           690/50

 

Enquiries

For further information on our light and laser microscopy capabilities, please contact: acmm.lo@sydney.edu.au