Transcript Quattro Presentation Part I--

Quattro Presentation
Part I---About
Photoluminescence
About Fluorescence
The phenomenon of temporary light absorption
and subsequent light emission is called
Photoluminescence
The Jablonski Diagram
• Absorption (10-15 s)
• Internal conversion (10-12 s)
• Intersystem crossing (heavyatom enhanced)
• Quenching by external
quenchers (diffusioncontrolled)
• Fluorescence (10-9 s)
• Phosphorescence/Inorganic
luminescence (10-5 - 101 s)
Kasha’s Rule: Photoluminescence always from S1 (fluorescence) or T1 (phosphorescence)
Important Characteristics of
Photoluminescence
• Each Photoluminescence substance has its own excitation
and emission spectra.
• The intensity of the Photoluminescence is proportional to
its absorption, which in turn is proportional to the
excitation light.
• The Photoluminescence emission of a substance is always
at a higher wavelength than the light used to excite the
substance (Stokes-shift phenomenon).
• The Photoluminescence of a substance is sensitive to many
factors and will change or even disappear under different
conditions.
Advantage of Using
Photoluminescence as Research Tool
• It is very sensitive
• It allows to measure very small amount of
substance in a given sample
• It allows to measure very small changes
• It allows to track conformational changes
• It is very safe
• It is very fast
• It is cost-effective
• It is widely used in every research
What Can be Measured of a
Photoluminescence Substance
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Photoluminescence intensity
Excitation spectra
Emission spectra
Photoluminescence lifetime
Anisotropy & Polarization
Time-based Photoluminescence intensity
Time-based Photoluminescence lifetime
Quattro II can measure them all!
Part II---General
Photoluminescence Instruments
and Instruments from PTI/
OBB Family
Photoluminescence System
Composition--General
• Excitation
– Excitation light source
– Excitation wavelength selection
– Optics for sample excitation
• Sample holder/ compartment
• Emission
– Emission wavelength selection
– Optics for sample emission
– Detector
• Computer interface
• Software
• Accessories & Attachments
Photoluminescence
Systems from OBB/ PTI
• Complete Integrated PLSystems:
– Quattro
– Easylife Series
– FluoDia T70 High Temperature fluorescence Plate Reader
• Complete open architecture design PL Systems:
– QuantaMaster (QM) system for steady-state luminescence
measurement
– RatioMaster (RM) system for Ratiometric fluorescent
measurement of microscopic samples using PMT detector(s)
– Easy-Ratio Pro (ERP) system for Ratio fluorescent imaging of
microscopic samples using camera as detector
– TimeMaster (TM) system for Time-resolved luminescence
measurement
• Optical Building Blocks: light sources, monochromator,
detectors, accessories, et al
Who are We?
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PTI is the oldest independent photoluminescence company,
specializing in manufacturing and selling complete spectroscopy
systems as well as their basic building blocks globally for nearly
thirty years.
The Company was established in 1983 in New Jersey, U.S.A. ,
where the current Headquarter is located
It has a Canadian subsidiary, where most of manufacturing is done
and also offices in UK and Germany.
It has international representatives in most countries worldwide.
PTI is entirely dedicated to Photoluminescence products.
In 2007, Optical Building Blocks was established from within PTI all Integrated Photoluminescence systems, along with optical
building blocks now form the core products of Optical Building
Blocks Corp., with headquarter in New Jersey, USA
Universal Spectrofluorometer
Quattro
Quattro
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Ultra-high Sensitivity
Exceptional precision
Super fast speed
Very low stray-light
Extremely easy to use
• Ultra-high sensitivity: S/N >
20,000:1 using water Raman
• Single-shot operation
• Exceptional precision, stability
and reproducibility
• Very low stray-light
• Super fast acquisitions: measure
up to 500 complete decays in a
second
• Fully automated: complete
spectral and lifetime
characterization with one click
• Easy to operate software.
• Low Maintenance
• Anti-photo-bleaching design
Unmatchable Sensitivity
S/N of 40000:1
measured with Water
Raman here on this
unit
Super Fast Acquisition
Decay of Terbium:
t1=422.4us (100%)
Offset=-0.000014V,
Chi-sq=1.13 (Auto
mode, Integration:
10000 Shots at 250
HZ
Obtain Decay Data
within several
seconds.
Optical Layout
Light Source
Emission Monochromator
Detector
Sample Compartment
Excitation Monochromator
Excitation Light Source
• High power pulsed xenon lamp with pre-aligned ellipsoidal
reflector
• Wavelength Range: 200 to 2000 nm
• Pulsed Repetition rate: 1 to 500 HZ (software control )
• Pulse Width 1 µs (FWHM)
• Energy per flash: up to 0.15 J
• Each pulse is excitation corrected by a silicon photodiode
• Lamp life time: 1 x 109 flashes - flashing only during
acquisition extends the lamp life time up to 10 years with 8 hr
operation per day.
Excitation Monochromator
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Asymmetrical Czerny-Turner, coma corrected for minimal stray light
Focal length: 250 mm
Anti-backlashed sine-drive
Precision, two stage auto-calibration
Range: -50 to 1100 nm
Computer selectable spectral bandwidth: 1.5, 2.5, 5, 10, 20 nm slits
Throughput: 70% at 300 nm – grating blazed @ 300nm
Grating type: ruled
Grating size: 50 x 50 mm
Grating groove density: 1200 lines/mm
Apertures: input - f/3.3; output - f/3.7
Reciprocal linear dispersion: 3.3 nm/mm
Stray light: <0.005% 20 nm away from 632.8 nm Helium Neon line
Accuracy: ±0.2 nm
Optical resolution: 1.5 nm
Maximum scanning speed: 50 nm/sec
Excitation Correction
RCQC:
• Type: Enhanced
silicon photodiode
• Gain: 1 to 1250
times
• Linearity: Linear
for the entire range
of gain values
• Range: 200 – 1100
nm
• Calibration at
factory: 250 – 600
nm vs. rhodamine
White: a lamp scan without excitation correction; Red: Same scan with
excitation correction activated.
Conclusion: standard deviation is better than 0.6 % (260 – 600 nm)
Sample Compartment
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Standard Peltier thermo-electrical temperature controlled 3-window cuvette
holder: 10° -70° C
Standard Variable speed magnetic stirrer
Aberration corrected demagnified optics result in a light spot <500 um at the
sample.
Sample light collection solid angle: 40
Beam splitter reflects a fraction of the excitation beam to a RCQC
(Reference Channel Quantum Counter) photodiode to monitor fluctuations
in the excitation beam, both temporal and wavelength.
Double sets of excitation and emission filter holders accommodate 1 inch.
diameter round filters
Gas purgeable compartment
Temperature Controlled Sample Holder
• Temperature ranges:
10 – 70 °C (nitrogen
purging is required when
<15°C)
• Maximum temperature
ramping speed:
20 °C per min (0.33°C/ sec)
• Accuracy: ±0.1 °C
• Temperature settings
resolution: 10 bit (0.06°C)
• Temperature stability (after
settling): 0.1°C
• Water cooled heat sink.
Emission Monochromator
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Asymmetrical Czerny-Turner, coma corrected for maximal throughput
Focal length: 250 mm
Anti-backlashed sine-drive
Precision, two stage auto-calibration
Range: -50 to 1100 nm
Computer selectable spectral bandwidth: 1.5, 2.5, 5, 10, 20 nm slits
Throughput: 70% at 500 nm – grating blazed @ 500nm
Grating type: ruled
Grating size: 50 x 50 mm
Grating groove density: 1200 lines/mm
Apertures: input - f/3.7; output - f/3.7
Reciprocal linear dispersion: 3.3 nm/mm
Stray light: <0.015% 20 nm away from 632.8 nm Helium Neon line
Accuracy: ±0.2 nm
Optical resolution: 1.5 nm
Maximum scanning speed: 50 nm/sec
Detector
• Detector: DC Electrical Coupling,
• Typical wavelength range 185-650nm
– Optional: 185-900 nm
• Instrument Response: 6 μs
Instrument Response Function
Digitizing: 0.8 us/ point
(Fastest)
Xe Lamp flash (FWHM):
1.1 us
PMT and electronic
amplifier response
(FWHM): 6 usec
Minimum lifetime:
50 usec (Auto Mode).
10 usec. (Manual mode)
Emission & Excitation Correction
White: synchronous scan of a lamp without excitation and emission correction ;
Green: Same scan with excitation and emission correction activated.
Conclusion: standard deviation of synchronous scan is better than 8 % (over
Teflon diffuser)
Software and Electronics
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Real time digital signal filtering (user selectable)
Transient digitizer acquisition sampling rate: 1.25 Ms/s (0.8 ms/data point)
Digitizing: 16 bits
FluoreScan software
All acquisitions are selected from the main screen of the software.
One click “water Raman” test to confirm system sensitivity
Automatic identification and optimization of excitation and
emission wavelengths for unknown samples.
• Multiple lifetime fitting 1 to 4 exponentials.
• Life times decays can be plotted in real time with automatic
exponential fitting
Analysis
• Automatic Analysis features can be applied in
real time:
– Digital filtering,
– Automatic multi-exponential fitting.
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Normalize, average, Integrate, differentiate
Multi peak finder, smooth
3D peak finder
Automatic water Raman calculation
Options
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Rotatable solid sample holders
Rotatable powder sample holders
Polarizers
Filters (Long pass, bandpass, neutral
Density filters)
• Semi-micro cuvette
Data Acquisition
• Corrected excitation/ Emission
spectra
• Time-based intensity scans
• Polarization (anisotropy)
excitation and emission spectra
• Synchronous scans
• Intensity vs temperature ramping
• 3-D excitation/ emission
mapping (EEM): Excitation vs
emission vs intensity
• Phosphorescence decay with
instant lifetime calculation (up
to 4)
• Time-based lifetime scan
• Anisotropy decay (rotational
correlation times)
• Lifetime temperature ramping
• 3-D excitation vs. decay vs.
intensity
• 3-D emission vs decay vs
intensity
Automatic Hardware Configuration
Automatic Calibration
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FluoreScan has four auto calibration
procedures:
Excitation monochromator: for
excitation wavelength calibration
Emission monochromator: for emission
wavelength calibration
PMT: automatic calculate best HV for
the PMT detector for each sample
RCQC: automatic calculate best HV for
the detector used for real-time correction
for each sample
Automatic Monochromator Calibration
Timebased Polarization
3D Display
Real time 3D scan of DYAG : Slits are 2.5 nm; repetition is 250 Hz; HV is 1000V
Applications
• General steady state fluorescence and
phosphorescence
• Phosphorescence lifetimes
• Inorganic crystals, semiconductors
and doped glasses
• Protein-protein Interactions
• Protein-peptides interactions
• Protein folding-unfolding
• Membrane Studies
• Photosynthesis
• Room temperature phosphorescence
of proteins (Trp)
• Lanthanide-based assays
development
• LRET based assays
• Room temperature phosphorescence
of micelle-stabilized aromatics
• Room temperature phosphorescence
of organics on solid substrate
• Optical sensor for carbon dioxide.
• Assay for clostridial toxin activity
• DNA hybridization assay
• Luminescence-based molecular
beacons
Luminescence Excitation and Emission Spectra of Sapphire Crystals
Luminescence Excitation and Emission Spectra of Sapphire Crystals
Luminescence Decay of Sapphire Crystals
Deconvolution with Felix 32 Software
Luminescence Decay of Sapphire Crystals
Deconvolution with Felix 32 Software
Luminescence Decay of chelated Tb3+
Deconvolution with Felix 32 Software