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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

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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

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Jana Doehrner

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Ro

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Zheng Chao

Alexia Loynton-Ferrand

Alexia Lo

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n-Ferran

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Leila Nah

idi

Thomas Start

Ro

nal

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Ch

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Zee

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Gert van Cappellen Thomas Start

Jana Doehrner

Ger

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app

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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

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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