A Research Blog

Antonius Van DongenAssociate Professor Antonius Van Dongen of the Neuroscience and Behavioural Disorders Programme at Duke-NUS Medical School is also Director of the SingHealth Advanced Bioimaging Core.Today, Dr Van Dongen shares with us more about microscopy and the Core's advanced bioimaging capabilities available to SingHealth and Singapore investigators.

The invention of the light microscope is usually attributed to Zacharias Jansen (1580 - 1638), while Anton van Leeuwenhoek (1632 - 1723) pioneered the first biology experiments using a microscope he built. Ever since its inception in the 16th and 17th centuries, it was believed that there is a fundamental lower limit to the smallest detail that can be resolved with a light microscope. This idea was given a theoretical foundation by Ernst Abbe in 1873, which allowed the resolution limit to be calculated from the wavelength of the light and the properties of the optics. For the best possible optics, the limit is approximately a quarter of a micrometer (250 nm). This Abbe limit had the status of an unbreakable physics law until the 1990s, when several investigators independently developed approaches that could break it. In 2014 the Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for "the development of super-resolved fluorescence microscopy".

The increase in resolution afforded by these new methods is substantial and now allows visualization of cellular components down to the molecular level. Many structures previously only visible with electron microscopy (EM) now can be visualized by light microscopy using antibodies or GFP-fusions, which has many significant advantages. A few examples are: nuclear pore complexes, axon cytoskeletonsynaptic contacts and virus particles. The super-resolution methodologies continue to be improved and now can be used to generate 3D images of cellular structures at the nanometer scale: the movie shows a single transcription complex in the nucleus of a cultured neuron consisting of three proteins, PHF8 (green),  PSF (blue), and Tip60 (red), obtained by 3D-STORM (Oey et al. 2015). Mutations in PHF8 cause mental retardation, while TIP60 has been implicated in the pathophysiology of Alzheimer’s disease. The PHF8/TIP60 chromatin-modifying complex regulates the expression of Arc, a gene whose function is critical for memory consolidation (Wee et al., 2015).

Legend: Green - PHF8; Blue - PSF; Red - Tip60

These super-resolution light microscopy approaches are now available to all investigators in the SingHealth Duke-NUS Academic Medical Centre (AMC) with the establishment of the SingHealth Advanced Bioimaging Core. The core is also accessible to academic researchers outside the AMC, as well as scientists from industry and the private sector. Users of the core have access to a wide variety of imaging platforms and services in both conventional and super-resolution light microscopy, as well as electron microscopy. The core provides guidance and training to investigators in image acquisition and analysis. We also provide services for electron microscopy sample preparation and imaging.

Each super-resolution imaging technique adopts a unique methodology for improving the resolution beyond the diffraction limit for light microscopy. The Table below summarizes the four super-resolution methods that are available at the Advanced Bioimaging Core, with their resolution and main features:

Method   Resolution Features
SIM Structured Illumination Microscopy 90 nm No restriction on wavelength: use any fluorophore.Medium acquisition speed.
STED Stimulated Emission Depletion 50 nm Fastest acquisition. Pure optical method. Restricted fluorophores.
STORM Stochastic Optical Reconstruction Microscopy 15 nm Highest resolution. Slowest acquisition. 
PALM Photo-activated Localization Microscopy 15 nm Highest resolution. Slowest acquisition. Requires photo-activatable GFP isoform.

 In addition to super-resolution imaging, the SingHealth Advanced Bioimaging Core Platform provides access and training for many conventional imaging techniques in electron and light microscopy:

1)      Laser scanning confocal (LSC)

2)      Spinning Disc Confocal (SDC)

3)      Bright field (BF)

4)      Differential Interference Contrast (DIC)

5)      Wide-Field fluorescence (WF)

6)      Total Internal Reflection Fluorescence (TIRF)

7)      Live-cell imaging (SDC, WF, TIRF, DIC)

8)      High-Content Screening (HCS)

9)      Laser-Capture Micro-Dissection (LCMD).

10)   Transmission Electron Microscope (TEM)

 

For more information, please refer to this website:

https://research.singhealth.com.sg/Pages/advanced-bioimaging.aspx

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