Confocal Microscopy

ZEISS LSM 800 Confocal Microscope plus Airyscan

The Bosch Institute Advanced Microscopy Facility welcomes a new addition to the family of microscopes – the ZEISS LSM 800 laser confocal microscope. This microscope supersedes the older technology the ZEISS LSM 510 Meta confocal microscope.

Zeiss LSM 800

Image description: ZEISS LSM 800 confocal microscope plus Airyscan. This confocal microscope is located in the Bosch Institute Advanced Microscopy Facility, Rm S448, Anderson Stuart Building F13, The University of Sydney.

Muller glia

Image description: Muller glia extending across the entire depth of the retina. Muller cells are the most prominent retinal glial cells. They display characteristic bipolar morphology, form architectural support structures, and ensheath all retinal neurons. In this tile-scan image, it is shown that, how Muller glia (red, stained with vimentin) extend across the entire thickness of human retina and interact with retinal neurons (blue, stained with DAPI) and retinal blood vessels (green, stained with UEA lectin). Sample slide kindly provided by Mohammad Nasir Uddin (Anatomy & Histology, The University of Sydney).

c-fos expression

Image description: This tile-scan image shows c-fos expression (in green) in the mouse hippocampus following context fear conditioning. Cell nuclei are labelled with DAPI. Sample slide kindly provided by Jessica Leake (School of Psychology, The University of Sydney).

Technical Specifications:

This confocal system is based on the Axio Observer.Z1 inverted research class optical microscope and is located in Room S448, Anderson Stuart Building F13 at the University of Sydney.

It is equipped with 405, 488, 561, 640 nm laser lines, the Airyscan super-resolution module plus two individual GaAsP PMT detectors, a transmitted light detector and an AxioCam 506 digital camera. Also included is the Shuttle & Find module for Correlative Microscopy, additional tiling and experiment designer software, as well as incubation equipment.

The two highly sensitive GaAsP detectors provide the highest signal-to-noise ratio and together with the unique AiryScan technology provide a pronounced improvement in x, y and z resolution. As a result of these features, the imaging speed is markedly increased so that more time is available for image acquisition at the microscope and for more users.


Additional Key features are outlined below:

Epi-Fluorescence illumination (HXP 120) set-up includes standard filters: DAPI, FITC, Cy3, PI and Cy5

Objectives included:

  • Objective Plan-Apochromat 10x/0.45
  • Objective Plan-Apochromat 20x/0.8 M27
  • Objective Plan-Apochomat 40x/1.3 Oil DIC M27
  • Objective Plan-Apochromat 63x/1.40 Oil

LSM 800 Confocal GaAsP-detection module consists of Confocal point scanning system with patented long pass (LP) variable secondary dichroic (VSD) for flexible spectral imaging and two highly sensitive GaAsP-PMT detectors.

Speed: Up to 8 frames/s at 512 x 512 pixel.

Continuous zoom optics from 0.5x to 40x.

Maximum frame resolution6144 x 6144 pixel (with Airyscan 4096 x 4096 pixel)

Main beam splitter (MBS) for 4 laser lines (405, 488, 561 and 640nm). MBS and VSD at 10 degree angle to incident beam for most effective excitation light suppression.

Digital Camera (Camera Axiocam 506 mono) integrated into ZEN 2.3 control software

More details about this high-end confocal microscope can be found at the following webpage.

Funding for this brand new microscope was provided by a 2016 Ian Potter Foundation Grant and a 2016 Cancer Institute NSW Research Equipment Grant as well as support funding from the DVCR, Faculty of Medicine, School of Medical Sciences, Bosch Institute, Physiology, Pathology, Anatomy & Histology, Pharmacology and the Advanced Microscopy Facility.


Zeiss LSM 510 Meta Spectral Confocal Microscope

The ZEISS LSM 510 Meta is the workhorse confocal microscope at the Bosch Institute Advanced Microscopy Facility. This spectral confocal microscope is based on an inverted Zeiss AxioObserver microscope. It has a number of lasers for excitation of fluorescent dyes and fluorescent proteins. These include a 405 nm laser for UV excitation, an Argon (458, 477, 488 and 514 nm lines), 561 nm DPSS and 633 nm HeNe laser. The LSM software allows for multi-channel imaging, z-sectioning, time-lapse imaging, live-cell imaging, quantitative co-localisation, FRET analyses and photo-bleaching methods. The scanning module has three conventional and one Meta detector with 32-spectral channels. The Meta detector enables Emission Fingerprinting (a technique developed by Carl Zeiss) to be carried out so that the emission spectra of different dyes is separated precisely. This can eliminate cross-talk, auto-fluorescence bleed-through and can even reliably separate dyes with close emission signals.

A new objective is on order for the ZEISS LSM 510 Meta confocal microscope; this ZEISS 25x/NA 0.8 Plan-Apochromat immersion objective is suitable for use with oil, water or glycerol. This objective has a working distance of 0.57 mm and will be particularly useful for imaging larger samples that have been mounted in a range of solutions, thereby allowing refractive-index matching. This objective should arrive by the end of March 2016. The purchase of this objective was supported by a Mason Foundation Grant 2015 (D. Johnstone, CIA). If you are interested in trying this objective on the ZEISS LSM 510 Meta confocal microscope at the Bosch AMF, please contact Louise Cole.

This microscope was purchased in February 2007. Funding for this microscope was supported by an NHMRC Equipment grant 2006, a University of Sydney Large Equipment grant 2007, RIBG funding and also the Rebecca L. Cooper Medical Research Foundation.

ZEISS LSM 510 Meta

Costaining of obscurin and α-actinin in human left ventricular tissue. Michael Kahramanian.

Objectives:

  • EC Plan-Neofluar 2.5x/ NA 0.075 M27/ WD 9.5mm
  • Plan-Apochromat 10x/ NA 0.45 M27/ WD 2mm
  • Plan-Apochromat 20x/ NA 0.8 M27/ WD0.55mm
  • EC Plan-Neofluar 40x/ NA 0.75 M27/ WD 0.71mm
  • Plan-Apochromat 63x/ NA 1.40 Oil DIC M27/ WD 0.19mm
  • Plan-Apochromat 100x/ NA 1.40 Oil DIC M27/ WD 0.17mm
  • C-Apochromat 40x/ NA 1.2 W Corr UV-VIS-IR M27/ WD 0.28mm
  • Plan-Apochromat 25x/NA 0.8 immersion objective Coming soon!

Filters: CFP, DAPI, YFP, FITC, GFP, Rhodamine, Texas red, Cy5

Accessories

  • Aqua-Stop
  • Heated stage and temperature controller (dual-control)
  • Heating collar for 63x oil objective
  • Multi-well plate holder
  • Heating platform
ZEISS LSM 510 Meta

Murine microglia under chronic inflammatory conditions. Carsten Minten, School of Molecular Bioscience.

Manuals Available:

ZEISS LSM 510 Meta

Colocalisation of Obscurin and Connexin-43 at the Cardiac Intercalated Disc. Michael Kahramanian.


Leica SPE-II Confocal Microscope

SPEII

This high resolution spectral confocal microscope is compact and easy-to-use. It consists of four solid-state lasers (405 nm, 488 nm, 532 nm and 635 nm) that allows a broad spectrum of dyes to be imaged. It has one spectral detector and allows spectral detection between 430-750 nm. The system runs on the Leica LAS AF software platform that is both easy-to-use and modular in design. Besides the one variable spectral detector, the system has a transmitted light detector which allows a bright-field image to be overlayed onto the fluorescence image. For full training on this system, please contact Dr Louise Cole, Bosch Institute Advanced Microscopy Core Facilities Manager.

This upright confocal microscope was purchased using RIBG and Bosch funding in November 2010. This is the "entry" confocal microscope in the Bosch Institute Advanced Microscopy Facility and it allows users increased confocal imaging hours together with the older workhorse Zeiss LSM 510 meta spectral confocal microscope. The Leica SPE-II confocal microscope is located in Rm S443, Anderson Stuart Building.

Mouse fibroblast by Leica

Objectives:

  • ACS APO 10x dry/ NA 0.3 CS/ WD 3.00mm
  • HCX PL Fluotar 20x dry/ NA 0.5/ WD 1.15mm
  • HCX APO L20x water immersion/ NA 0.50 W U-V-I/ WD 3.50mm
  • ACS APO 40x oil/ NA 1.15 Oil CS/ WD 0.27mm
  • ACS APO 63x oil/ NA 1.30 Oil CS/ WD 0.16mm

A Leica DFC500 camera is also attached to this microscope platform so
that both wide-field fluorescence and confocal images of the same sample
can be captured.

Manuals Available:

Leica SPE2

Vertical interneuron of the dorsal horn labelled with biocytin. Denise Tam.