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13/12/2017
1
New modalities in Re-scan Confocal Microscopy (RCM)
Erik Manders
Innovative Microscopy Lab
Swammerdam Institute for Life Science
University of Amsterdam
and
Confocal.nl BV
The historical Brakenhoff-confocal (1989)
Prof. Brakenhoff
Confocal Microscopy
Unit 1: Confocal microscope
Unit 2: Pinhole + PMT
PMT
Standard confocal
Pinhole
0.2 AU
1.0 AU
4.0 AU
Sectioning Resolution SNR
Super-resolution
SIM
STORM
STED Confocal
Wide-field
Abbe’s limit
Optical sectioning
Labeling density
RCM
%
Unit 1: Confocal microscope
Unit 2: Re-scanner + CCD
CC
D
Re-scan Confocal Microscopy (RCM)
Giulia De Luca De Luca, et al. (2013) Biomedical Optics Express, 4(11), 2555–2569
13/12/2017
2
Re-scan Confocal Microscopy (RCM)
RCM Standard
Super resolution: from 245 ± 15 nm to 170 ± 10 nm
100 nm beads, 100x objective (CFI Apo TIRF 100X Oil, NA 1.49, Nikon)
De Luca, et al. (2013) Biomedical Optics Express, 4(11), 2555–2569 Giulia De Luca
Pinhole
0.2 AU
1.0 AU
4.0 AU
Sectioning Resolution SNR
Standard confocal
Pinhole Sectioning Resolution SNR
0.2 AU
1.0 AU
4.0 AU
RCM Sensitivity helps to see resolution
Giulia De Luca
Pinhole Sectioning Resolution SNR
0.2 AU
1.0 AU
4.0 AU
RCM Confocal Lateral resolution:
240 nm
RCM Lateral resolution:
170 nm
13/12/2017
3
Jana Doehrner
Ch
rist
iaan
Zee
len
ber
g
Ro
nal
d B
reed
ijk
Zheng Chao
Alexia Loynton-Ferrand
Alexia Lo
ynto
n-Ferran
d
Leila Nah
idi
Thomas Start
Ro
nal
d B
reed
ijk
Ch
rist
iaan
Zee
len
ber
g
Gert van Cappellen Thomas Start
Jana Doehrner
Ger
t va
n C
app
elle
n
Zheng Chao
Live cell RCM imaging
Ronald Breedijk, Christiaan Zeelenberg and Giulia De Luca
FRET-RCM
T=0 is after ~2 hr after adding Staurosporine (2 μM) to a Tag-GFP Tag-RFP Caspase sensor Sample courtesy: Mark Hink, Linda Joosen, UvA
00.5
11.5
0 50 100 150
time (min)
FRET ratio
Giulia De Luca
NIR-RCM (for deep-tissue imaging)
50%
Ronald Breedijk
Super-resolution
SIM
STORM
STED Confocal
Wide-field
Abbe’s limit
Optical sectioning
Labeling density
RCM RCM
13/12/2017
4
Deconvolution 130 nm
Mika Ruonala
Raw RCM Deconvolved
RCM-SIM 110 nm
Ronald Breedijk
Confocal RCM-SIM
Rescan Confocal Microscopy (RCM)
2013 2014
2015 2016
Axial: 600 nm @ 488 nm
Lateral : 170 nm (FWHM @ 488 nm)
resolution
up to 4 lasers
e.g. 405, 488, 561, 638 nm
four colours
typical 1 fps @ 512x512 images.
Higher speed possible for smaller images.
scanning speed
C-mount low cost EMCCD/SCMOS QE: 80-95 %
camera based
Innovative Microscopy Lab University of Amsterdam
Ronald Breedijk Giulia De Luca Christiaan Zeelenberg Venkat Kishnaswami Rick Brandt Irene Stellingwerf Emilie Desclos Erik Manders Fred Brakenhoff
Department of Cell Bio. and Histology, Academic Medical Center Venkat Krishnaswami Ron Hoebe
Department of Imaging Science & Technology Delft University of Technology Bernd Rieger Sjoerd Stallinga
Stichting Technologie en Wetenschap “ Ultra-sensitive Conf. Microscopy”
STW-perspectief progr:am “Nanoscopy”
Confocal.nl BV, Amsterdam
Paula Onrust Ronald Breedijk Irene Stellingwerf Stan Hilt Carla Kalkhoven Erik Manders Peter Drent
Laboratory of Functional and Structural Tissue Imaging, Nencki Institute, Warsaw, Poland Tytus Bernas