gated Stimulated Emission Depletion (gSTED) Microscopy

The gated Stimulated Emission Depletion (gSTED) microscope was installed within the institute in 2012 with the aim of achieving greater resolution and clarity. Under gSTED illumination a resolution of less that 30nm can be attained thus it is possible to visualise biology at the molecular level. It must be remembered however that the smallest object that can be discerned under and microscope and resolution are not describing the same function. 

 

 

gated Stimulated Emission Depletion (gSTED) microscopy at the Cancer Research UK Manchester Institute


Imaging Modes

The system operates in several imaging modes as well as super resolution

  • Confocal Microscopy (excitation via a white light laser)
  • Two Photon Microscopy
  • Second Harmonic Imaging
  • Reflection Imaging
  • gated STED

 

Super-Resolution

To view objects under a microscope a fluorescent molecule, or fluorophore, is attached to a biological structure often via an antibody linkage. The fluorophore is excited by a light source at a particular wavelength and emits light at a longer wavelength, which is then recorded. With a suitable detection system a fluorophore/antibody linkage can be detected singularly, which has an approximate size of around 30-60nm. When imaging multiple fluorophores the resolution becomes important, this is the measure of the minimum distance between two fluorophores where the objects remain as separate and discernable entities.

Under a widefield microscopy, based around a UV source (Bulb or LED), the resolution of the instrument is around 220nm whereas under confocal microscopy with a laser source the resolution is around 200nm. Please note that the resolution stated previously is lateral resolution (xy) and not axial (z). Under gSTED the resolution of the instrument is around 30nm in the lateral and 100nm in the axial.

gated Stimulated Emission Depletion (gSTED) Microscopy

Simply described gSTED reduces the time that a fluorophore spends in the excited state (and emitting photons) when compared to widefield and confocal microscopy. Two lasers are utilised in tandem, one for the excitation (on-state) of the fluorophore and the other for depletion (off-state). The depletion beam is ring-doughnut in profile whilst the excitation laser is perfectly aligned to the centre of the ring. The depletion of fluorescence with the off-beam forces the relaxation of the fluorophore molecule thus reducing the volume of light that is excited hence emitted, which in turn increases the resolution.

actin labeled with Alexa 488 under gSTED 

The image above shows fluorescent Histone H3 in nucleus of a HeLa cell visualised under confocal microscopy (left) and super resolution microscopy using gated stimulated emission depletion, gSTED (right). Both sides of the above image has been captured by the same methods, no image processing has been applied, the only varying factor is the method of laser illumination. As the image clearly demonstrates, there is a vast improvement in clarity and resolution.   Image: S Bagley, AIFC

In addition the gSTED utilises a technique it borrowed from Fluorescence Lifetime Imaging (FLIM) technology. If a fluorophore is excited, the emitted light has an initial burst which is not planar, the emitted light travels in 360 degrees around the fluorophore, thus the physical size of the molecule/label when recorded appears larger than the object in real life. The emitted light then decays or fades over time. By using an electronic time-gate the system can control what part of the light decay will contribute towards the final image. Rather than visualise the initial burst but the fall away of light intensity, resolution is further improved.

Another method within gSTED to improve the resolution is to increase the power of the depletion laser. As the depletion laser is manipulated to form the ring-doughnut profile, as outlined earlier, by increasing the power of the laser there is a subsequent reduction in the size of the ring at the centre. Consequently the profile of the excitation beam is lessened, so diminishing the volume of excitation thus increasing the resolution. As this method increases the exposure of laser light to the biological specimen (and the attached fluorophore) consideration must be given to the damage to the signal.

In the laboratory gSTED is achieved by manipulating the power of the depletion and excitation beams, the gating strategy and amplification of the signal at the detector. Futher improvment of the image is achived by modeling the light path and sources of electronic and photon based noise to this end the Huygens software (Scientific Volume Imaging) is routinely applied to the data to imporve clarity and resolution.

actin labeled with Alexa 488 under gSTED illumination

To demonstrate the super resolution technique, actin within the cell has been labeled with Alexa488-Phalloidin and then imaged under gated stimulated emission depletion (gSTED) microscopy. Each pixel of the image is 19nm in size and the whole image took twelve seconds to capture. After image capture, deconvolution has been applied with the Huygens software resulting in an overall resolution of around 28-30nm. The spotted nature of the image is probably due to the size of the labeling complex which blocks additional binding sites.   Image: S Bagley, AIFC.

When considering the technique

Caveats to this or any other super resolution technology include:
1. Over-labeling, if a sample emits too much light due to high concentration of fluorophore the possible improvement in resolution is reduced.
2. Suitable for only certain labels and pairs of labels (see below)
3. Loss of signal via photo-bleaching can occur as with all laser based illumination systems. With multiple laser beams under gSTED illumination photo-bleaching could be an issue, increasing the laser power of the depletion beam will increase resolution but will result in laser damage to the fluorophore.

Labeling Technologies

With the equipment available at the Cancer Research UK: Manchester Institute the following labels have been utilised successfully.

Single labels

Abberior STAR 440SX, Alexa 488, ATTO 488, BD Horizon V500, Chromeo 505, Citrine, Dylight 488, eGFP, Oregon Green 488, , Oregon Green 514, eYFP and Venus

Dual-colour

  • Abberior STAR 440SX & Chromeo 505
  • Abberior STAR 440SX & Oregon Green 488
  • Abberior STAR 440SX and Abberior STAR 488
  • BD Horizon V500 & Chromeo 505
  • BD Horizon V500 & Oregon Green 488

For more information

Sample preparation and labelling for gSTED imaging (Leica) [pdf link]

Leica Science lab [link]

STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects
Vicidomini G, Schönle A, Ta H, Han KY, Moneron G, Eggeling C and Hell SW. PLoS One. 2013; 8(1): e54421 [link]

Abberior (supply of the STAR labels) [link]