Future equipment

With the introduction of the College Imaging Steering Group (ISG) we try to capture the need within the College for replacement or new imaging systems. Below is a short overview of some of the relatively new imaging techniques available.


Strategic Review of Bioimaging in the UK 2018 by the BBSRC
Imagining the future of bioimage analysis.

Quick links:
Structured Illumination Microscopy (SIM)
Stimulated Emission Depletion Microscopy (STED)
Stochastic Optical Reconstruction Microscopy (STORM)
Photo Activated Localization Microscopy (PALM)
Grounds State Depletion Microscopy (GSD)
Light Sheet Fluorescence Microscopy (LSFM)
Microscope Slide Scanner
Coherent anti-Stokes Raman Scattering microscopy (CARS)
Force-Sensing Optical Tweezers and Optical Trapping

 If you would like additional systems to be considered for the AIF please contact Dr Kees Straatman (krs5).

Systems previously on this list and purchased:

In December 2015 we received funding for a high speed VisiTech confocal laser microscope. This system is a 2D array scanner (spinning disk like).

Two disadvantages of a confocal laser scanning microscope is that it has to use a relatively high powered focused laser beam and has to scan the sample pixel by pixel. This gives high resolution images but at the cost of speed. To improve the speed of image acquisition but keep part of the resolution improvements one can make use of a spinning disk microscope. These systems in general use a laser to scan the sample.

  • High speed; 2,000 frames per second are possible.
  • High sensitivity due to use of EM-CCD or sCMOS camera instead of PMTs.
  • Relatively low photo-toxicity.

 

1. Super-resolution microscopy

See also: use of Super-Resolution Radial Fluctuations and Super-resolution Imaging in Leicester.

On 8th October 2014 The Royal Swedish Academy of Sciences announced that the Nobel Prize in Chemistry 2014 has been awarded to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy".

The resolution of a normal fluorescence microscope is limited by diffraction to around 200 nm radial and to around 750 nm axial. Recently several imaging techniques have been introduced to break this diffraction limit what should benefit us as many biological processes occur at scales much smaller than this limit. This development is ongoing and improvements and variations on existing techniques are reported almost every month in the literature. Below is a brief overview of the main techniques.

For a recent review article see: Requejo-Isidro (2013) J Chem Biol. 6:97-120.

NEW 1: New techniques for super-resolution imaging are regularly published and new products released. Olympus has released a super-resolution add-on for their FV1000/FV1200 confocal laser scanning microscopes, the FV-OSR (Olympus Super Resolution) unit. Information receiced from Olympus shows that the technique relies on reducing the pinhole and using a fourier transform of the intensities to visualise the high spatial frequency components. The xy resolution is around 120nm. At the moment it is not clear if the axial resolution is also improved. The scan speeds are the same as the normal FV1000 however you have to perform around 15 - 20 iterations to get a good signal to noise. 

NEW 2: An other interesting systems to hit the market recently is the Zeiss LSM 880 with Airyscan. This system offers enhanced resolution in x, y and z, combined with high acquisition speeds. Zeiss has claimed a resolution of 140 nm laterally and 400 nm axially for 480 nm. For more information visit the Zeiss website.

A. Structured Illumination Microscopy (SIM)

SIM is a wide-field fluorescence imaging technique in which a striped pattern of light illuminates the whole imaging field generating interference patterns know as moiré fringes followed by extensive software calculations. In general 7-9 images are used to build up a single high resolution image.

 SIM microscopy
Source: Nikon brochure N-SIM/N-STORM

 SIM

 

 

 

 

 

Source: http://ebi.cbst.ucdavis.edu/Projects/Fast-super-resolution-microscopy

  • SIM increases the resolution by a factor 2 in X, Y and Z. The X-, Y-resolution is around 110 nm, the Z-resolution around 350 nm (depending on light wavelength used). The depth of imaging is at the moment limited to around 10 microns as the structured illumination becomes degraded in thicker samples.
  • The technique works with standard fluorophores.
  • 3 and 4 colour imaging is possible.
  • The images can be acquired in less than 1 second (depending on signal) so live cell imaging is possible.
  • Image reconstruction algorithms are “black boxes”. At a recent meeting different results were shown, especially in Z, obtained for the same sample using different SIM systems.

Commercial systems are available from Nikon, Zeiss and GE Healthcare (Applied Precision).

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B. Stimulated Emission Depletion (STED)

In a STED microscope the improved resolution is achieved by restricting the volume from which fluorescence occurs by using a donut shaped depletion laser beam. Fluorescent molecules in the outer part of the donut are forced back to the ground state by stimulated-emission without emitting a photon. This results in an x-y resolution of around 70 nm. The only company providing STED is Leica.

Further improvement to the resolution has been achieved by selecting in time only those photons which contribute to the high spatial resolution using time gated detection, gSTED. The gSTED upgrade will improve the resolution to 50-70 nm. An additional advantage of gSTED is that the depletion power can be reduced opening the way for live cell imaging.

gSTED Source: Leica

Recently, 3D STED has been introduced as well as the possibility to use up to 3 depletion lasers (two continuous wave lasers at 592 nm and 660 nm and a pulsed laser at 775 nm) allowing more choice in the fluorophores that can be used.

 

STED

3D STED. Source: Leica

We cannot upgrade our Leica SP5 system to a 3D gSTED system but would have to purchase a new Leica SP8 system.

  • Resolution below 50 nm in radial resolution and around 80 nm in axial resolution.
  • You can choose between best lateral resolution, best vertical resolution or anything in between as STED light can be distributed between lateral and axial resolution.
  • Purely optical imaging technique.
  • Works with standard fluorophores.
  • Multi-colour STED is possible.
  • System more or less works like a standard confocal.
  • Can penetrate deeper into tissue than any other (commercial) super-resolution technique availalbe at the moment.
  • For optimal outcome the wizard driven software can be used which adjusts all necessary settings like STED laser intensity, pixel size, z step size, the pinhole, gate settings and averaging.

Commercial systems are available from Leica, Abberior Instruments and PicoQuant.

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C. Localization microscopy

The highest resolution currently available in fluorescence microscopy is obtained by techniques that use the principle of single fluorophore detection. The imaging process consists of many cycles and during each cycle only a subset of the fluorescent labels is switched on, such that each of the active fluorophores is optically resolvable from the rest. By building up images of thousands of isolated molecules sequentially over time a detailed image can be assembled. 3D localization is achieved by introduction of an astigmatic lens in the system which produces a distortion in the single molecule image PSF what correlates with its axial position.

  • Resolutions of up to 10-20 nm lateral and ~50 nm axial have been reported.
  • Most if not all systems are based on TIRF microscopy what means that only fluorophores  ~250 nm above the cover glass can be imaged.

 

-       Stochastic Optical Reconstruction Microscopy (STORM)

STORM uses often a pair of fluorophores. First the imaged fluorophore is brought into a dark state and a few fluorophores are activated exciting a activator fluorophore for a very short time; for example Cy2-Cy5 or Cy3-Cy5 in which Cy 5 is the  photoswitchable reporter dye and Cy2 or Cy3 the activator. This also allows for multi colour imaging as the activation laser is a different laser for Cy2-Cy5 and Cy3-Cy5 combination. On and off switching of single fluorophores has also been reported.

STORMLakadamyali et al. Plose ONE (2012) 0030826

 

-       Photo Activated Localization Microscopy (PALM)

PALM uses photoswitchable fluorescent proteins or fluorophores to activate a small subset of fluorophores, like PA-GFP. When the fluorophore enters a dark state or becomes deactivated by photobleaching a new subset of fluorophores is activated. In principle any photoswitchable fluorophore can be used.

 

PALM and STORM are closely related techniques. Commercial systems are available from Nikon, Zeiss, Leica and Vutara.

 

-       Grounds State Depletion (GSD)

In GSD microscopy the majority of fluorphores are brought into the dark state, which is used as the off-state. This leaves only a few fluorophores available that can emit fluorescence. This system is commercially available from Leica.

  • The commercial Leica system has a lateral resolution of 20 nm and axial of 50 nm.

  • GSD works with standard fluorophores.

  • Dual coloured GSD has been shown, maybe triple colour is possible.
  • Is used on a fully motorised TIRF based system (Leica).
  • 160x high-performance objective (Leica).
  • Uses psf for 3D calculation of signal.

 

 Overview super resolution

Above is an overview of the resolution obtained with the different systems. Adapted from: Schermelleh et al. J. Cell Biol. (2010) 190: 165-175.

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2. Light Sheet Fluorescence Microscopy (LSFM)

 SPIM

This technique is also known as selective or single plane illumination microscopy (SPIM). The basic principle is to illuminate the sample with a sheet of light that comes at 90 ° angle to the detection objective. The lightsheet allows imaging of optical sections of relative large structures at high speed with little photo damage using sensitive sCMOS cameras. To make it possible to image the sample from all sides the sample is often rotated during image acquisition. SPIM systems can be used for time-lapse imaging of developmental biology processes in intact organisms, like zebrafish, Drosophila or mouse embryo development. In recent years several different optimalisations to this scheme have been developed. Commercial systems has been introduced to the market by Zeiss and LaVision BioTec and several websites are dedicated to home build systems.

  • Reduced photo-toxicity.
  • Fast acquisition speed.
  • Imaging of larger samples/deep tissue imaging.

  
Four views of dual coloured live zebrafish embryo imaged with ZEISS Lightsheet Z.1 (Source: Zeiss Microscopy).

Review article: Huisken and Stainier (2009) Development. 136:1963-75.
A more recent development:Olarte et al (2012) Biomed Opt Express. 3:1492-505.

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3. Microscope Slide Scanner

 

These systems are also known under the name virtual slide microscope or scanner. Microscope slide scanners are imaging systems that capture images of histology and pathology slide samples at low and high resolution often followed by automatic analysis of the tissue samples. Systems are available for scanning in bright-field and/or fluorescence mode and several systems can handle tissue microarrays (TMAs). Often the system will scan the slide at low resolution to find the location of the material on the slide followed by a scan at higher resolution of the area with sample material. The results can be viewed at low resolution and the user can zoom in on areas of interest in some cases in a 'Google-Earth' fashioned way.
Scan

 

 

 

An example of a results window using a slide scanner viewing program. (A) zoom slider, (B) thumbnail image, (C) magnified field, (D) circled area is the annotation layer information used to mark up areas of interest, (E) drawing tool bar. (Khalbuss et al. (2011) Path. Res. Int. Article ID 264683)

 

Advantages:  

  • Avoids loss of staining intensity after long term storage of physical slides.
  • Electronically archive all slides.
  • Remote access for teaching and second opinion consultation.
  • Automatic image analysis.
  • Convenient viewing of results on a computer monitor.
  • Easily retrieve, review and re-analyse slide images years after the study is completed.
  • Simplifies R&D project audit or regulatory requests.
  • All main microscope companies and several specialized companies sell this type of system.

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    4. Coherent anti-Stokes Raman Scattering (CARS) microscopy

     

    Another new development in cell imaging is the use of a label-free, chemical-specific imaging technique based on the Raman spectrum of a molecule but with an increased signal compared with Raman imaging. CARS requires the input of two synchronized ultrafast laser pulses whose typically near-infrared frequencies avoid photochemical damage and allow for deep penetration into tissue.

    CARS tissue

    CARS images of tissue sections from normal colon (left), inflammatory colon (middle) and colon carcinoma (right). The color code indicates high (red) to low (blue) intensities. Bar = 50 µm. Source: Krafft at el. Analyst (2009); 134: 1046-1057.

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    5. Force-Sensing Optical Tweezers and Optical Trapping

    JPK Instruments have introduced an inverted confocal laser scanning microscope equipped with dual beam force-sensing optical tweezers, the NanoTracker 2. This system allows you to trap and track particles with a size of several µm down to 30nm with the ability to control, manipulate and observe them in real time with nanometer precision and femtoNewton resolution for direct single-molecule manipulations, force-measurements, and 3D tracking. For more information see: Integration of Confocal Laser Scanning Microscopy (CLSM) with the JPK NanoTracker™ 2.

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