Applications

Biological Optical Microscopy Platform

The Biological Optical Microscopy Platform provides access to high-end fluorescence microscopy systems with a wide range of possible applications. The glossary below provides a brief description of the applications possible with our technology. If you would like to discuss your experiments further then don't hesitate to contact us.

CONFOCAL APPLICATIONS

Laser Scanning Confocal Microscopy
Laser scanning confocal microscopy produces very sharp images by excluding out of focus light using an iris in the lightpath known as a pinhole. Images are produced by scanning a laser beam across the sample and collecting emitted light on PMT or GaAsP detectors.
This technique can be applied on fixed or live samples and when combined with motorised stages can be used to build up large 3D datasets.
Systems Available: Click here for more info
Live Cell Imaging
We have microscopes equipped with incubation chambers to allow timelaspe recording of live cells/samples. Most instruments are located within PC2 certified facilities enabling experiments on human pathogens such as parasites and viruses.
Within BOMP, live imaging experiments can be performed on laser scanning confocal, spinning disk confocal, super-resolution and TIRF microscopes.
Systems Available: Click here for more info
Zeiss LSM980 (Peter Doherty Institute)
Zeiss Elyra LSM880 (Bio21 Institute)
Zeiss LSM900 (Medical Building)
Leica Stellaris 5 (Eastern Hill Precinct)
Nikon Spinning Disk (Biosciences 2)
Nikon A1R (Biosciences 4)
Operetta High Content Imaging (Medical Building)
Olympus FV3000 (Physics)
Spinning Disk Confocal Microscopy
Like the laser scanning confocal microscope this microscope excludes out of focus light using pinholes. However, in the spinning disk confocal microscope there are hundreds of pinholes arranged on a spinning disk.
This produces key advantages in acquisition speed & reducing photo-toxicity, making spinning disk confocal microscopes ideal for fast and/or long-term imaging experiments.
Systems Available: Click here for more info
Nikon Spinning Disk (Biosciences 2)
Airyscan Confocal Microscopy
This super-resolution confocal microscopy technique works on the knowledge that narrowing the pinhole can increase your resolution. However, this is not normally performed, as it results in much less light reaching the detectors.
The Airyscan system gets around this limitation by using a hexagonal array of 32 GaAsP detectors to image as the laser scans across the sample. Each detector is the equivalent of imaging with a pinhole 5 times smaller than normal. The data from all 32 detectors is added together and a linear deconvolution is applied resulting in a 1.7-fold increase in resolution in xyz (5-fold increase in volume resolution). The new Airyscan Fast module enables 4x faster acquisition while providing a 1.5 fold increase in resolution.
Systems Available: Click here for more info
Zeiss LSM980 (Peter Doherty Institute)
Zeiss LSM900 (Medical Building)
Zeiss LSM880 (Medical Building)
Zeiss LSM800 (Medical Building)
Zeiss Elyra LSM880 (Bio21 Institute)
Spectral unmixing
Spectral unmixing allows researchers to stain samples with multiple fluorophores and separate the signals. This application is possible on confocals fitted with a spectral detector. Reference spectra for each of the fluorophores are used to perform a linear unmixing of the fluorescence signals. This application has been used to separate up to 10 different fluorpohpres in a single sample.;
Systems Available: Click here for more info
Multi-channel detectors (simultaneous scanning)
Zeiss LSM880 (Medical Building)
Nikon A1R (Eastern Hill Precinct)
Zeiss LSM780 (Peter Doherty Institute)
Zeiss LSM980 (Peter Doherty Institute)
Zeiss LSM880 (Bio21 Institute)
Spectral scanning (sequential scanning)
Leica SP8 (Bio21 Institute)
Nikon A1R (Biosciences 4)
Nikon C2 (Biosciences 2)
Olympus FV3000 (Physics)
Zeiss LSM800 (Medical Building)
Zeiss LSM900 (Medical Building)
Leica Stellaris (Eastern Hill Precinct)
F-Experiments
These are a group of quantitative methods that allow researchers to investigate cellular dynamics.
Fluorescence Recovery After Photobleaching (FRAP): This technique provides a measure of the diffusion or lateral movement of your molecule of interest. The experiment uses a short pulse of intense laser to photobleach a region of interest within a cell. By quantifying the recovery of fluorescence to the region of interest over time researchers can calculate the diffusion rates of their molecules of interest.
Forster Resonance Energy Transfer (FRET): This technique provides a measure of the proximity of two fluorophores. The experiment requires two specially selected fluorophores, the donor and the acceptor. When the two fluorophores are within 10nm of each other excitation of the donor will result not in emission of the donor, but in the acceptor fluorophore. Depending of FRET efficiency researchers can measure the interaction between two molecules.
Fluorescent Fluctuation Spectroscopy (FFS): This group of techniques can provide information about protein interaction, aggregation, binding kinetics and diffusion within living cells. Techniques used include Correlation Spectroscopy (e.g. STICS, RICS), Pair Correlation Spectroscopy (e.g. pCF, pCOMB) and Moment-based brightness analysis (e.g. N&B, PCH)
Systems Available: Click here for more info
All live-cell confocals - FRAP & FRET
Olympus FV3000 (Physics) - FFS & FLIM-FRET
Fluorescence Lifetime Microscopy (FLIM)
Detects the lifetime of a fluorophore (the time between excitation and emission) rather than its wavelength dependent emissions. The system can be used to for a number of applications including;
1) FLIM-FRET: Using FLIM for FRET analyses can remove a number of technical issues that occur using spectral-FRET applications
2) FLIM imaging: Can be used to image samples with spectrally similar staining e.g. Image samples with a high number of fluorophores without spectral unmixing.
3) Autofluorescence imaging: Can image autofluorescence such as NADH or tissue based autofluorescence
4) Local environment sensing: The fluorescence lifetime of a molecule can be modified by its surrounding and changes in pH, polarity, temperature, ion concentration
Systems Available: Click here for more info
Olympus FV3000 (Physics)
Image Analysis
Quantitative analyses of fluorescence images enable researchers to extrapolate extra information from their experiments. Metrics such as fluorescence intensity, area, volume, shape, distance, velocity, colocalisation etc can be fairly easily calculated using either freeware or commercially available packages.
The platform can provide researchers with access to Imaris, Huygens, Volocity, Metamorph and FIJI/Image J. If you would like to request access to a BOMP computer or request software access on your labs analysis computer then please contact us.
Systems Available: Click here for more info
Diamond Computer (Medical Building or online access)
Gold Computer (Medical Building or online access)
Green Computer (Medical Building or online access)
Cherry Computer (Bio21 Institute or online access)
Cyan Computer (Bio21 Institute or online access)

SPECIALIST APPLICATIONS

High-Content Screening
Enables fluorescence and brightfield/phase contrast imaging of samples grown in multi-well plate formats (such as 12-well, 96-well, 384 well plates). This enables researchers to perform experiments such as drug or RNAi screen with fluorescent probes for markers such as cell viability.
This can be combined with timeplapse imaging and automated image analysis to provide quantitative readouts in high-throughput assays.
Systems Available: Click here for more info
Perkin Elmer Operetta (Medical building)
Lightsheet Microscopy
The lightsheet microscope is capable of rapid imaging of larger biological samples (10x10x10mm) at the macro scale (2x-12x magnification). The system uses sheets of light to illuminate samples in the imaging plane while simultaneously acquiring the data.
The system is commonly used with samples that have been optically cleared. Tissue clearing is a developing area but most methods are based around four clearing techniques;
1) Solvent based: Sample dehydration followed by refractive index matching (e.g. BABB, DISCO, Ethyl Cinnamate)
2) Simple Immersion: Equilibration of sample with high refractive index media (e.g. SeeDB, FRUIT, ClearT, TDE)
3) Hyper-Hydration: The sample is treated with Urea/Formamide to reduce the refractive index (e.g. Scale, CUBIC)
4) Hydrogel embedding: The sample is embedded in a hydrogel, before lipids are removed and the embedded in a aqueous clearing solution (e.g. Clarity, PACT)
Systems Available: Click here for more info
UltraMicroscope II (Medical Building)
Super-Resolution Microscopy
The resolution of standard fluorescence microscopes is limited by the wavelength of light used to image the samples. Super-Resolution Microscopy refers to a group of techniques enable researchers to image beyond this resolution limit by using a number of different methods.
BOMP has two super-resolution techniques available to researchers:
1.Single Molecular Localisation Microscopy
2.Airyscan Confocal Microscopy
See individual sections below for more details
Single Molecule Localisation Microscopy
Using special fluorophores it is possible to image emission events from single molecules by ensuring that most of the fluorophores do not emit at the same time. By performing a Gaussian fit to the emission events we can narrow down their positions and use this build up a super-resolution over hundreds of images. This can result in up to a 10-fold increase in resolution in xy
Two of the more common localisation microscopy techniques are:
dSTORM: Uses reducing conditions and strong laser power to turn most of the fluorophores in a sample into a long lived dark (non-emitting) state.
PALM: Uses fluorescence proteins that are capable of photo-conversion or photo-switching to produce the blinking effect required.
Systems Available: Click here for more info
Zeiss Elyra LSM880 (Bio21 Institute)
Airyscan Confocal Microscopy
This confocal microscopy technique works on the knowledge that narrowing the pinhole can increase your resolution. However, this is not normally performed, as it results in much less light reaching the detectors.
The Airyscan system gets around this limitation by using a hexagonal array of 32 GaAsP detectors to image as the laser scans across the sample. Each detector is the equivalent of imaging with a pinhole 5 times smaller than normal. The data from all 32 detectors is added together and a linear deconvolution is applied resulting in a 1.7-fold increase in resolution in xyz (5-fold increase in volume resolution). The new Airyscan Fast module enables 4x faster acquisition while providing a 1.5 fold increase in resolution.
Systems Available: Click here for more info
Zeiss LSM980 (Peter Doherty Institute)
Zeiss LSM900 (Medical Building)
Zeiss LSM880 (Medical Building)
Zeiss LSM800 (Medical Building)
Zeiss LSM880 (Bio21 Institute)
Multi-Photon Microscopy
Multi-photon microscope enables researchers to penetrate much deeper (up to 1mm) into samples than standard confocal microscopy. This technique is often used for imaging experiments looking at tissue explants or intra vital experiments (experiments on anaesthitised animals).
The microscope uses pulses of longer wavelength (>800nm) lasers to produce fluorophore excitation (when two pulses are simultaneously absorbed by the target fluorophore). Due to their longer wavelengths, the excitation light can penetrate deeper and is less damaging to the sample.
Systems Available: Click here for more info
Thorlab Bergamo II (Peter Doherty Institute)
Olympus FVMPERS (Peter Doherty Institute)
Total Internal Reflection Microscopy
Total Internal Reflection Flurescence Microscopy is used mostly to image cell surface, cell cytoskeletal or cytoplasmic compartments. It is also used in some super-resoltuion microscopy techniques, such as PALM or dSTORM.
This technique produces an evanescent wave that illuminates only the first 100-150nm past the coverslip and therefore is much less phototoxic to the cell than standard widefield or confocal microscopy
Systems Available: Click here for more info
Nikon Spinning Disk (Biosciences 2)
Zeiss Elyra LSM880 (Bio21 Institute)
Image Analysis
Quantitative analyses of fluorescence images enable researchers to extrapolate extra information from their experiments. Metrics such as fluorescence intensity, area, volume, shape, distance, velocity, colocalisation etc can be fairly easily calculated using either freeware or commercially available packages.
The platform can provide researchers with access to Imaris, Huygens, Volocity, Metamorph and FIJI/Image J. If you would like to request access to a BOMP computer or request software access on your labs analysis computer then please contact us.
Systems Available: Click here for more info
Diamond Computer (Medical Building or online access)
Gold Computer (Medical Building or online access)
Green Computer (Medical Building or online access)
Cherry Computer (Bio21 Institute or online access)
Cyan Computer (Bio21 Institute or online access)