Confocal Microscopes

Confocal microscopy offers advantages over conventional widefield optical microscopy by allowing optical sections to be collected. Out of focus light is removed from the immediate plane of focus by using an adjustable pinhole. For a good introduction to confocal microscopy, see the Olympus Microscopy Resource Centre.
There are five laser scanning confocal microscopes and one spinning disk confocal microscope within the Imaging Facility. Details of the laser lines and objectives available on each system are given below. For more information about these systems, please see the microscope comparison table and the commercial websites listed on the right. Refer to the resources section for detailed guides to specific confocal imaging techniques.

Widefield Microscopes

There are two widefield systems in the facility which are based on Olympus IX81 widefield microscopes together with Deltavision Softworx software. These systems are designed for deconvolution. Optical deconvolution is the process of reversing the effects of the point spread function on the image recorded by a light microscope.

https://www.gurdonphotonics.org/

Computational Image Processing and Analysis

Image processing is any form of signal processing where the input and output signals are images, and is commonly used to improve the signal:noise ratio and allow more accurate analysis of biological imaging data. Image analysis is the extraction of data from images and includes simple methods such as plotting intensity profiles as well as complex ones such as those used for object recognition and tracking of objects over time. There are two dedicated image processing and analysis workstations in the imaging facility with commonly used software packages installed:

Fiji (Fiji Is Just ImageJ) is an open-source image processing package based on ImageJ which includes a large number of the most useful plugins available. Fiji can be used for almost any image processing or analysis task, and additional plugins can be added to enable new functions. The Imaging Facility Team can assist with finding appropriate plugins or writing custom scripts and plugins to meet your specific requirements.

GIIF Fiji Plugins are available on our GitHub Page

CellProfiler is an open-source application designed for quantitative analysis of 2D imaging data using a pipeline construction approach.

icy is an open community platform for visualisation, annotation and quantification of bioimaging data.

BioImageXD is an open-source software package for analysing, processing and visualising multi-dimensional microscopy images. It includes versatile tools which can be built into pipelines and aims to provide a user friendly interface.

The closed-source, commercial applications Huygens, Volocity and Imaris are also available on the Imaging Facility workstations. These applications provide a set of generic tools for image analysis with a user-friendly interface, but have limited options available to customise their proprietary algorithms to meet the requirements of specific experiments. When considering the software to be used for biological image analysis, the situation is best summarised by Andrew C. Oates: "The most fundamental element is the openness; if you can't see the code of a piece of commercial software, then you cannot say what the software really does, and this is not scientific."

Huygens is an image processing package which is designed to allow automated deconvolution of images through a black-box pipeline.

Volocity displays 4D data and allows 3D object mapping to output quantitative data easily when known methods are not required. It is not supported by current versions of Mac OS or Windows 10.

Imaris is a commercial visualisation and data presentation package which also allows simple processing and analysis within a narrowly defined framework using hidden algorithms. We have a legacy license with no support from Bitplane.

Resources

Fluorophores

Tutorials

• Microscopy Acronyms
• iBiology Microscopy Course
• Choosing the right microscopy technique
• A beginners' guide to practical pitfalls in image acquisition
• AMMRF Interactive Training
• Microscopy from the very beginning
• A practical explanation of confocal pinhole
• A computational image analysis glossary for biologists
• Nikon MicroscopyU
• Leica Science Lab
• Molecular Expressions Microscopy Primer
• Olympus Microscopy Resource Center
• Zeiss Online Campus
• EAMNET Teaching Modules




Confocal Guides

• FRET
• FRAP




Super Resolution Guides

• OMX Startup and Shutdown Procedures
• OMX Sample Preparation
• Troubleshooting OMX SI Images




Quality Control

• Confocal Evaluation
• PSFs




Reference

• Clearing of fixed tissue
• Subcellular colocalisation analysis in light microscopy
• Image Processing for Biologists - Methods 2017
• Bayesian localization microscopy reveals nanoscale podosome dynamics

Microscope Comparison

Microscopy Acronyms

1. AFM - Atomic Force Microscopy
2. AOTF - Acousto-Optic Tunable Filters
3. AOBS - Acousto-Optical Beam Splitter
4. CCD - Charge-Coupled Device
5. DIC - Differential Interference Contrast
6. EMCCD - Electron Multiplying Charge-Coupled Device
7. FCS - Fluorescence Correlation Spectroscopy
8. FLAP - Fluorescence Localisation After Photobleaching
9. FLIP - Fluorescence Loss In Photobeaching
10. FOV - Field Of View
11. FRAP - Fluorescence Recovery After Photobleaching
12. FRET - Foerster Resonance Energy Transfer
13. GaAsP - Gallium Arsenide Phosphide
14. GSD - Ground State Depletion
15. LASER - Light Amplification by Stimulated Emission of Radiation
16. LIMON - LIght MicroscOpical Nanosizing
17. LSCM - Laser Scanning Confocal Microscopy
18. LSM - Laser Scanning Microscopy
19. LUT - Look-Up Table
20. NA - Numerical Aperture
21. OMX - Optical Microscope, eXperimental
22. PALM - Photo-Activated Localization Microscopy
23. PMT - Photo-Multiplier Tube
24. PSF - Point Spread Function
25. ROI - Region Of Interest
26. SIM - Structured Illumination Microscopy
27. SMI - Spatially Modulated Illumination
28. SOFI - Super-resolution Optical Fluctuation Imaging
29. SPDM - Spectral Precision Distance Microscopy
30. SPIM - Spectral Plane Illumination Microscopy
31. STED - STimulated Emission Depletion
32. STORM - Stochastic Optical Reconstruction Microscopy
33. TD - Time Domain
34. TIRF - Total Internal ReFlection
35. TL - Transmitted Light
36. VBDC - Variable Bright-Darkfield Contrast