Erasmus Optical Imaging Centre Zacherias (Hans) (OIC) Jansen 1595 - - PDF document

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Erasmus Optical Imaging Centre (OIC)

Gert van Cappellen Erasmus Optical Imaging Centre (OIC) ErasmusMC

The beginning

Zacherias (Hans) Jansen 1595 Leeuwenhoek (1632-1723) microscope

Green Fluorescent Protein (GFP)

Protein isolated from jellyfish Can be fused to other proteins Produced by the cell Very stable, less harmful Spectral variants: BFP (Blue) CFP (Cyan) YFP (Yellow/Green)

Nanoscopy Different transmission light microscopic techniques

(A) Bright Field (B) Phase Contrast (C) Nomarski Differential Interference Contrast, DIC (D) Dark Field

Bright field

HE (Hematoxylin Eosin) staining Rat ovary with ovulating oocyte

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Immunohistochemistry

Rat ovarian follicle Immunostaining BrdU (5-bromo-2- deoxyuridine) DAP (DiAmidono-2- Phenyl indole4,6) HE (Hematoxylin Eosin)

Introduction

Leeuwenhoek (1632-1723) microscope Modern confocal microscope

Different colors

Blue - DAPI nucleus (direct) Green - α-tubulin (anti-body) Red - phalloidin actin (direct)

GFP labelled Luteinizing Hormone (LH) receptor 2D Stack with Hoechst staining (DNA) 3D scanning

2D Stack 3D Reconstruction Volume rendering

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Hela cells H2b-GFP

H2b - Histone 2b protein skeleton for the DNA. One image every 10 min 14h total Hela - Henrietta Lacks (August 18, 1920 – October 4, 1951)

Testicular tubule

Distance (µm) 587 474 619 274 368 441 431 542 445 (µm) Stage I-IV I-IV I-IV I-IV I-IV V-VI VIII VIII IX-XI IX-XI Stage

Total size 4182 µm, image size 259x259µm, images rotated in advance, Andrea Enquita

Living living cells

Clip170-GFP Microtubule plus ends Tatiana Stepanova

Fluorescent Recovery After Photobleaching (FRAP)

CHO cells transfected with GFP-NLS 10 sec experiment

movie

EXPERIMENTAL SURGICAL ONCOLOGY

Intra-vital microscopy

Timo ten Hagen

EXPERIMENTAL SURGICAL ONCOLOGY

Intra-vital microscopy / two-photon microscopy

eNOS-GFP mouse: expression of green fluorescent protein under control of the eNOS promotor

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Neovasculature of the graft is connected to the recipient ciculation (eNOS-GFP)

5 minutes after tail vein injection of fluorescent microspheres, Timo ten Hagen

Lateral (XY) and Axial (Z) resolution

Axial (Z) resolution: Zmin=2*wavelength*refractive index/(Numerical aperture)2 Zmin=2*488*1.518/(1.4)2=756 nm Numerical aperture = refractive index * sin (µ) Lateral (XY) resolution: XYmin= 0.61*wavelength/Numerical aperture XYmin= 0.61*488/1.4 = 213 nm

213 nm 756 nm

µ

Convolution

RAD52-PSmOrange, U2OS, Fixed cell, γ-irradiated (10 Gy), Maarten Paul

Single molecule imaging Image analysis

Distance (µm) 587 474 619 274 368 441 431 542 445 (µm) Stage I-IV I-IV I-IV I-IV I-IV V-VI VIII VIII IX-XI IX-XI Stage

Total size 4182 µm, image size 259x259µm, images rotated in advance, Andrea Enquita

Image analysis (segmentation)

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Number of cells Visualisation 3D surface rendering Visualisation 4D surface rendering Erasmus Optical Imaging Centre (OIC)

www.erasmusmc.nl/oic

Where Courses Once a month introduction course, new users, 2 x 1/2 day Twice a year in depth course, one week (MSc, PhD), October/November 2018 Once a year Image analysis course, 2018

OIC microscopes

MPL311 Zeiss LSM510NLO FCS MP, 488, 543, 633 Meta311 Zeiss LSM510Meta 266, 405, 488, 543, 633 Meta116 Zeiss LSM510Meta 488, 543, 633 Intra-Vital Meta729 Zeiss LSM510Meta 405, 488, 561, 633 LSM700_311 Zeiss LSM700, 405, 488, 555, 641 Upright LSM700_760 Zeiss LSM700, 405, 488, 555, 641 Upright Elyra Zeiss Elyra PS1, LSM780, SIM, PALM 405, 488, 561, 641, GASP 4Pi Leica 4Pi SP2, MP, 488, 561, 633 SP5_1315 Leica SP5, 405, 488, 561, 633, APD SP5_778 Leica SP5, 266, MP, 488, 561, 633, APD SP5_604 Leica SP5, 405, 488, 561, 594, 633, HyD SP5_Intravital Leica SP5, 405, 588, 561, 633, MP, OPO, NDD 4x SpinD_1454 Roper/Nikon Hamamatsu, 405, 488, 561 TIRF_1454 Roper/Nikon 405, 488, 561 LaDis2 Zeiss PALM Laserdissection microscope Perkin Elmer Opera – High content screening (CTSF)

The “OIC” team

Tsion Martijn Alex Bart Martin Johan Gert-Jan Adriaan Gert

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Erasmus Optical Imaging Centre (OIC)

Expert centre for advanced imaging

www.erasmusmc.nl/oic