Super-resolution microscopy for cellular imaging

M. Erdlyi1,2, A. Knight1, E. Rees2, D. Metcalf1, G. S. Kaminski Schierle2, C. F. Kaminski2

1. Analytical Science Division, National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK 2. Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museum Site, Pembroke Street, Cambridge CB2 3RA, UK

Abstract
The spatial resolution of traditional optical microscopy methods such as epifluorescence and confocal is limited (typically by half of the applied wavelength) due to optical diffraction. Super-resolution imaging methods have been proposed to break this resolution barrier down and to provide molecular scale imaging. We have built instruments based on direct stochastic optical reconstruction microscopy (dSTORM), which can achieve a resolution of 20 nm using commercially available organic dyes. The optical system is based on total internal reflection fluorescence (TIRF) illumination by a fibre-coupled 642 nm diode laser. Image stacks are captured by an EMCCD and processed by an inhouse developed software program to give super-resolution images. We are initially investigating the clathrin mediated endocytosis of EGF receptor proteins.

Localization Microscopy
White light image Singe frame image
15000 frames

dSTORM image
15000 frames

2 m

Single frame with isolated fluorophores

Selection of region of interests

Localization

Post-processing

In localization microscopy techniques the fluorescence molecules can be turned on and off (active and passive states). In a single frame just a few molecules are on (active) with spatial separation smaller than the spatial resolution, and therefore their exact position can be given by a fitting algorithm. The final image is generated by repeating the process several thousand times.

Optical Diffraction Limit

Measured PSF

Mechanical stability
30 min
Stability (nm) 100

1 h 45 min

R 0.61
Calculated PSF

NA
Pixel size=16nm

3 h 30 min 5 h 30 min

50 20 nm 0

100

200 Time (min)

300

Calculated PSF

DOF 2

NA2

Normalized calculated PSF

Data acquisition takes several minutes. Mechanical stability of the system is crucial; spatial drift must be smaller than the localization precision (1020nm). The main source of drift is temperature-related.

Optical microscopy methods are limited by optical diffraction. The spatial (R) and axial (DOF) resolution is given by the Rayleigh limits. Their typical values are 250nm and 600nm, respectively.

Applications
Clathrin Mediated Endocytosis
TIRF image on unresolved receptor-bound EGF dSTORM image on resolved receptor-bound EGF

TIRF and Highly Inclined Illumination

TIRF illumination is ideal for single molecule detection because it provides excellent sectioning capability (100 nm) and thus eliminates background fluorescence effectively. As above, but with the addition of an anticlathrin heavy chain antibody to highlight areas of clathrin coated pit and vesicle formation.
TIRF (Clathrin-Alexa-555)

HeLa cells serum-starved for 30 minutes, followed by incubation with EGFAlexa 647 for 30 minutes on ice to allow binding to EGF receptor whilst preventing its internalization (endocytosis).

TIRF (Clathrin-Alexa-555)

Normalized TIRF

TIRF

TIRF (EGF-Alexa-647)

TIRF (EGF-Alexa-647)

Highly Inclined illumination

dSTORM (EGF-Alexa-647)

White light
Temperature block

TIRF illumination requires samples to be attached to the surface. However, fixed cells usually detach from the surface by several m, therefore highly inclined illumination is more practical.

Signalling and gene transcription modification

Endocytosis resulting in silencing of signalling and receptor degradation

Clathrin Epidermal growth factor (EGF) EGF receptor

References
M. Heilemann, S. van de Linde, M. Schuttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer: Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes, Angew. Chem. Int. Ed. 47, 6172 6176 (2008). G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjrner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, C. F. Kaminski: In Situ Measurements of the Formation and Morphology of Intracellular -Amyloid Fibrils by Super-Resolution Fluorescence Imaging, revised manuscript in review for publication in JACS

Acknowledgements
This work was funded by the National Measurement System, the EPSRC-NPL Postdoctoral Research Partnership scheme and by the CamBridgeSens via the Innovation Competition Program. The authors also acknowledge the help of Prof. Markus Sauer from Julius-Maximilians-University, Wuerzburg.

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