Widefield and TIRF Microscopy

EPI and TIRF FacilitiesEpifluorescence (widefield) microscopy allows for fast acquisition of 3D or 4D data sets as an EMCCD camera is used and an entire field of view is captured at one time. TIRFM uses the unique properties of an evanescent wave to selectively excite fluorophores within a restricted region i.e. the plasma membrane of a cell.

ESRIC houses an epifluorescent Olympus IX81 fully motorised microscope (see specification) controlled by Olympus CellR software, which is equipped with total internal reflection fluorescence microscopy (TIRFM) capabilities. 

Live cell recordings can be aquired at high temporal resolution (10 ms frame rates). This system is capable of live cell imaging on a heated stage with optional CO2 perfusion. This system uses a coverslip chamber which fits 25 mm coverslips (SLS Cat No. MIC3350). In addition a stage for mounted slides is provided.

This system is available at the Heriot-Watt University site, for all enquires please contact This email address is being protected from spambots. You need JavaScript enabled to view it..

A Large Dense Core Vesicle tracked over time at the plasma membrane of a cell using Imaris (Bitplane) software available in ESRIC. 

Confocal Laser Scanning Microscopy (CLSM)                                                                         

Confocal laser scanning microscopy (CLSM) is an optical imaging technique used to acquire highly detailed information of thin optical sections, a process known as optical sectioning. This is possible through the rejection of out

Confocal our facilities

 of focus light using pinholes and the pixel by pixel illumination of the sample.

Techniques possible on this system include Fluorescence correlation spectroscopy (FCS), time correlated single photon counting - fluorescence lifetime imaging microscopy (TCSPC-FLIM) and gated-stimulated emission depl

ESRIC houses a Leica SP5 SMD laser scanning confocal microscope (see specification) equiped with a super continuum white light laser. This system is capable of live cell imaging on a heated stage with optional CO2 perfusion. This system uses a coverslip chamber which fits 25 mm coverslips (SLS Cat No. MIC3350). In addition a stage for mounted slides is provided.etion microscopy (gSTED).

This microscope is available at the Heriot-Watt University site, contact This email address is being protected from spambots. You need JavaScript enabled to view it.

A reconstructed 3D stack of intestinal tissue from a mouse stained with two fibrous structural proteins in red and green.

SRRF-Stream

Andor SRRF-Stream is a commercial implementation of the SRRF algorithm (Super Resolution Radial Fluctuations) developed by Ricardo Henriques at University College London.  By capturing many images of the same field it is possible to calculate a probabilistic estimate of the position of fluorophores by analyzing the radial intensity gradient and the intensity fluctuations characteristic of these molecules.

The advantage of SRRF is that it is one of the few super resolution methods conducive to live cell imaging.  Conventional fluorophores can be used with comparatively low excitation energies from widefield or laser based sources with image capture rates of 1-2 seconds per frame.

SRRF is best suited to samples where the fluorophore density is low or higher density samples where photoswitchable fluorophores are used in the presence of a reducing buffer environment.  Depending on the sample, the equivalent lateral resolution afforded by SRRF can be 50-150nm.

SRRF-Stream is available on the Andor Dragonfly Spinning Disk confocal microscope utilizing widefield, TIRFM or confocal modalities (see specification).

The microscope is available at the University of Edinburgh (IGMM) site.  Please contact Ann Wheeler This email address is being protected from spambots. You need JavaScript enabled to view it. for more information.

A live capture of mitochondrial movement in a COS 7 cell.