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Cameron J. Nowell

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Widefield microscopy is useful for the examination of thin tissue sections or whole cells, be they stained or unstained. All widefield microscopes in the Centre are fitted with high sensitivity monochrome CCD cameras that have the ability to capture colour images as well (SPOT RT3 Slider and Pursuit Slider).

The widefield platforms available are:

  • Stereo microscope (Nikon SMZ1500) with fluorescence capability. Fitted with a Nikon DMX1200C colour camera.
  • Upright widefield microscope (Nikon 90i) with fluorescence, DIC and Phase capability. Fitted with 3 cameras (SPOT RT3 Slider, Colormetrics Coolsnap HQ2 and Nikon DMX1200C). This microscope also has a motorised stage allowing for the automated acquisition of large tile scans (montage images made up of multiple fields of view).
  • Inverted widefield microscope (Nikon TE2000-E) with fluorescence, DIC and phase capability. Fitted with SPOT Pursuit Slider camera. This microscope is also fitted with an incubator and gas control allowing for live cell/tissue/animal imaging to be performed.

 

 

Confocal microscopy allows thin optical sections of thick tissue or cells to be captured. These thin slices can then be combined to provide detailed 3D reconstructions of the scanned sample. Multiphoton microscopy gives the added advantage of increased penetration depth (up to 10 times that of single photon confocal) and decreased phototoxicity to live samples.

The confocal platforms available are:


Single Photon

  • Nikon C1plus attached to a Nikon Ti-E with 405nm, 488nm and 532nm laser lines (all solid state).
  • Olympus FV1000 with spectral and transmitted light detection attached to a Olympus IX81 with 405nm, 473nm, 559nm and 635nm laser lines (all solid state). This microscope is also fitted with a SIM scanner for bleaching based experiments and a zero drift automatic focus system for long term live experiments.
  • Olympus FV1000 with spectral and transmitted light detection attached to a Olympus BX61W with 405nm, 473nm, 559nm and 635nm laser lines (all solid state).

Multi Photon

One Spectra-Physics Mai Tai® DeepSee® laser is shared between the two Olympus FV1000 platforms. This laser provides pulse widths of less than 100fs and is tunable from 690nm-1020nm providing multi photon excitation to a broad range of fluorescent proteins. Each FV100 system is equipped with two non-descanned detectors allowing simultaneous acquisition of two fluoropohres

 

All the confocal microscope platforms are equipped with incubators and gas control, allowing for live cell or tissue imaging to be performed. Additionally the Olympus BX61W microscope has anesthesia attached for imaging live animals.

 

 

Analysis of captured brightfield and fluorescent images can provide large amounts of useful information from simple percentage stain present, to complex diffusion measurements, through to more complicated and intensive image correlation. The facility provides and supports the following applications:


MetaMorph (v7.6.1)

One of the industry standard analysis packages. This software allows for a large range of analysis to be performed. Some examples are:

  • Determine the area and intensity of a given stain in a tissue or cell.
  • Count the number and type of cells.
  • Analyse cell cycle characteristics of a given sample.
  • Segment out individual cells and provide information on each cells morphological characteristics
  • Generate 3D models and measure volumes of objects within the model.
  • Track objects (e.g. live cells) over time in 2D and 3D.

All functions can be automated and ran with minimal user input if required.

 

Imaris (v6.3)

The industry standard 3D/4D analysis and visualisation software package. This software can generate detailed 3D/4D models from confocal data that can be analysed in a range of ways. Some examples are:

  • Create animations of rotation, fly through, cropping etc..
  • Track objects in 3D over time (4D analysis).
  • Measure volume, shape, texture etc of objects.

 

ImageJ

Free, open source software that has many powerful function available through a large list of user written plugins.

 

BioImageXD

Free, open source 3D/4D rendering software

 

Olympus FV1000

Software provided with Olympus confocal systems allowing basic image processing options as well as advanced function such as 3D/4D movie generation and analysis of co-localisation.