Spinning Disc Confocal Microscope



Cell division in live nematod captured on spining disc confocal microscope
Picture courtesy of David Smith, Fryer Company

Most confocal microscopes use scaning laser technology for image acquisition. However, scanning laser severely lacks the temporal resolution required to monitor very rapid biological process (one example is microtubule dynamics). In order to provide users of this facility an instrument capable of rapid confocal image acquisition, we have purchased the Yokogawa spinning disc confocal microscope from Perkin Elmer, fitted on a Nikon TE2000-U fluorescent inverted microscope.

Using the Nipkow disc technology, the spinning disc consists of a thin wafer with hundreds of pinholes that are arranged in a spiral pattern. When a portion of the disc is placed in the internal light path of the confocal microscope, the spinning disc produces a scanning pattern of the subject. As the subject is inspected, light is reflected back through the microscope objective. The light, that was reflected from in front of or behind the focal plane of the objective approaches the disc at an angle rather than perpendicularly. The pinholes of the disc permit only perpendicularly oriented rays of light to penetrate. This enables the microscope to view a very thin optical section of tissue, at extremely high speed because it is not limited by the speed of a scanning laser. Instead, the combination of AOTFs (acousto-optical tunable filters) will allow micro-seconds switch of excitation wavelengths.


Time-resolved keratin network 3-D remodeling under fluid shear stress. Images were taken using the spinning disc and 4D iso-surface image reconstruction performed using Volocity 2.0 software. Video clip courtesy of Karen Ridge lab.

The Yokogawa microlens excitation technology splits the laser beam into 1,000 beams to simultaneously scan the entire field in less than three milliseconds – at a rate of 360 times per second. This high frequency, low intensity illumination substantially decreases harmful photobleaching and phototoxicity to ensure cells continue functioning. And, the GFP or fluorescent probe signal is maintained at an acceptable imaging level – for the duration of the experiment. Even hours into the process, living cell images are still strong.

The facility has also fitted a Piezo stage controller that is capable of auto- focusing to a sub-micrometer range, thus allowing users to perform very long term live cell imaging, with or without the presence of the users.

Equipped with a powerful Innova-70 laser system from Coherent, the system is capable of delivering up to 5-watt laser power to the specimen, thus allowing users to perform ultra-rapid image acquisition. The following excitation lasers are available: 442nm, 488nm, 514nm, 568nm, and 647nm. Please click here to check your fluorophores excitation and emission wavelengths before using the scope. For those using Bioptech Live cell imaging chamber, the facility also has a stage adaptor for the Nikon TE2000-U microscope.



Real time live cell applications

Protein production and transportation within live cells using fluorescent protein probes (GFP)

Intracellular calcium measurements in living cells and organs

Receptor turnover and localization with fluorescently labeled ligands

Vesicle trafficking and structural dynamics of intracellular organelles

Structural dynamics of cell components

Co-localisation studies of protein, DNA, carbohydrate and lipids within cells

Cell-cell and cell-matrix interactions

Fixed cell applications

Structural studies of cell components

Pathological and histological specimens

Whole mount embryo studies

This instrument will require substantial amount of training, and at least some fluorescent imaging experience on the part of the users. Please contact Teng-Leong Chew for initial technical consultation and subsequent training and experimental design.