Leica TCS SP5 The Multi-User Confocal Microscope

Transcription

1 Leica TCS SP5 The Multi-User Confocal Microscope

2 A microscopia confocal emprega luz entre UV e IR e baseia-se na varredura da amostra ponto a ponto.

3 Princípio da microscopia confocal

4 Topics Confocal Principle Element # 1: AOTF Element # 2: Beam Splitter / AOBS Element # 3: SP Detector TCS SP5: Tandem Scanner

5 Confocal Principle Detector Benefits of Confocal Microscopy: Optical Sectioning, 3D High Contrast Confocal Pinhole (detection) Beam Splitter Laser Confocal Pinhole (excitation) Microscope Objective Light from above the focal plane Light from within the focal plane Light from below the focal plane

6 WideField vs. Confocal Drosophila melanogaster (larvae) Widefield Confocal Green: Nuclei (RNA binding protein), Alexa 488; Red: Axons, Cy 3; Blue: Axon endings (of MJ94-positive neurons), Cy 5 Courtesy: Dr. Christoph Melcher, Research Center Karlsruhe

7 Confocal Principle XY y x y x y z Beam is scanned in x and y direction by galvanometer driven scan mirrors

8 Confocal Principle XYZ y x Begin z End Beam is scanned in x and y and the sample is moved in z galvanometer driven stage electronic focus of the microscope

9 Super Z: Fine Focusing Stage Online cross-section 3T3 fibroblasts Blue: Nuclei, DAPI; Green: Actin, Phalloidin-Alexa 488; Red: Paxilin, TexasRed Courtesy: Dr. Sinikka Eskelinen, Biocenter Oulu, Finland Parallel stroke

10 3D Reconstruction Sample: Convallaria majalis (Lily of the Valley) More than 40 different cardiac glycosides have been found in the plant. In former times it was used in the alternative medicine for treatment of cardiac insufficiency and cardiac arrhythmia. Channel1: Green: Fast green (Starch Granules) Channel2: Red: Safranine (Cell walls)

11 3D Reconstruction Sample: Convallaria majalis (Lily of the Valley) More than 40 different cardiac glycosides have been found in the plant. In former times it was used in the alternative medicine for treatment of cardiac insufficiency and cardiac arrhythmia. Channel1: Green: Fast green (Starch Granules) Channel2: Red: Safranine (Cell walls)

12 Leica TCS SP5: Hardware Setup

13 Leica TCS SP5: Software Set Up LAS AF: Leica Application Suite Advanced Fluorescence

14 Beam Path Spectral detection AOTF Laser Beam splitter Excitation selection Specimen

16 AOTF AOTF: Acousto Optical Tunable Filter Laser Excitation selection Specimen

17 Acousto-Optic Tunable Filters (AOTFs)

18 Acousto-Optic Tunable Filters (AOTFs)

19 Laser Set: Merge Module

20 AOTF: Excitation Selection Principle: Laser Selection Specimen

21 LAS AF: AOTF Set Up

23 Beam Splitter Beam splitter Laser Excitation selection Specimen

24 AOBS: Acoust Optical Beam Splitter

25 Leica AOBS vs. Beam Splitter Leica AOBS Triple dichroic 488/543/633 Transmission Wavelength

26 Leica AOBS - TOTAL Flexibility 1 0,9 0,8 transmission 0,7 0,6 0,5 0,4 0,3 0,2 0, wavelength

27 Properties of Leica AOBS Optical neutral element High selectivity (0,6-2 nm bandwidth) More space for detection of fluorescence Fast switchable (sequential scanning) No moving elements

29 Spectral Detection Spectral detection Laser Excitation selection Specimen

30 Conventional Beam Splitter Vs Leica SP Detector Filter based system: Spectrometer System: Selectivity? Transparency?

31 Conventional Beam Splitter Vs Leica SP Detector TCS NT (sold out 1997) TCS SP5

32 Leica TCS SP5 - SP Detector 5 true confocal channels simultaneously All dyes: Freely tuneable emission bands Low sample photodamage: high efficiency Intuitive operation Highly selective: no gaps Frexibility: Create any bandpass-filter Up to 5 PMT s individual adjustment

33 Leica TCS SP5 - SP Detector Principle

34 Leica TCS SP5: Multicolour Imaging 5-Colour Imaging of Neurons in C. elegans Hutter, H. J. Microsop. 215, (2004)

35 Leica TCS SP5: SP Detector & AOBS Highly Selective: no gaps High Frexibility: Create any bandpass-filter High Sensitivity: no beam splitters New laser lines / line combinations New Dyes / fluorophore combinations

36 Leica TCS SP5: Broad range of lasers combinations 355 and/or 405 Correction optics VIS Lasers Ar Multiline DPSS 442 NeNe 543 DPSS 561 HeNe 594 HeNe 633 MP lasers Tunable ( nm)

38 Leica TCS SP5 Tandem Scanner: Always Innovating All in one system: Live Cell Imaging - Dynamics & Kinetics High Resolution - Morphology and structure The fastest True Confocal System Leading in Multispectral Imaging Ergonomic and Intuitive handling

39 Leica TCS SP5 Conventional Scanner: High Resolution 8k x 8k imaging (64 Megapixel) Full resolution transfer Overview images without stitching Detailed view: - Electronic zoom viewer - Electronic scrollbars -Crop-tool Drosophila melanogaster (larval stadium) Blue: Nuclei, DAPI; green: Feb211 positive neurons + their axons, Alexa 488; Red: fibrous part of the cns, Cy3; grey Nuclei of neurons, Alexa 594 Courtesy: Dr. Christoph Melcher, Forschungszentrum 2 Karlsruhe, Germany

40 Leica TCS SP5 Conventional Scanner: High Resolution 8k x 8k imaging (64 Megapixel) Full resolution transfer Overview images without stitching Detailed view: - Electronic zoom viewer - Electronic scrollbars -Crop-tool Drosophila melanogaster (larval stadium) Blue: Nuclei, DAPI; Green: Feb211 positive neurons + their axons, Alexa 488; Red: fibrous part of the cns, Cy3; Grey Nuclei of neurons, Alexa 594 Courtesy: Dr. Christoph Melcher, Forschungszentrum Karlsruhe, Germany

41 Confocal Microscopy: Two worlds Structure Dynamics Mouse-embryo (detail of the heart) Cyan: Nuclei, DAPI; Green: Actin, Cy2; Red: Myosin, Cy3; Blue: uncharacterised Protein, Cy5 Courtesy: Dr. Elisabeth Ehler, King s College London, UK Medaka spec. ( Flow of red blood cells and migration of macrophages) Green: Macrophages, RFP; Yellow: Macrophages, YFP Grey: Red blood cells, TLD Courtesy: Clemens Grabner and Jochen Wittbrodt, EMBL, Heidelberg, Germany

42 Resonant Scanner: Imaging at High Speed Frames x512 pixels x16 pixels Very good SNR Paramecium bursaria Blue: reflection; Green: chloroplasts, autofluorescence; Grey: Cilia, TLD (with DIC) Courtesy: Leica Microsystems CMS

43 Resonant Scanner: Excellent depth discrimination Resonant scanner: Confocal point illumination Perfect optical sectioning Spinning disc/grid systems: Spatial crosstalk Depth discrimination impaired Line scanner: Slit illumination & detection Depth discrimination impaired Organisms: Elodea spec. Channel 1: Cell wall & Mitochondria (Blue)/ Channel 2: chloroplasts/ Autofluorescence of chlorophyll (Red) / TLD/DIC: Grey Acquisition Data: xyt series, 3ch, 512x512 bidir, 29fps, 1000 images, 34 sec Lens: HCX PL APO 63.0x1.20 WATER UV

44 Resonant Scanner: Multifluorescence Resonant scanner: SP detector and AOBS available Perfect fit for multifluorescence Spinning disc/grid systems: Single dichroic/chromatic aberrations Limited or no multifluorescence capabilities Line scanner: 2 channels only Limited multifluorescence Caenorhabditis elegans Cyan: sensory and interneurons, CFP; Green: GABAergic neurons, GFP; Yellow: excitatory motoneurons, YFP; Red: glutamatergic interneurons, DsRed; Grey: sensory neurons, DiD Courtesy: Dr. H. Hutter, MPI for medical research, Heidelberg, Germany

45 Resonant Scanner: Zoom, Pan & Rotation Resonant scanner: Zoom by amplitude Panning by offset (y) motor (x) Rotation without loss of speed Fully functional Spinning disc/grid systems: No zoom or panning No rotation Cardiac muscle cell (Calcium waves & sparks) Green: Calcium, Fluo 3; Grey: TLD Courtesy: Prof. Neumann, University Halle, Germany Line scanner: Zoom limited Panning unavailable No rotation

46 Resonant Scanner: Summary Dynamic live cell imaging and kinetic measurements Crisp thin optical sections Full multiparameter fluorescence performance Cardiac muscle cell (Calcium waves & sparks) Green: Calcium, Fluo 3; Grey: TLD Courtesy: Prof. Neumann, University Halle, Germany Overview and details by zoom and pan

47 Gain and offset/bl

48 Fotomultiplicador

49 Hybrid Detectors

50 Trad x Hybrid detectors

51 A microscopia por varredura de feixe é um processo essencialmente seqüencial e digital. Iluminando-se um ponto por vez, o microscópio confocal ganha um aumento de intensidade enorme quando comparado ao de campoaberto.

52 O microscópio confocal varre o ponto (ou a amostra) sobre uma área (tela) quadrada. No exemplo a tela ou matriz é de 16x16 onde cada quadrado individual é denominado pixel (picture element). Figura: Um microscópio de varredura gera uma imagem pelo movimento de um ponto de laser sobre (PIXEL) numa matriz. Esta matriz de 16x16 pixels é muito grosseira e produz uma imagem 'boxed' e "pixelada". Imagens são mais comumente obtidas em matrizes de 256 x 256 e 512 x 512.

53 Formação de Imagens Num sistema de 8 bits: 1 byte pode armazenar 2 8 valores inteiros (em um sistema binário poderá ser 0 ou 1) de 0 a 255 (2 8 = 256).

54 Isso implica que a intensidade de luz que será representada por um pixel poderá variar entre 0 e 255 (sem unidades). Esta propriedade intrínseca nos permitirá verificar se uma imagem está ou não saturada. No monitor do computador, 1 pixel (picture element) corresponde a 1 Byte. Portanto, uma imagem de 512 x 512 pixels vai ocupar bytes.

55 Leica Microsystem: Always Innovating SP detector (Leica 1997) AOBS (Leica 2002) Tandem Scanner (Leica 2005) Leica STED (2008)

56 Leica TCS SP5: The Real Multi-User/ Broadband Confocal High Speed High Resolution Multi-spectral Multi-channel Extreme Sensitivity Configurable Ergonomic Application wizards Zoom, Pan, Rotation SuperZ Stage

57 Thank You