Confocal microscopy is a fluorescence imaging technique which uses a pinhole in the detection light path to remove out-of-focus haze, generating an image with improved contrast and finer detail. The removal of signal above and below the focal plane creates an image that represents a slice or "optical section" through the sample. By stepping through a series of focal planes and acquiring a series of optical sections it is possible to generate a three dimensional representation of the sample which can be analysed as a volume. Confocal microscopes can be used to image both fixed and live specimens and are generally considered the work-horse instruments of a light microscopy facility.
High content imaging (HCI, also called High Content Screening) describes an automated workflow that can be divided into three-steps: image acquisition, image processing and data analysis. HCI can massively increase the throughput of imaging experiments, reducing user bias and improving the quantitation of image data. HCI can be used in large-scale assays such as CRIPSR screens and drug library screens.
Wide-field fluorescence is the most straightforward method of fluorescence microscopy. Excitation light is directed through the objective lens to illuminate the sample and the same lens used to capture the emitted fluorescence. The emitted light can be looked at by eye or captured using a high-sensitivity camera. Wide-field systems have the benefits of ease of use, speed and sensitivity, making them suitable for a broad range of different applications including live cell imaging, imaging tissue sections and imaging samples prepared in multi-well plates.
The resolution of standard light microscopy is limited due to diffraction, with an optimised configuration unable to resolve two points separated by a distance less than ~250 nm. Reducing this distance will result in the points appearing as a single object meaning that it is not possible to use standard light microscopy to directly visualise the details of biological structures that are less than ~250 nm in size.
Super resolution microscopy (SRM), sometimes called optical nanoscopy, is a term covering a variety of light microscopy techniques that enable imaging below the diffraction limit. The SRM methods available at Babraham enable our researchers to capture images with a resolution in the range of 20-180 nm.
Multi-photon imaging (MPI) uses short pulses of high intensity near-infrared (NIR) laser light to drive fluorescent molecules into their excited state. NIR light is absorbed and scattered less than visible light in biological tissue, providing improved ability to image into samples at depth. This makes the technique very appropriate for imaging into living tissues where MPI has been used to study the dynamics of individual cells in living animals.
Fluorescence imaging uses different colours to label different targets in the same sample. This is referred to as multi-plex imaging, and typically uses a maximum of five different colours to identify five different targets. Using special modes of imaging it is possible to increase number this to around ten (sometimes referred to as high-plex). Certain applications require the identification of many more targets, for example in the tumour microenvironment or in the germinal centre, where it is necessary to understand the interaction of multiple populations of different cell types. This can be achieved with sequential rounds of labelling and imaging, with the possibility of imaging limitless numbers of targets in the same sample. A variety of different "ultra-plex" methods have evolved to facilitate this, with varying degrees of complexity and automation.
Biology is dynamic and understanding the processes of life requires the study of living systems. Light microscopy, particularly fluorescence microscopy, is well-suited for this purpose and live cell imaging is a routine method of scientific investigation. Most of the imaging platforms in our Facility have been adapted to accommodate living specimens with full enclosure or stage-top environmental control systems regulating temperature, CO2, O2 and humidity. For longer term studies it is possible to image living samples inside the tightly-regulated environmental control of a tissue culture incubator using Incucyte technology.
Laser capture micro-dissection (LCM) is a tool that allows isolation of selected regions from tissue sections. A brightfield and/or wide-field fluorescence image is used to identify and select the target area(s) of interest. A UV laser cuts around the selected areas and a high energy laser pulse projects the isolated section(s) into a collection device for downstream analysis. In this way it is possible to, for example, isolate RNA from selected cells that are present in a tissue section.
Electron Microscopy (EM) uses a beam of electrons to illuninate a sample rather than light, with electro-magnetic lenses shaping and focussing the beam rather than lenses and mirrors. Electron micrsoscopes can generate images with much higher resolution than those acquired using standard light microscopy, making it an ideal technique to study fine surface details or sub-cellular ultrastructure.
The Imaging Facility has all the tools required for standard histological techniques including a wax embedding station and microtome, a cryostat for sectioning frozen tissue and a vibratome for cutting thicker sections. The Facility's Nikon Ti2 wide-field microscope is equipped with a Nikon Ri2 camera to detect colour contrast and has a motorised stage to enable rapid acquisition of large area scans; all of the Facility's confocal and wide-field systems can image fluorescence labelling in tissue sections.