3. How to detect light 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 we need 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 a detector
sensitive?
Two key parameters…
• Quantum Efficiency
• Noise floor
Detectors must be designed to ensure
these parameters are optimized.
8. Making sense of sensitivity
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)
13. What makes a detector
sensitive?
Two key parameters…
• Quantum Efficiency
• Noise floor
14. Primary sources of noise 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 - reduced read 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 extensive cooling 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.
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. sCMOS image comparison
490 68 8
photons photons photons
per pixel per pixel per pixel
sCMOS
2x2 binned
(13 µm)
EMCCD
(13 µm)
24. Field of View comparisons
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 Plane Illumination 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 through a 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
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.
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.
