CONFOCAL SENSORS
Description and how to use the HyDs
Light sensor
• It resembles the retina, converting light into a signal that is
subsequently transferred into a storable and processable
ensemble of information.
• Sensors for single point scanning confocal microscopes:
• PMT (photomultiplier tube).
• Avalanche detectors (silicon pendants of PMTs, used
especially single-molecule measurements).
• Hybrid detectors (HyD) which unite the benefits of the two
technologies while eliminating the disadvantages.
Detectors: Different types
Type
Photo Multiplier Tube
GaAsP detector
Hybrid Detector (HyD)
Amplification detector
(vacuum technology)
Amplification detector
(vacuum technology)
Photocounting avalanche detector
(vacuum and silicon technologies)
Principle
Gallium Arsenide
Multi-alkali photocathode
Sequence of dynodes
Photoelectric effect + secondary emission
Anode Current Measurement (“analog
imaging”)
Similar to a PMT but with a more sensitive
photocathode (Gallium Arsenide
phosphide)
Cathode for light conversion (as GaAsP)
One single acceleration step with high
voltage (~8kV) + avalanche effect
Collect photons then digitalize them.
1 photon = 1 grey value
Influence of Gain
statistical noise in the generation of
secondary electrons
Same as for standard PMT
No influence
Pros
• High dynamic range
• “Indestructible”
More sensitive than standard PMT (QE
~40%)
•
•
•
•
Cons
• Not very sensitive (QE ~30%)
• First devices to allow photon counting,
but only suitable for very low light
levels (few photons per unit time)
High instability - Susceptible for damage
More sensitive than std PMT (QE ~45%)
Reduced sensor’s vulnerability
Fast detector
Precise and fast measurements over a
wide dynamic range
• Lower dark current than PMT
Detectors: Different types
Type
Photo Multiplier Tube
GaAsP detector
Hybrid Detector (HyD)
Amplification detector
(vacuum technology)
Amplification detector
(vacuum technology)
Photocounting avalanche detector
(vacuum and silicon technologies)
Principle
Gallium Arsenide
Multi-alkali photocathode
Sequence of dynodes
Photoelectric effect + secondary emission
Anode Current Measurement (“analog
imaging”)
Similar to a PMT but with a more sensitive
photocathode (Gallium Arsenide
phosphide)
Cathode for light conversion (as GaAsP)
One single acceleration step with high
voltage (~8kV) + avalanche effect
Collect photons then digitalize them.
1 photon = 1 grey value
Influence of Gain
statistical noise in the generation of
secondary electrons
Same as for standard PMT
No influence
Pros
• High dynamic range
• “Indestructible”
More sensitive than standard PMT (QE
~40%)
•
•
•
•
Cons
• Not very sensitive (QE ~30%)
• First devices to allow photon counting,
but only suitable for very low light
levels (few photons per unit time)
High instability - Susceptible for damage
More sensitive than std PMT (QE ~45%)
Reduced sensor’s vulnerability
Fast detector
Precise and fast measurements over a
wide dynamic range
• Lower dark current than PMT
PMT
Detectors: Different types
Type
Photo Multiplier Tube
Avalanche detector
Hybrid Detector (HyD)
Amplification detector
(vacuum technology)
Avalanche photo diodes
(silicon technology)
Photocounting avalanche detector
(vacuum and silicon technologies)
Multi-alkali photocathode
Sequence of dynodes
Photoelectric effect + secondary emission
Anode Current Measurement (“analog
imaging”)
hν absorbed in the insertion layer
Brief and intense electric pulse upon
absorption of a single photon
Outer photoelectric effect + emultiplication (avalanche effect)
Cathode for light conversion (as GaAsP)
One single acceleration step with high
voltage (~8kV) + avalanche effect
Collect photons then digitalize them.
1 photon = 1 grey value
Principle
Influence of Gain
statistical noise in the generation of
secondary electrons
No influence
Pros
• High dynamic range
• “Indestructible”
• Ideal for single photon detection and
photon counting measurements
• More sensitive than standard PMT (QE
~45%)
Cons
• Not very sensitive (QE ~30%)
• First devices to allow photon counting,
but only suitable for very low light
levels (few photons per unit time)
• Device sensitive to high currents
• Low dynamic range
More sensitive than std PMT (QE ~45%)
Reduced sensor’s vulnerability
Fast detector
Precise and fast measurements over a
wide dynamic range
• Lower dark current than PMT
•
•
•
•
Detectors: Different types
Type
Photo Multiplier Tube
Avalanche detector
Hybrid Detector (HyD)
Amplification detector
(vacuum technology)
Avalanche photo diodes
(silicon technology)
Photo counting avalanche detector
(vacuum and silicon technologies)
Multi-alkali photocathode
Sequence of dynodes
Photoelectric effect + secondary emission
Anode Current Measurement (“analog
imaging”)
hν absorbed in the insertion layer
Brief and intense electric pulse upon
absorption of a single photon
Outer photoelectric effect + emultiplication (avalanche effect)
Cathode for light conversion (as GaAsP)
One single acceleration step with high
voltage (~8kV) + avalanche effect
Collect photons then digitalize them.
1 photon = 1 grey value
Principle
Influence of Gain
statistical noise in the generation of
secondary electrons
No influence
Pros
• High dynamic range
• “Indestructible”
• Ideal for single photon detection and
photon counting measurements
• More sensitive than standard PMT (QE
~45%)
Cons
• Not very sensitive (QE ~30%)
• First devices to allow photon counting,
but only suitable for very low light
levels (few photons per unit time)
• Device sensitive to high currents
• Low dynamic range
More sensitive than std PMT (QE ~45%)
Reduced sensor’s vulnerability
Fast detector
Precise and fast measurements over a
wide dynamic range
• Lower dark current than PMT
•
•
•
•
Leica HyD for confocal
The road to super-sensitivity
How to avoid HyD overloads (Sequential Scan)
How to avoid HyD overloads (Notch filter)
How to avoid HyD overloads (Notch filter)
How to avoid HyD overloads (Notch filter)
How to avoid HyD overloads (Notch filter)
 Matching
 Not Matching
HyD operation modes in LASAF
Standard
BrightR
Photon
Counting
• Operating mode for image acquisition.
• Can set the Gain of the detector as usual with PMTs
• Mapping absolute photon counts to a look-up table means one has to introduce a scaling factor
• Specifically for dynamic samples with dark and bright in one image.
• In this operating mode, the Gain should be set to the lowest possible value.
• Direct translation of photon counts into grey value
• No further amplification
• In this operating mode, the Gain of the light signals is set to a fixed value in order to ensure constant
detection conditions for photon counting.
HyD: Standard mode
Mapping absolute
photon counts to a
look-up table means
one has to introduce a
scaling factor.
HyD: Photon Counting mode
Each photon is
represented as digital
number „one“ .
Photon counting vs Standard
HyD Standard mode; gain 10%-50%-100%
10%
50%
100
%
HyD: fast detector
Short transit time spread in HyD
Time of flight dispersion in PMT’s
Differences between PMT and HyD
Differences between
PMT
HyD
analog imaging
and
photon counting
• This technique is often called Anode Current Measurement (“analog imaging”)
• PMTs have a high dynamic range
• PMTs are not very sensitive
• Dark current (noise) is generated during the e- amplification
• Photons are registered individually as binary events (“Photon counting”)
• HyDs have a wide dynamic range
• HyDs are more sensitive than PMTs
• No noise generated during the e- amplification
• Fast detector
Quantum efficiency: Capability of detector to translate photons into electrical signals
To go further…
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http://www.leica-microsystems.com/science-lab/gates-open-for-improvedconfocal-fluorescence-and-super-resolution-sted/
http://www.laserfocusworld.com/articles/2008/03/photomultipliers-hybriddetector-combines-pmt-and-semiconductor-diode-technologies.html
http://www.leica-microsystems.com/science-lab/sensors-for-true-confocalscanning/