The different components and configuration of a Confocal laser scanning microscope (CLSM)

Transcription

1 The different components and configuration of a Confocal laser scanning microscope (CLSM) Internal training, 29 Mars 2013 Optical microscopy cell cell imaging platform Amandine Durand-Terrasson

2 Confocal microscope anatomy : laser Optical path Detection And scanning system Scan head Excitation system Detection and scanning system - Laser -AOTF - Optical fiber - Scanning system

3 Excitation system

4 LASER = Light Amplification by Stimulated Emission of Radiation HBO lamp LASER illumination Polychromatic Monochromatic Essential for HFT Every directions Single direction Incoherent Coherent Generation of interferences (objective) Unpolarized The electric vector have a randomed orientation Linealy polarized The electric vector have a fixed orientation Very significant for DIC, anisotropy, SGH

5 Optical fiber/beam expander : Fiber optic laser coupler Objective lenses + Handy (vs rigid alignment) - Power decrease and beam divergeance Laser beam expander The laser light beams can : - travel over long distances - be expanded to fill apertures => focused to a very small spot with a high level of brightness

6 The stimulated Emission Jablonski diagram for a Helium/Neon laser with 4 levels Stimulated emission in a mirrored laser cavity FAST non radiative transition metastable state N in exciting state >> N in groud state FAST non radiative transition Groud state E1 -> E4 : Excitation E4 -> E3 : Instable State E3 -> E2 : Stimulated emission E2 -> E1 : Back to the lower level

7 LASER sources for Confocal microscopy Laser Hélium Néon Kastler-Brossel lab. Pierre Marie-Curie and University (Paris VI) Continuous-Wave Lasers -Gaz : He/Ne, Multi raies : Argon -DPSS (Diode Pumped Solid State) -Dye Laser (Rhodamine ) Ti:Sa Pulsed Laser Laser power : 1mW - 4 Watts Continuous-Wave Lasers Pulsed Laser Temporal domain Spectral domain Temporal domain Spectral domain Example: laser Hélium-Néon λ = nm +/- 0.2nm Temporal window : 140 fs Repetition rate : 80 MHz

8 Continuous wave laser Vs pulse laser 1 photon 2 photons Fluorescent solution Continuous-Wave Lasers : Cone of illumination / pinhole / Photobleaching of adjacent planes Pulsed Laser : Small focal volume / no pinhole / decrease photobleaching

9 Acousto Optic Tunable Filters (AOTF) The diffraction angle depends on sin(θ) = λ2/λ Spectral selection Advantages : - The power of each line is controlled independently : Variation of the amplitude of the acoustic wave - Switching between lines on the order of a few µs - Transmission is greater than 90 % - Selection the wavelength : Variation of the frequency of the acoustic wave Evolution of the laser beam intensity under modifications of the AOTF settings Linearity zone

10 Scanning system Differents kind of galvanometric mirror Movement of the mirrors Two orthogonal galvanometric mirrors control the scanning Need to be calibrated Linearity zone

11 Detection system

12 Beam splitter/ spectrale selection Emission filter Secondary beam splitter Primary beam splitter - Splitting laser beams of different color - The main beam splitter reflects 90% of the laser light - Filters improve S/N

13 PMT : Photomultiplier tube Vs APD : Avalanche Photo Diode Low Quantum Efficiency Hight internal gain Hight background : No photon counting Amplified electrical signal Numerisation (image) Hight Quantum Efficiency Photon Counting : Photon counting imaging, Time resolved detection

14 Spectral detector : After passing the detection pinhole, the light emitted from the focal plan is passed through a prism which diffracts the emitted light. The entire spectrum can be image onto the window of the PMT A spectral image requires the creation of a three-dimensional data set that contains a collection of images of the same viewfield captured at different wavelengths.

15 Microscope settings

16 Power modulation Vs AOTF LSM 510 Interface ZEN Interface Select laser Switch on required laser lines Select laser Switch on required laser lines Slider AOTF Selection hide line Sliders AOTF

17 Scanning system modulation LSM 510 Interface ZEN Interface

18 Set gain and Offset

19 Set gain and Offset Poor dynamic The better dynamic

20 Adjusting pinhole Beads : Green/red Pinhole centered: Superposition of the two channels Maximum intensity Pinhole offset Offset channels Decrease in intensity

21 Adjusting of collimator beads : blue core/red ring collimated beam Superposition of the two axial channels Poorly collimated beam Axial offset of the two channels