Leica TCS SP5 The Multi-User Confocal Microscope

Leica TCS SP5 The Multi-User Confocal Microscope

Leica TCS SP5
The Multi-User Confocal Microscope
A microscopia confocal emprega luz entre UV e IR e
baseia-se na varredura da amostra ponto a ponto.
Princípio da
microscopia
confocal
Topics
Confocal Principle
Element # 1: AOTF
Element # 2: Beam Splitter / AOBS
Element # 3: SP Detector
TCS SP5: Tandem Scanner
Confocal Principle
Benefits of Confocal Microscopy:
• Optical Sectioning, 3D
Detector
• 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
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
Confocal Principle XY
y
x
y
x
zy
Beam is scanned in x and y
• direction by galvanometer
• driven scan mirrors
Confocal Principle XYZ
y
Beam is scanned in x and y and
the sample is moved in z
x
Begin
z
End
• galvanometer driven stage
• electronic focus of the
microscope
Super Z: Fine Focusing Stage
• Online cross-section
• Parallel stroke
3T3 fibroblasts
Blue: Nuclei, DAPI; Green: Actin, Phalloidin-Alexa 488;
Red: Paxilin, TexasRed
Courtesy: Dr. Sinikka Eskelinen, Biocenter Oulu, Finland
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)
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)
Leica TCS SP5: Hardware Setup
Leica TCS SP5: Software Set Up
LAS AF: Leica Application Suite Advanced Fluorescence
Beam Path
Spectral detection
AOTF
Laser
Beam
splitter
Excitation
selection
Specimen
Topics
Confocal Principle
Element # 1: AOTF
Element # 2: Beam Splitter / AOBS
Element # 3: SP Detector
TCS SP5: Tandem Scanner
AOTF
AOTF:
Acousto Optical Tunable Filter
Laser
Excitation
selection
Specimen
Acousto-Optic Tunable Filters (AOTFs)
http://micro.magnet.fsu.edu/primer/java/filters/aotf/index.html
Acousto-Optic Tunable Filters (AOTFs)
Laser Set: Merge Module
AOTF: Excitation Selection
Principle:
Laser
Selection
Specimen
LAS AF: AOTF Set Up
Topics
Confocal Principle
Element # 1: AOTF
Element # 2: Beam Splitter / AOBS
Element # 3: SP Detector
TCS SP5: Tandem Scanner
Beam Splitter
Beam
splitter
Laser
Excitation
selection
Specimen
AOBS:
Acoust Optical Beam Splitter
Leica AOBS® vs. Beam Splitter
Leica AOBS
Transmission
Triple dichroic
488/543/633
Wavelength
Leica AOBS® - TOTAL Flexibility
1
0,9
0,8
transmission
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
450
500
550
600
wavelength
650
700
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
Topics
Confocal Principle
Element # 1: AOTF
Element # 2: Beam Splitter / AOBS
Element # 3: SP Detector
TCS SP5: Tandem Scanner
Spectral Detection
Spectral detection
Laser
Excitation
selection
Specimen
Conventional Beam Splitter Vs Leica SP Detector
Filter based system:
Spectrometer System:
• Selectivity?
• Transparency?
Conventional Beam Splitter Vs Leica SP Detector
TCS NT
(sold out 1997)
TCS SP5
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
Leica TCS SP5 - SP Detector Principle
Leica TCS SP5: Multicolour Imaging
5-Colour Imaging of Neurons in C. elegans
Hutter, H. J. Microsop. 215, 213 – 218 (2004)
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
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 (690-1040nm)
Topics
Confocal Principle
Element # 1: AOTF
Element # 2: Beam Splitter / AOBS
Element # 3: SP Detector
TCS SP5: Tandem Scanner
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
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
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
Confocal Microscopy: Two worlds
Structure
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
Dynamics
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
Resonant Scanner: Imaging at High Speed
• Frames
25 frames/sec @ 512x512 pixels
250 frames/sec @ 512x16 pixels
• Very good SNR
Paramecium bursaria
Blue: reflection; Green: chloroplasts,
autofluorescence;
Grey: Cilia, TLD (with DIC)
Courtesy: Leica Microsystems CMS
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
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
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
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
Gain and offset/BL
Fotomultiplicador
http://micro.magnet.fsu.edu/primer/flash/photomultiplier/index.html
Hybrid Detectors
Trad x Hybrid detectors
• 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.
• 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.
Formação de Imagens
Num sistema de 8 bits:
1 byte pode armazenar 28
valores inteiros
(em um sistema binário poderá
ser 0 ou 1)
de 0 a 255 (28 = 256).
• 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.
Byte
• Portanto, uma imagem de 512 x 512 pixels vai ocupar
262.144 bytes.
Leica Microsystem: Always Innovating
SP detector (Leica 1997)
AOBS® (Leica 2002)
Tandem Scanner (Leica 2005)
Leica STED (2008)
Leica TCS SP5: The Real Multi-User/ Broadband Confocal
High Speed
High Resolution
Configurable
Multi-spectral
Multi-channel
Ergonomic
Application wizards
Extreme
Sensitivity
SuperZ Stage
Zoom, Pan,
Rotation
Thank You