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.
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.
Light-sheet microscopy (also known as Selective Plane Illumination Microscopy) is a fluorescence imaging technique which uses a thin sheet of light to illuminate a specimen, generating optical sections by restricting the illuminated volume. Compared to other optical sectioning methods such as confocal, light-sheet microscopy is rapid and "gentle", making it appropriate for imaging larger specimens and specimens which are sensitive to light.
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.