SIM is an imaging technique that employs patterned excitation light to generate interference patterns (moiré patterns) that contain high frequency spatial information. By collecting a series of images with different interference patterns, specialized algorithms are able to extract super-resolution detail to produce a reconstructed final image that has approximately twice the resolution of traditional diffraction limited microscopes, ie. approx. 110 nm (XY), 300 nm (Z). This technique is ideally suited for 3D imaging of thin (<20 um), fixed samples that have excellent fluorescence labeling.

Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) function by stochastically cycling fluorophores between ‘on’ and ‘off’ states in a controlled manner, followed by precise localization of the centre of each fluorescent molecule to within tens of nanometers. By processing thousands of images, the molecular coordinates of all labeled molecules are obtained with a resolution ten times better than traditional diffraction limited microscopes, i.e. approximately 20 nm (XY), 50 nm (Z). This technique is best suited for imaging the stoichiometric architecture of proteins within defined complexes, and is largely limited to fixed, cellular samples.

STED microscopy overcomes the diffraction limit by overlapping the primary excitation laser with a secondary, donut-shaped, high-power laser beam. Irradiation by the secondary laser forces molecules in the outer region of the primary excitation area to return to the ground state, while leaving fluorophores in the centre of the excitation focus to form the high resolution image. This application can achieve resolutions up to 50 nm laterally, and up to 120 nm axially, and is best suited for 2D or 3D imaging of thin samples (<50 um) that have excellent fluorescence labeling.

Confocal microscopy is a powerful technique in which a pinhole is used to block out-of-focus light in image formation, thereby generating improved contrast and resolution compared to widefield microscopes. Generally speaking, there are two types of confocal microscopes: point scanning and spinning disk. Although slower than a spinning disk confocal, point scanning confocal microscopes tend to achieve higher resolutions, and have more integrated solutions for specialized techniques such as fluorescence recovery after photobleaching (FRAP). On the other hand, spinning disk confocals tend to have significant advantages in both speed and phototoxicity, making them ideally suited for live cell imaging.

In recent years, the resolution of traditional point scanning confocal microscopes has been extended to the super-resolution realm by implementing three key components to a workflow:

1. Closing the pinhole diameter below 1 Airy Unit,
2. Using low-noise, high-sensitivity detectors,
3. Combining pixel oversampling with modern deconvolution algorithms.

Taken together, these implementations can improve resolution by up to 40% compared to traditional confocal microscopes, i.e. 120 nm (XY), 400 nm (Z), and can be applied to most standard confocal applications.

A specialized application of confocal microscopy is multiphoton imaging. In this technique, a specialized infrared laser is used to selectively excite molecules located within a femtoliter volume of space, up to one millimetre deep within a sample. The primary advantage of multi photon imaging is reduced phytotoxicity and deep sample penetration, making it ideal for intravital microscopy.

Widefield imaging is the method of choice for basic fluorescence imaging. It is most often applied to examining protein expression levels in cells and tissue, general patterns of localization, and population studies. It is especially powerful for high-speed imaging, as well as ratiometric measurements of intracellular pH, or other ion levels. Finally, it is the tool of choice for researchers looking to image cellular samples with white light (eg. DIC imaging of wound assays, beating cilia, etc.).

In light sheet imaging, samples are illuminated perpendicularly to the direction of observation using a thin sheet of light that is projected through the sample. Although the lateral resolution of a light sheet microscope is comparable to a widefield microscope, its key advantages are greatly reduced phototoxicity, rapid imaging, and the ability to image large samples from multiple directions for increased clarity and resolution. This technique has been masterfully adapted for use with cleared tissues, such as brain, kidney, and other organs measuring up to several millimetres in thickness

FLIM is an excellent technique for mapping the local environment and molecular interactions of a fluorophore. A fluorophore’s lifetime (ie. the amount of time spent in the excited state) is a quantitative signature that can be used to extract information about its surrounding environment: ion concentrations, pH, viscosity, and molecular interactions. Furthermore, because fluorescence lifetime measurements are not affected by probe concentrations or photobleaching, and are independent of excitation, FLIM is an ideal tool for measuring FRET in living cells.

FCS is a correlative analysis of fluorescence fluctuations over time within a defined volume. As fluorophores enter and exit the observed volume, the resulting fluctuations in intensity encode parameters such as diffusion rates, molecular volume, binding affinities, and colocalization. This tool is especially useful for measuring the mobility of rapidly diffusing cytosolic proteins.

TIRF microscopy is a specialized technique that allows for imaging of fluorescent molecules located within 200 nm of the coverslip. It accomplishes this by the generation of a fluorescent evanescent wave that decays exponentially as it projects upward from the surface of the coverslip. In doing so, only fluorophores located 100-200 nm from the coverslip are excited, resulting in very high signal-to-noise imaging of the basolateral surface of cells. This technique is especially useful for observing membrane associated processes such as ligand binding, cell adhesion, and vesicle trafficking from the cell surface.

Digital slide scanning is the ideal solution for imaging whole tissue sections. Up to 250 slides can be automatically scanned in a high-throughput fashion using high-resolution 20x and 40x objectives, in both brightfield and fluorescence modes. The resulting images can be analyzed to provide area quantification, complex colour analysis, cytonuclear quantitation, etc. This technology is featured as a service in the Facility, with the turnaround typically being 24 hours.

All Imaging Facility equipment can be booked through QReserve.