Part of the Faculty of Infectious and Tropical Diseases, our facility is set up to enhance the research work of staff at the School. Our aim is to provide training and access to state-of-the-art equipment for the pursuit of excellence within laboratory-based research. These facilities are open to both staff and students of the School and external users.
|Theresa Ward||Academic in Charge of Imaging Facility|
|Liz McCarthy||Imaging, Biacore and Real Time PCR|
|Rachel Gregory||Imaging, Biacore and Real Time PCR|
|Liz King||Flow cytometry and Luminex|
Zeiss LSM510 Confocal
The LSHTM confocal microscope is an Zeiss axiovert contained within a incubation chamber, which can be temperature and CO2 regulated. The microscope has an electronic stage, and uses Zeiss LSM510 software for image acquisition. The microscope is equipped with x10, x20, x40, x63 and x100 lenses, and the following lasers;
Laser Diode: 405nm i.e. DAPI
Argon laser: 458, 488, 514nm i.e.FITC/Alexa 488
HeNe1 laser: 543nm i.e. rhodamine/CY3/Alexa 546
HeNe2 laser: 633nm i.e. CY5/Alexa 633
Laser scanning microscopy (confocal) enables images on a single plane to be taken within a larger section. It uses light from a laser to excite the sample which is passed though a pinhole which is much smaller than those used in a conventional microscope allowing light from a narrow plane of focus to pass through to the photomultiplier tube. The photomultiplier tube is an extremely sensitive device for converting photons into an electrical signal. The confocal microscope can also take multiple sections through cells to produce 3D images of both live and fixed samples.
This technique is useful for determining the subcellular location of stained molecules within the cell and to establish whether molecules collocate. Examples of applications used at the LSHTM are: observing cell component rearrangement and protein expression following exposure to an organism, cell-cell interactions, observing expression of recombinant proteins often linked to GFP within cells or parasites.
The microscope is also equipped with a Femtojet microinjection unit allowing the introduction of DNA into individual cells in culture.
- Laser Capture Miccrodissection
Laser Capture Microdissection (LCM) is a technique whereby a population of pure cells can be isolated from a heterogenous tissue sample.
We have an Arcturus Pixcell IIe Laser Capture Microdissection System. This system uses an infra-red (IR) laser to capture cells of interest onto a transparent film placed above the sample. The microscope is equipped with x5, x10, x20, and x40 lenses, with fluorescence capabilities. There are filters available to differentiate between fluorescence in red, blue and green spectra.
LCM can be used on a variety of sample preparations such as blood smears, cell cultures and tissue sections.A cap containing the 'capture film' is placed on top of the sample. The IR laser is aligned over the area of interest and a laser pulse fired. The energy from the laser pulse is absorbed by the film, and does not affect the sample itself. The absorbed energy causes the film to melt, this creates contact between the film and the cells underneath which then adhere to the film.
We allow the use of our LCM system by users outside of the School. This is by arrangement only, and charges apply. We provide training on the machine itself, and at least 1 session of supervised training is compulsory prior to usage regardless of previous experience.
- CCD Fluorescence Microscope
The CCD microscope is an Axioplan 2 upright microscope with a CCD camera connected to a computer with Perkin Elmer Volocity software. The microscope is equipped with x5, x10, x20, x40 and x100 lenses, as well as red, blue and green fluorescence filters. This microscope is used for imaging histology sections, as well as taking fluorescence images, z stacks and movies.
- Flow Cytometry
Becton Dickenson LSR-II
3 laser (B/R/V), 2 scatter, 14 fluorochrome (6/3/6) LSR-II flow cytometer for Multiparameter analysis. This model has a High Throughput System (HTS) for acquiring data from 96 well plates and FACSFlow Supply system for ease of use in operation. Cytometer setup and tracking (CST) and FACSDiva 6.1.3 software are used to QC the instrument and acquire the data.
Becton Dickinson FACSCalibur
The 488nm (Ar) and 635nm red diode lasers allow 4 fluorescence and 2 light scatter parameter detection. The FACS Calibur is located in our Containment Level 3 laboratory for work with Category 3 pathogens. This model has a Carousel option, and the Macintosh Computer runs CellQuest v3.3 and Worklist Manager software for acquisition.
Flow Cytometric Data Analysis
There is a dedicated Macintosh Workstation located on the 6th floor specifically for the Analysis of Flow Cytometric data using FlowJo and Spice*. USB dongles for the use of FACSDiva and FCAP Array software on User’s own computers are available for short-term loan.
The Biacore 3000 is an opitical biosensor which monitors real-time interaction of macromolecules via measuring their surface plasmon resonance. There are many potential applications which include affinity and binding kinetics, epitope mapping and concentration determination. This piece of equipment is at present being used to measure interactions between various peptides and receptors.
We have a number of manuals, protocols and training CD roms for potential users. We also offer training and advice on use, experimental design and data analysis.
- 7500 Fast RT PCR machine
The 7500 Fast is a real-time PCR machine which enables detection and quantification of PCR products in real-time using either SYBR green reagents or Taqman probes. It is possible to monitor PCR reactions cycle by cycle enabling quantification and rapid analysis of many different targets.
Most users within the school use SYBR Green reagents which emit a fluorescent signal upon binding to double-stranded DNA molecules. This allows the amount of each PCR product being synthesised to be measured, giving either relative or actual measurements of mRNA (via RT-PCR) or DNA levels. The results are plotted as a sigmoidal curve in which the number of cycles are plotted against fluorescence. The threshold cycle (CT), is used to calculate the starting amounts of template DNA.
This technology can be used in number of applications including measuring gene expression, pathogen detection and in post PCR non quantitative assays for allelic discrimination. Within the School it is being widely used to measure gene expression.
- Luminex 100
The Luminex® 100 System is a laser based system that can be used to develop multi-analyte assays. It allows the simultaneous assay of up to 100 analytes in a single well of a microtiter plate by using dyed antibody coated beads which bind the analytes. A reporter antibody with another dye then quantifies the amount of analyte on the bead.
This system can be used for a number of different applications including nucleic acid assays, receptor-ligand assays, immunoassays and enzymatic assays.
Within the unit it is currently being used to determine cytokine concentrations by fluorescence linked immunosorbent assays (Fluorescence ‘ELISAs’) and to quantitatively measure multiple RNA targets.
The advantages over traditional methods are the low volume of sample required (50μl), the ability to perform multiple analyte detection on one sample, sensitivity and speed.