STED

STED = STimulated-Emission-Depletion

Basic photophysics
S1*
S1

Leutenegger et al. (2010) Optics Express 18, 26417-26429.

Principles of STED

Yamanaka et al. (2014) Microscopy 63, 177-192.

Klar et al. (2000) PNAS 97, 8206-8210.

Principles of STED

Hell (2007) Science 316, 1153-1158.

Yamanaka et al. (2014) Microscopy 63, 177-192.

Klar et al. (2000) PNAS 97, 8206-8210.

Factors that Affect Spatial Resolution

IS: intensity that makes stimulated emission rate equal spontaneous dec
ay rate

Imax/IS cannot be made arbitrarily large

Photobleaching
Damage to sample
Finite spontaneous decay rate

Maximal spatial resolution of biological samples:

~30nm (until 2013.9)
Lakadamyali. (2014) ChemPhysChem 15, 630-636.

Generalization: RESOLFT
REversible Saturable Optical F GSD(Ground State Depletion)
Luorescence Transitions
Super-resolution microscopy th
at involves saturation/depolariz
ation of dark/fluorescent state
Generalization of STED

SSIM (Saturated Structured Ill

umination Microscopy, SPE
M)

Temporal Resolution of STED

Spatial resolution & field of view scanning rate
Current implementations allow 35ms long video-rate(28fps) recordings wit
h minimal resolvable distance of ~60nm and 5m2 field of view

Continuous wave laser

Improved scanning rate
>5fps, 190ms, ~65nm, 72m2

Parallelization
116,022 doughnuts
12,000m2 of area scanned
in 2~4s with minimal resolvable
distance of ~80nm
Moneron et al. (2010) Optics Express 18, 1302-1309.
Lakadamyali. (2014) ChemPhysChem 15, 630-636.

Chmyrov et al. (2013) Nature Methods 10, 737-740.

Westphal et al. (2008) Science 320, 246-249.

Temporal Resolution of STED

Spatial resolution & field of view scanning rate
Current implementations allow 35ms long video-rate(28fps) recordings wit
h minimal resolvable distance of ~60nm and 5m2 field of view

Continuous wave laser

Winter, Shroff (2014) Curr. Opin. Chem. Bio. 20, 46-53.

Improved scanning rate

>5fps, 190ms, ~65nm, 72m2

Parallelization
116,022 doughnuts
12,000m2 of area scanned
in 2~4s with minimal resolvable
distance of ~80nm
Moneron et al. (2010) Optics Express 18, 1302-1309.
Lakadamyali. (2014) ChemPhysChem 15, 630-636.

Chmyrov et al. (2013) Nature Methods 10, 737-740.

Westphal et al. (2008) Science 320, 246-249.

Applications of STED in biomedical research

Subcellular structure imaging
Probing distribution of molecules
Possible new diagnostic tool?
Sub-neuronal structures and dynamics
May lead to better understanding of neura
l physiology, diagnosis, and/or treatment

Molecular dynamics & interactions

Blom & Widengren (2014) Curr. Opin. Chem. Bio. 20, 127-133.
Yamanaka et al. (2014) Microscopy 63, 177-192.

Blom & Brismar (2014) J. Intern. Med. 276, 560-578.

Spine neck plasticity regulates compartment

alization of synapses
Detailed and accurate measureme
nt of dendritic spine morphology
(100~1000nm)
Relationship between morphology
and electrical & chemical compart
mentalization in the spine
Influence of synaptic activity on m
orphology of the dendritic spine

Tonnesen et al. (2014) Nature Neuroscience 17, 678-685.

STED Super-Resolution Microscopy of Clinical Pa

raffin-Embedded Human Rectal Cancer Tissue
Formalin-fixed, paraffin-embedded rectal cancer tissue sampl
e observed under STED
Unlike conventional wide-field light microscopy, STED can ma
p detailed distribution of proteins within a cell(e.g. HERG2)

STED Super-Resolution Microscopy of Clinical Pa

raffin-Embedded Human Rectal Cancer Tissue

50m

50m

2m

0.5m