Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope’s resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5–180 μm.

1.
E.
Abbe
, “
Beiträge zur theorie des mikroskops und der microskopischen wahrnehmung
,”
Arch. Mikrosk. Anat.
9
,
413
468
(
1873
).
2.
S. W.
Hell
and
J.
Wichmann
, “
Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy
,”
Opt. Lett.
19
(
11
),
780
782
(
1994
).
3.
B.
Harke
,
J.
Keller
,
C. K.
Ullal
,
V.
Westphal
,
S. W.
Hell
, and
A.
Schönle
, “
Resolution scaling in STED microscopy
,”
Opt. Express
16
(
6
),
4154
4162
(
2008
).
4.
S.
Galiani
,
B.
Harke
,
G.
Vicidomini
,
G.
Lignani
,
F.
Benfenati
,
A.
Diaspro
, and
P.
Bianchini
, “
Strategies to maximize the performance of a STED microscope
,”
Opt. Express
20
(
7
),
7362
7374
(
2012
).
5.
T. A.
Klar
,
S.
Jakobs
,
M.
Dyba
,
A.
Egner
, and
S. W.
Hell
, “
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
,”
Proc. Natl. Acad. Sci. U. S. A.
97
(
15
),
8206
8210
(
2000
).
6.
K. I.
Willig
,
J.
Keller
,
M.
Bossi
, and
S. W.
Hell
, “
STED microscopy resolves nanoparticle assemblies
,”
New J. Phys.
8
(
6
),
106
(
2006
).
7.
F.
Gorlitz
,
P.
Hoyer
,
H.
Falk
,
L.
Kastrup
,
J.
Engelhardt
, and
S. W.
Hell
, “
A STED microscope designed for routine biomedical applications (Invited paper)
,”
Prog. Electromagn. Res.
147
,
57
68
(
2014
).
8.
J. G.
Danzl
,
S. C.
Sidenstein
,
C.
Gregor
,
N. T.
Urban
,
P.
Ilgen
,
S.
Jakobs
, and
S. W.
Hell
, “
Coordinate-targeted fluorescence nanoscopy with multiple off states
,”
Nat. Photonics
10
(
2
),
122
128
(
2016
).
9.
B.
Harke
,
C. K.
Ullal
,
J.
Keller
, and
S. W.
Hell
, “
Three-dimensional nanoscopy of colloidal crystals
,”
Nano Lett.
8
(
5
),
1309
1313
(
2008
).
10.
D.
Wildanger
,
R.
Medda
,
L.
Kastrup
, and
S. W.
Hell
, “
A compact STED microscope providing 3D nanoscale resolution
,”
J. Microsc.
236
(
1
),
35
43
(
2009
).
11.
J.
Chojnacki
,
T.
Staudt
,
B.
Glass
,
P.
Bingen
,
J.
Engelhardt
,
M.
Anders
,
J.
Schneider
,
B.
Müller
,
S. W.
Hell
, and
H.-G.
Kräusslich
, “
Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy
,”
Science
338
(
6106
),
524
528
(
2012
).
12.
J.
Hanne
,
F.
Göttfert
,
J.
Schimer
,
M.
Anders-Össwein
,
J.
Konvalinka
,
J.
Engelhardt
,
B.
Müller
,
S. W.
Hell
, and
H.-G.
Kräusslich
, “
Stimulated emission depletion nanoscopy reveals time-course of human immunodeficiency virus proteolytic maturation
,”
ACS Nano
10
(
9
),
8215
8222
(
2016
).
13.
T.
Matkovic
,
M.
Siebert
,
E.
Knoche
,
H.
Depner
,
S.
Mertel
,
D.
Owald
,
M.
Schmidt
,
U.
Thomas
,
A.
Sickmann
,
D.
Kamin
,
S. W.
Hell
,
J.
Bürger
,
C.
Hollmann
,
T.
Mielke
,
C.
Wichmann
, and
S. J.
Sigrist
, “
The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles
,”
J. Cell Biol.
202
(
4
),
667
683
(
2013
).
14.
S.
Galiani
,
D.
Waithe
,
K.
Reglinski
,
L. D.
Cruz-Zaragoza
,
E.
Garcia
,
M. P.
Clausen
,
W.
Schliebs
, and
C.
Eggeling
, “
Super resolution microscopy reveals compartmentalization of peroxisomal membrane proteins
,”
J. Biol. Chem.
291
,
16948
16962
(
2016
).
15.
N.
Mazkereth
,
F.
Rocca
,
J.-R.
Schubert
,
C.
Geisler
,
Y.
Hillman
,
A.
Egner
, and
Z.
Fishelson
, “
Complement triggers relocation of Mortalin/GRP75 from mitochondria to the plasma membrane
,”
Immunobiology
221
(
12
),
1395
1406
(
2016
).
16.
L.
Grosse
,
C. A.
Wurm
,
C.
Bruser
,
D.
Neumann
,
D. C.
Jans
, and
S.
Jakobs
, “
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis
,”
EMBO J.
35
(
4
),
402
413
(
2016
).
17.
G.
Donnert
,
J.
Keller-Findeisen
,
R.
Medda
,
M. A.
Andrei
,
S. O.
Rizzoli
,
R.
Lührmann
,
R.
Jahn
,
C.
Eggeling
, and
S. W.
Hell
, “
Macromolecular-scale resolution in biological fluorescence microscopy
,”
Proc. Natl. Acad. Sci. U. S. A.
103
(
31
),
11440
11445
(
2006
).
18.
F.
Bottanelli
,
E. B.
Kromann
,
E. S.
Allgeyer
,
R. S.
Erdmann
,
S. W.
Baguley
,
G.
Sirinakis
,
A.
Schepartz
,
D.
Baddeley
,
D. K.
Toomre
,
J. E.
Rothman
, and
J.
Bewersdorf
, “
Two-colour live-cell nanoscale imaging of intracellular targets
,”
Nat. Commun.
7
,
10778
(
2016
).
19.
V.
Westphal
,
S. O.
Rizzoli
,
M. A.
Lauterbach
,
D.
Kamin
,
R.
Jahn
, and
S. W.
Hell
, “
Video-rate far-field optical nanoscopy dissects synaptic vesicle movement
,”
Science
320
(
5873
),
246
249
(
2008
).
20.
J.
Tønnesen
,
F.
Nadrigny
,
K. I.
Willig
,
R.
Wedlich-Söldner
, and
U. V.
Nägerl
, “
Two-color STED microscopy of living synapses using a single laser-beam pair
,”
Biophys. J.
101
(
10
),
2545
2552
(
2011
).
21.
E.
Wagner
,
M. A.
Lauterbach
,
T.
Kohl
,
V.
Westphal
,
G. S. B.
Williams
,
J. H.
Steinbrecher
,
J.-H.
Streich
,
B.
Korff
,
H.-T. M.
Tuan
,
B.
Hagen
,
S.
Luther
,
G.
Hasenfuss
,
U.
Parlitz
,
M. S.
Jafri
,
S. W.
Hell
,
W. J.
Lederer
, and
S. E.
Lehnart
, “
Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction
,”
Circ. Res.
111
(
4
),
402
414
(
2012
).
22.
S.
Berning
,
K. I.
Willig
,
H.
Steffens
,
P.
Dibaj
, and
S. W.
Hell
, “
Nanoscopy in a living mouse brain
,”
Science
335
(
6068
),
551
(
2012
).
23.
N. T.
Urban
,
K. I.
Willig
,
S. W.
Hell
, and
V.
Nägerl
, “
STED nanoscopy of actin dynamics in synapses deep inside living brain slices
,”
Biophys. J.
101
,
1277
1284
(
2011
).
24.
K. T.
Takasaki
,
J. B.
Ding
, and
B. L.
Sabatini
, “
Live-cell superresolution imaging by pulsed STED two-photon excitation microscopy
,”
Biophys. J.
104
(
4
),
770
777
(
2013
).
25.
L.
Kastrup
,
H.
Blom
,
C.
Eggeling
, and
S. W.
Hell
, “
Fluorescence fluctuation spectroscopy in subdiffraction focal volumes
,”
Phys. Rev. Lett.
94
,
178104
(
2005
).
26.
C.
Ringemann
,
B.
Harke
,
C.
von Middendorff
,
R.
Medda
,
A.
Honigmann
,
R.
Wagner
,
M.
Leutenegger
,
A.
Schönle
,
S. W.
Hell
, and
C.
Eggeling
, “
Exploring single-molecule dynamics with fluorescence nanoscopy
,”
New J. Phys.
11
,
103054
(
2009
).
27.
J.
Keller
,
A.
Schönle
, and
S. W.
Hell
, “
Efficient fluorescence inhibition patterns for RESOLFT microscopy
,”
Opt. Express
15
(
6
),
3361
3371
(
2007
).
28.
A.
Schönle
and
S. W.
Hell
, “
Calculation of vectorial three-dimensional transfer functions in large-angle focusing systems
,”
J. Opt. Soc. Am. A
19
(
10
),
2121
2126
(
2002
).
29.
M. J.
Booth
,
M. A. A.
Neil
, and
T.
Wilson
, “
Aberration correction for confocal imaging in refractive-index-mismatched media
,”
J. Microsc.
192
(
2
),
90
98
(
1998
).
30.
S.
Hell
,
G.
Reiner
,
C.
Cremer
, and
E. H. K.
Stelzer
, “
Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index
,”
J. Microsc.
169
(
3
),
391
405
(
1993
).
31.
T. J.
Gould
,
D.
Burke
,
J.
Bewersdorf
, and
M. J.
Booth
, “
Adaptive optics enables 3D STED microscopy in aberrating specimens
,”
Opt. Express
20
(
19
),
20998
21009
(
2012
).
32.
B. R.
Patton
,
D.
Burke
,
D.
Owald
,
T. J.
Gould
,
J.
Bewersdorf
, and
M. J.
Booth
, “
Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics
,”
Opt. Express
24
(
8
),
8862
(
2016
).
33.
M. O.
Lenz
,
H. G.
Sinclair
,
A.
Savell
,
J. H.
Clegg
,
A. C. N.
Brown
,
D. M.
Davis
,
C.
Dunsby
,
M. A. A.
Neil
, and
P. M. W.
French
, “
3-D stimulated emission depletion microscopy with programmable aberration correction: 3-D STED microscopy with programmable aberration correction
,”
J. Biophotonics
7
(
1–2
),
29
36
(
2014
).
34.
G.
Lukinavičius
,
L.
Reymond
,
E.
D’Este
,
A.
Masharina
,
F.
Göttfert
,
H.
Ta
,
A.
Güther
,
M.
Fournier
,
S.
Rizzo
,
H.
Waldmann
,
C.
Blaukopf
,
C.
Sommer
,
D. W.
Gerlich
,
H.-D.
Arndt
,
S. W.
Hell
, and
K.
Johnsson
, “
Fluorogenic probes for live-cell imaging of the cytoskeleton
,”
Nat. Methods
11
(
7
),
731
733
(
2014
).
35.
F.
Göttfert
,
T.
Pleiner
,
J.
Heine
,
V.
Westphal
,
D.
Görlich
,
S. J.
Sahl
, and
S. W.
Hell
, “
Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent
,”
Proc. Natl. Acad. Sci. U. S. A.
114
,
2125
2130
(
2017
).
36.
B.
Richards
and
E.
Wolf
, “
Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system
,”
Proc. R. Soc. A
253
,
358
379
(
1959
).
37.
P.
Török
,
P.
Varga
,
Z.
Laczik
, and
G. R.
Booker
, “
Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: An integral representation
,”
J. Opt. Soc. Am. A
12
(
2
),
325
332
(
1995
).
38.
T.
Wilson
, “
Resolution and optical sectioning in the confocal microscope
,”
J. Microsc.
244
(
2
),
113
121
(
2011
).
39.
B.
Neupane
,
F.
Chen
,
W.
Sun
,
D. T.
Chiu
, and
G.
Wang
, “
Tuning donut profile for spatial resolution in stimulated emission depletion microscopy
,”
Rev. Sci. Instrum.
84
(
4
),
043701
(
2013
).
40.
J.
Bückers
,
D.
Wildanger
,
G.
Vicidomini
,
L.
Kastrup
, and
S. W.
Hell
, “
Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses
,”
Opt. Express
19
(
4
),
3130
3143
(
2011
).
41.
G.
Vicidomini
,
A.
Schönle
,
H.
Ta
,
K. Y.
Han
,
G.
Moneron
,
C.
Eggeling
, and
S. W.
Hell
, “
STED nanoscopy with time-gated detection: Theoretical and experimental aspects
,”
PLoS One
8
(
1
),
e54421
(
2013
).
42.
A. N.
Butkevich
,
G.
Lukinavičius
,
E.
D’Este
, and
S. W.
Hell
, “
Cell-permeant large Stokes shift dyes for transfection-free multicolor nanoscopy
,”
J. Am. Chem. Soc.
139
,
12378
12381
(
2017
).
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