Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

1.
S.
Moreno Flores
and
J. L.
Toca-Herrera
, “
The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques
,”
Nanoscale
1
,
40
49
(
2009
).
2.
R.
Kassies
,
K. O.
Van Der Werf
,
A.
Lenferink
,
C. N.
Hunter
,
J. D.
Olsen
,
V.
Subramaniam
, and
C.
Otto
, “
Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology
,”
J. Microsc.
217
,
109
116
(
2005
).
3.
A. B.
Mathur
,
G. A.
Truskey
, and
W. M.
Reichert
, “
Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells
,”
Biophys. J.
78
,
1725
1735
(
2000
).
4.
D.
Hu
,
M.
Micic
,
N.
Klymyshyn
,
Y. D.
Suh
, and
H. P.
Lu
, “
Correlated topographic and spectroscopic imaging beyond diffraction limit by atomic force microscopy metallic tip-enhanced near-field fluorescence lifetime microscopy
,”
Rev. Sci. Instrum.
74
,
3347
3355
(
2003
).
5.
F.
Dubreuil
,
N.
Elsner
, and
A.
Fery
, “
Elastic properties of polyelectrolyte capsules studied by atomic-force microscopy and RICM
,”
Eur. Phys. J. E
12
,
215
221
(
2003
).
6.
C.
Picart
,
B.
Senger
,
K.
Sengupta
,
F.
Dubreuil
, and
A.
Fery
, “
Measuring mechanical properties of polyelectrolyte multilayer thin films: Novel methods based on AFM and optical techniques
,”
Colloids Surf., A
303
,
30
36
(
2007
).
7.
J.
Yu
,
J.
Yuan
,
X.
Zhang
,
J.
Liu
, and
X.
Fang
, “
Nanoscale imaging with an integrated system combining stimulated emission depletion microscope and atomic force microscope
,”
Chin. Sci. Bull.
58
,
4045
4050
(
2013
).
8.
J. V.
Chacko
,
C.
Canale
,
B.
Harke
, and
A.
Diaspro
, “
Sub-diffraction nano manipulation using STED AFM
,”
PLoS One
8
,
e66608
(
2013
).
9.
A.
Monserrate
,
S.
Casado
, and
C.
Flors
, “
Correlative atomic force microscopy and localization-based super-resolution microscopy: Revealing labelling and image reconstruction artefacts
,”
ChemPhysChem
15
,
647
650
(
2014
).
10.
W. F.
Heinz
and
J. H.
Hoh
, “
Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope
,”
Trends Biotechnol.
17
,
143
150
(
1999
).
11.
A.
Touhami
,
B.
Nysten
, and
Y. F.
Dufrêne
, “
Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy
,”
Langmuir
19
,
4539
4543
(
2003
).
12.
T.
Wilson
,
Confocal Microscopy
(
Academic Press Inc
,
1990
), p.
448
.
13.
M.
Petráň
,
M.
Hadravský
,
M. D.
Egger
, and
R.
Galambos
, “
Tandem-scanning reflected-light microscope
,”
J. Opt. Soc. Am.
58
,
661
664
(
1968
).
14.
R.
Juskaitis
,
T.
Wilson
,
M. A. A.
Neil
, and
M.
Kozubek
, “
Efficient real-time confocal microscopy with white light sources
,”
Nature
383
,
804
806
(
1996
).
15.
R.
Heintzmann
, “
Handbook of biological confocal microscopy
,” in
Handbook of Biological Confocal Microscopy
(
Springer
,
USA
,
2006
), pp.
265
279
.
16.
M. A. A.
Neil
,
R.
Juskaitis
, and
T.
Wilson
, “
Method of obtaining optical sectioning by using structured light in a conventional microscope
,”
Opt. Lett.
22
,
1905
(
1997
).
17.
T.
Wilson
,
R.
Juskaitis
,
M. A. A.
Neil
, and
M.
Kozubek
, “
Confocal microscopy by aperture correlation
,”
Opt. Lett.
21
,
1879
1981
(
1996
).
18.
M. A. A.
Neil
,
T.
Wilson
, and
R.
Juškaitis
, “
A light efficient optically sectioning microscope
,”
J. Microsc.
189
,
114
117
(
1998
).
19.
P. A. A.
De Beule
,
A. H. B.
de Vries
,
D. J.
Arndt-Jovin
, and
T. M.
Jovin
, “
Generation-3 programmable array microscope (PAM) with digital micro-mirror device (DMD)
,”
Proc. SPIE
7932
,
79320G
(
2011
).
20.
G. M.
Hagen
,
W.
Caarls
,
M.
Thomas
,
A.
Hill
,
K. A.
Lidke
,
B.
Rieger
,
C.
Fritsch
,
B.
van Geest
,
T. M.
Jovin
, and
D. J.
Arndt-Jovin
, “
Biological applications of an LCoS-based programmable array microscope (PAM)
,”
Proc. SPIE
6441
,
64410S
(
2007
).
21.
D.
Sarid
,
Scanning Force Microscopy
(
Oxford University Press
,
New York
,
1994
).
22.
M.
Boudaoud
,
Y.
Haddab
,
Y.
Le Gorrec
, and
P.
Lutz
, “
Study of thermal and acoustic noise interferences in low stiffness atomic force microscope cantilevers and characterization of their dynamic properties
,”
Rev. Sci. Instrum.
83
,
013704
(
2012
).
23.
F.
Gittes
and
C. F.
Schmidt
, “
Thermal noise limitations on micromechanical experiments
,”
Eur. Biophys. J.
27
,
75
81
(
1998
).
24.
J. L.
Hutter
and
J.
Bechhoefer
, “
Calibration of atomic-force microscope tips
,”
Rev. Sci. Instrum.
64
,
1868
1873
(
1993
).
25.
H.
Amick
,
M.
Gendreau
,
T.
Busch
, and
C.
Gordon
, “
Evolving criteria for research facilities: Vibration
,”
Proc. SPIE
5933
,
593303
(
2005
).
26.
R.
Heintzmann
,
Q. S.
Hanley
,
D.
Arndt-Jovin
, and
T. M.
Jovin
, “
A dual path programmable array microscope (PAM): Simultaneous acquisition of conjugate and non-conjugate images
,”
J. Microsc.
204
,
119
135
(
2001
).
27.
J.
Teng
and
P. D.
Prewett
, “
Focused ion beam fabrication of thermally actuated bimorph cantilevers
,”
Sens. Actuators, A
123-124
,
608
613
(
2005
).
28.
S.
Timoshenko
, “
Analysis of bi-metal thermostats
,”
J. Opt. Soc. Am.
11
,
233
255
(
1925
).
29.
T.
Ludwig
,
R.
Kirmse
,
K.
Poole
, and
U. S.
Schwarz
, “
Probing cellular microenvironments and tissue remodeling by atomic force microscopy
,”
Pflugers Arch. Eur. J. Physiol.
456
,
29
49
(
2008
).
30.
R. P.
Richter
and
A. R.
Brisson
, “
Following the formation of supported lipid bilayers on mica: As study combining AFM, QCM-D, and ellipsometry
,”
Biophys. J.
88
,
3422
3433
(
2005
).
31.
S.
Garcia-Manyes
and
F.
Sanz
, “
Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective
,”
Biochim. Biophys. Acta
1798
,
741
749
(
2010
).
You do not currently have access to this content.