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Detectors for light microscopy
- 1. Detectors for lightmicroscopy Marcin Barszczewski PhD 1
- 2. • Single MoleculeDetection „Low Light Imaging‟ • Live cell confocal Application examples • TIRF • Ion signalling • Selective Plane Illumination Microscopy (SPIM) • Intracellular luminescence • Cell Motility • FRET •Single photon counting • Fluorescence Correlation Spectroscopy (FCS) • Super-resolution PALM/ STORM • Adaptive optics
- 3. How to detectlight from your samples? - PMTs Point detectors - APDs “1D”, Low QE, uniformity, repeatability, Speed, low noise factor (PMTs) high noise factor (APD) - CCDs Array (2D) detectors - CMOS High sensitivity, low noise, 2D Historically low speed, dynamic range, “packaging”
- 4. Scientific imaging trade-offs Today‟s imaging detectors exhibit trade-offs between key performance parameters • Low noise/sensitivity • Speed • Wide dynamic range • High resolution • Large field of view
- 5. When do weneed sensitivity? • Low dye concentrations / single molecule • Short exposures / fast frame rates • High photon loss / rejection • Lower excitation power • Greater magnifications • Low quantum yield / Raman scatter
- 6. What makes adetector sensitive? Two key parameters… • Quantum Efficiency • Noise floor Detectors must be designed to ensure these parameters are optimized.
- 7. Typical Quantum EfficiencyCurves Back-illuminated 100 „Virtual Phase‟ 90 MicroLens Front-illuminated 80 front-illuminated 70 Front-illuminated QE (%) 60 ICCD 50 40 30 20 10 0 200 300 400 500 600 700 800 900 1000 Wavelength (nm)
- 8. Making sense ofsensitivity Shot Noise • Read noise Variation ‘Usual’ camera detection limit. • Dark noise Dependent on temperature Average • Shot Noise Signal Intensity QE and signal dependent. Noise Floor (Read Noise and Dark Noise)
- 9. Camera-based imaging technologies Sony interline EMCCD sCMOS Sensor format 1.4 MP 1 MP (max.) 5.5 MP Pixel size 6.45 m 8 to 24 m 6.5 m Max. frame rate 12 fps @ 20MHz > 30 fps 100 fps Read noise 4 – 8 e- Negligible (<1 e-) 1 e- @ 30 fps 1.3 e- @ 100 fps QE ~ 60% (FI) 65% (FI) / >90% (BI) ~ 57% (FI) (excellent red response) Dynamic range ~ 3,000:1 ~ 8,500:1 30,000:1 (@ 11 frames/sec) (@ 30 frames/sec) (@ 30 frames/sec) Darkcurrent (TE cooled) 0.0003 e/pix/sec 0.001 e/pix/sec 0.07 e-/pix/sec @ -55 0C @ -85 0C @ -30 0C
- 10. Frame transfer EMCCD– gain register
- 11. Effect of EMGain on signal-to-noise EMCCD Gain Gain x1 Gain x10 Gain x100 Gain x500
- 12. CMOS vs. CCDArchitecture
- 13. What makes adetector sensitive? Two key parameters… • Quantum Efficiency • Noise floor
- 14. Primary sources ofnoise within imaging sensors 1. READ NOISE - Caused by electronic noise in the CCD output transistor and in the external circuitry 2. DARK CURRENT - Caused by thermally generated electrons in the CCD 3. PHOTON NOISE / SHOT NOISE - It is due to the fact that the CCD detects photons + other noise types
- 15. CCDs - reducedread noise 1MHz (offering ~ 1 fps) - 2.4 e- read noise but slower frame rate - Read Noise is a fundamental trait of CCDs - Read noise can be accounted and corrected - Its influence on images 20MHz (offering ~ 11 fps) – 5.5 e- read noise can be decreased by reducing frame rates
- 16. Impact of extensivecooling on weak signals -70 0C -95 0C - All CCDs build up “dark current” whether the CCD is being exposed to light or not - The rate of dark current build up can be reduced by a factor of 100 or more by cooling the CCD - The remaining dark current • Extremely weak signal –low-light is subtracted from an image luminescence experiment using dark frames. • High EM Gain
- 17. External noise source- background photon Sources? • Out of focus fluorescence background – counter with confocal, TIRF, SPIM • Non-optimal optical filters • Stray background light • Non-specific binding of fluorophore.
- 18. Application Considerations -which camera for what application?
- 19. Scientific imaging trade-offs Today‟s imaging detectors exhibit trade-offs between key performance parameters • Low noise/sensitivity • Rapid frame rates • Wide dynamic range • High resolution • Large field of view
- 20. Scientific CMOS (sCMOS) is unique in simultaneously offering: • Extremely low noise (without multiplication) • Rapid frame rates • Wide dynamic range • High QE • High resolution • Large field of view
- 21. The Electron Multiplying Charge Coupled Device (EMCCD) Eliminates read noise detection limit High Quantum Efficiency Fast frame rates with the lowest noise
- 22. Single Molecule Detection • extremely low light regime - a back-illuminated EMCCD domain • pushing typical exposure times even shorter Live cell imaging • sCMOS – greater flexibility with FOV, resolution, speed • Some low light modalities will still need the sensitivity of EMCCD, e.g. spinning disk confocal
- 23. EMCCD vs. sCMOSimage comparison 490 68 8 photons photons photons per pixel per pixel per pixel sCMOS 2x2 binned (13 µm) EMCCD (13 µm)
- 24. Field of Viewcomparisons sCMOS Sony ICX285 interline Field of view comparison of two technologies; x60 magnification; 1.25 NA; 5.5 megapixel sCMOS vs 1.4 megapixel interline CCD (each have ~ 6.5 m pixel pitch).
- 25. SPIM (Selective PlaneIllumination Microscopy) Neo sCMOS Dr. Lars Hufnagel, Developmenta l Biology Unit, EMBL Heidelberg. Resolution Field of View Speed • Optical sectioning even with lenses that have a large working distance and a relatively low numerical aperture • Especially well suited for the investigation of large samples (e.g. embryos) to study features such as growth, migration, morphological changes and gene expression patterns, that require high resolution, while being extended over a large volume. • Single plane illumination significantly reduces Mouse Embryo photobleaching/phototoxicity
- 26. Optical cross-sections througha developing Drosophila melanogaster embryo in stage 5/6. Two Neos are used to capture this 3-D structure and one of these can be captured every 20 seconds. 26
- 27. Thank you for yourattention! 27
Editor's Notes
- #13 Explain why CMOS is attractiveCCD = only for highend detectorsCMOS is used for more than 99% of all electronic manufactute. Therefore you can do more for cheap.
- #27 Themovie shows optical cross-sections through a developing Drosophila melanogaster embryo in stage 5/6. This H2B-mCherry mutant has fluorescently labeled nuclei. The left two images show recordings from the front sCMOS camera (good images at the beginning of the stack), the right two show images collected by the back sCMOS (good images at the end). The top images were illuminated from the top (top part of the images is sharp), while the bottom images were illuminated from the bottom (the bottom part of the images looks sharp). All four stacks together provide complete information about the location and speed of every single cell in the embryo. Due to the 4-lens setup and fast frame rates made possible by the sCMOS, we are able to record such a 3-dimensional image of the embryo every 20 seconds. (Stefan Guenther & Uros Krzic, Hufnagel lab, EMBL Heidelberg)They have one computer per camera and another "master computer" that controls the whole experiment, in total three PCs.Unfortunately we had to write the software from scratch (in LabView) to control such a complex machine, including the modules that talk to the sCMOS cameras. A typical experiment produces around 2-3 Tb of data. We have a dedicated RAID computer with approx 60Tb storage to store the data while it's being analysed, and a fiber-optic 10gbase network to the microscope computers.