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Silicon photodiodes• A silicon photodiode produces current when photons
impinge upon it (example are solar cells)• Does not require an external power source to operate• Peak sensitivity is about 900 nm• At 900 nm the responsivity is about 0.5
amperes/watt, at 500 nm it is 0.28 A/W• Are usually operated in the photovoltaic mode (no
external voltage) (alternative is photoconductive mode with a bias voltage)
• Have no gain so must have external amps• quantum efficiency ()% = 100 x (electrons out/(photons in)
PMT• Produce current at their anodes when photons impinge upon
their light-sensitive cathodes• Require external powersource• Their gain is as high as 107 electrons out per photon in• Noise can be generated from thermionic emission of electrons
- this is called “dark current”• If very low levels of signal are available, PMTs are often cooled
to reduce heat effects• Spectral response of PMTs is determined by the composition of
the photocathode• Bi-alkali PMTs have peak sensitivity at 400 nm• Multialkali PMTs extend to 750 nm • Gallium Arsenide (GaAs) cathodes operate from 300-850 nm
The PMTs in an Elite. 3 PMTs are shown, the other 2 have been removed to show their positions. A diode detector is used for forward scatter and a PMT for side scatter.
The Bio-Rad Bryte cytometer uses PMTs for forward and wide angle light scatter as well as fluorescence
High Voltage on PMTs• The voltage on the PMT is applied to the dynodes• This increases the “sensitivity” of the PMT• A low signal will require higher voltages on the
PMT to measure the signal• When the voltage is applied, the PMT is very
sensitive and if exposed to light will be destroyed• Background noise on PMTs is termed “dark
noise”• PMTs generally have a voltage range from 1-
2000 volts• Changing the gain on a PMT should be linear
over the gain range• Changing the voltage on the PMT is NOT a linear
• Combines the best features of PMTs and photodiodes• High quantum efficiency, good gain• Gain is 102-103 (much less than PMTs)• Problem with high dark current
Summary so far….• Photodiodes can operate in two modes - photovoltaic
and photoconductive• PMTs are usually used for fluorescence measurements• Photodiodes are usually used for scatter• PMTS are sensitive to different wavelengths according
to the construction of the photocathode• PMTs are subject to dark current• Voltages and gain are not linear• Photodiodes are more sensitive than PMTs but because
of their low gain, they are not as useful for low level signals (too much noise)
• Based upon differential pressure between sample and sheath fluid. • Require balanced positive pressure via either air or nitrogen• Flow rate varies between 6-10 ms-1
+ + ++ + ++ + +
Positive Displacement Syringe Systems
• 1-2 ms-1 flow rate• Fixed volume (50 l or 100 l)• Absolute number calculations possible• Usually fully enclosed flow cells
• The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing
[RFM]
FluidicsThe figure shows the mapping between the flow lines outside and inside of a narrow tube as fluid undergoes laminar flow (from left to right). The fluid passing through cross section A outside the tube is focused to cross section a inside.
From V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3[RFM]
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.
Notice also how the position of the inner ink stream is influenced by the position of the ink source.
• As cells (or other particles) are hydrodynamically focused, they experience different shear stresses on different points on their surfaces (an in different locations in the stream)
• These cause cells to orient with their long axis (if any) along the axis of flow
“a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow.
b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.”
Image fromV. Kachel, et al. – Melamed Chapt. 3[RFM]
Fluorescence Detectors and Optical TrainBrytec.mpg
Shown above is the Bryte HS optical train - demonstrating how the microscope-like optics using an arc lamp operates as a flow detection system. First are the scatter detectors (left side) followed by the central area where the excitation dichroic can be removed and replaced as necessary. Behind the dichroic block is the arc lamp. To the right will be the fluorescence detectors.