The beginning Erasmus Optical Imaging Centre Zacherias (Hans) (OIC) Jansen 1595 Gert van Cappellen Leeuwenhoek (1632-1723) Erasmus Optical Imaging Centre (OIC) microscope ErasmusMC Green Fluorescent Protein (GFP) Nanoscopy 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) Different transmission light Bright field microscopic techniques (A) Bright Field (B) Phase Contrast (C) Nomarski Differential Interference Contrast, DIC (D) Dark Field HE (Hematoxylin Eosin) staining Rat ovary with ovulating oocyte 1
Immunohistochemistry Introduction Rat ovarian follicle Immunostaining BrdU (5-bromo-2- deoxyuridine) DAP (DiAmidono-2- Leeuwenhoek Phenyl indole4,6) (1632-1723) microscope HE (Hematoxylin Eosin) Modern confocal microscope GFP labelled Luteinizing Hormone (LH) Different colors receptor Blue - DAPI nucleus (direct) Green - α -tubulin (anti-body) Red - phalloidin actin (direct) 2D Stack with Hoechst staining (DNA) 3D scanning 2D Stack 3D Reconstruction Volume rendering 2
Hela cells H2b-GFP Testicular tubule Stage I-IV I-IV I-IV I-IV I-IV Distance (µm) 587 474 619 274 368 441 431 542 445 (µm) V-VI VIII VIII IX-XI IX-XI Stage H2b - Histone 2b protein skeleton for the DNA. One image every 10 min 14h total Hela - Henrietta Lacks (August 18, 1920 – October 4, 1951) Total size 4182 µm, image size 259x259µm, images rotated in advance, Andrea Enquita Fluorescent Recovery After Living living cells Photobleaching (FRAP) CHO cells transfected with GFP-NLS 10 sec experiment movie Clip170-GFP Microtubule plus ends Tatiana Stepanova Intra-vital microscopy / two-photon microscopy Intra-vital microscopy eNOS-GFP mouse: expression of green fluorescent protein under control of the eNOS promotor EXPERIMENTAL EXPERIMENTAL ONCOLOGY ONCOLOGY SURGICAL SURGICAL Timo ten Hagen 3
Neovasculature of the graft is connected to the Lateral (XY) and Axial (Z) resolution recipient ciculation (eNOS-GFP) Numerical aperture = refractive index * sin (µ) Lateral (XY) resolution: XY min = 0.61*wavelength/Numerical aperture XY min = 0.61*488/1.4 = 213 nm µ Axial (Z) resolution: 756 nm Z min =2*wavelength*refractive index/(Numerical aperture) 2 Z min =2*488*1.518/(1.4) 2 =756 nm 213 nm 5 minutes after tail vein injection of fluorescent microspheres, Timo ten Hagen Convolution Single molecule imaging RAD52-PSmOrange, U2OS, Fixed cell, γ -irradiated (10 Gy), Maarten Paul Image analysis Image analysis (segmentation) Stage I-IV I-IV I-IV I-IV I-IV Distance (µm) 587 474 619 274 368 441 431 542 445 (µm) V-VI VIII VIII IX-XI IX-XI Stage Total size 4182 µm, image size 259x259µm, images rotated in advance, Andrea Enquita 4
Number of cells Visualisation 3D surface rendering Visualisation 4D surface rendering Erasmus Optical Imaging Centre (OIC) Where www.erasmusmc.nl/oic 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 The “OIC” team 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 Tsion Martijn Alex 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 Bart Martin Johan 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) Gert-Jan Adriaan Gert 5
Erasmus Optical Imaging Centre (OIC) www.erasmusmc.nl/oic Expert centre for advanced imaging 6
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