The document provides an overview of basic instrumentation components used in fluorescence spectroscopy, including light sources, detectors, wavelength selection components, and polarization optics. It discusses common light sources such as arc lamps, lasers, and LEDs. Detectors described include photomultiplier tubes, avalanche photodiodes, and photon counting modules. Wavelength selection is achieved using optical filters, tunable filters, and monochromators. Polarizers discussed are Glan Taylor and sheet polarizers. Time-resolved instrumentation for measuring fluorescence lifetimes is also summarized.
The detailed information of UV Visible Spectroscopy, it includes the information regarding electronic transitions, Electromagnetic radiations, Various shifts.
UV - Visible Spectroscopy detailed information is included .The Spectroscopy study provide the information and the absorbance as well the concentration of the drugs is studied.
A spectrophotometer is an instrument containing a monochromator, a device which produces a light beam containing wavelengths in a narrow band around a selected wavelength, and a means of measuring the ratio of that beam's intensity as it enters and leaves a cuvette 99 This describes a single-beam photometer.
Introduction to Fourier Transfer Infrared SpectroscopyRahulVerma550005
The preferred method of infrared spectroscopy is known as Fourier Transform InfraRed (FT-IR). Infrared spectroscopy involves passing IR photons through a sample. The sample absorbs some of the infrared light and passes some of it through (transmitted). The resulting spectrum depicts the sample's molecule absorption and transmission, resulting in a molecular fingerprint.
The detailed information of UV Visible Spectroscopy, it includes the information regarding electronic transitions, Electromagnetic radiations, Various shifts.
UV - Visible Spectroscopy detailed information is included .The Spectroscopy study provide the information and the absorbance as well the concentration of the drugs is studied.
A spectrophotometer is an instrument containing a monochromator, a device which produces a light beam containing wavelengths in a narrow band around a selected wavelength, and a means of measuring the ratio of that beam's intensity as it enters and leaves a cuvette 99 This describes a single-beam photometer.
Introduction to Fourier Transfer Infrared SpectroscopyRahulVerma550005
The preferred method of infrared spectroscopy is known as Fourier Transform InfraRed (FT-IR). Infrared spectroscopy involves passing IR photons through a sample. The sample absorbs some of the infrared light and passes some of it through (transmitted). The resulting spectrum depicts the sample's molecule absorption and transmission, resulting in a molecular fingerprint.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
7. Lamp Light Sources
1. Xenon Arc Lamp (wide
range of wavelengths)
2. High Pressure Mercury Lamps
(High Intensities but
concentrated in specific lines)
3. Mercury-Xenon Arc Lamp
(greater intensities in the UV)
4. Tungsten-Halogen Lamps
5. Light emitting diodes (LEDs)
Multiple color LEDs can be
bunched to provide a broad
emission range)
Xenon Arc Lamp Profiles
Mercury-Xenon Arc Lamp Profile
UV
Ozone Free
Visible
12. MCP & Electronics
(ISS Inc. Champaign, IL USA)
APD
The silicon avalanche photodiode (Si APD) has
a fast time response and high sensitivity in the
near infrared region. APDs can be purchased
from Hamamatsu with active areas from 0.2
mm to 5.0 mm in diameter and low dark
currents (selectable). Photo courtesy of
Hamamatsu
13. Dynodes
Photocathode
l
High Voltage Supply
(-1000 to -2000 V)
Ground
e-
Anode
Current Output
e-
e-
e-
Constant Voltage
(use of a Zenor Diode)
resister series
(voltage divider) capacitor series
(current source)
e-
e-
e-
e-
e-
e-
e-
e-
e-
Vacuum
The Classic PMT Design
Window
21. Tunable Optical Filters
An electrically controlled liquid crystal elements to select a specific visible wavelength of
light for transmission through the filter at the exclusion of all others.
Liquid Crystal Filters:
AO Tunable Filters:
The AOTF range of acousto-optic devices are solid state optical filters. The wavelength
of the diffracted light is selected according to the frequency of the RF drive signal.
Isomet (http://www.isomet.com/index.html)
22. Monochromators
Czerny-Turner design
Mirrors
Rotating Diffraction Grating
(Planar or Concaved)
Entrance slit
Exit Slit
1. Slit Width (mm) is the
dimension of the slits.
2. Bandpass is the FWHM of
the selected wavelength.
3. The dispersion is the factor
to convert slit width to
bandpass.
23. Zero Order
(acts like a mirror)
Nth Order
(spectral distribution)
Mirrors
Grating
The Inside of a Monochromator
26. Monochromator Polarization Bias
No Polarizer
Parallel Emission
Perpendicular Emission
Wood’s Anomaly
Adapted from Jameson, D.M., Instrumental Refinements in Fluorescence
Spectroscopy: Applications to Protein Systems., in Biochemistry,
Champaign-Urbana, University of Illinois, 1978.
250
250
800
800
Fluorescence
Fluorescence
Tungsten Lamp Profile Collected on an SLM Fluorometer
27. 300 350 400 450 500 550 600
vertical
horizontal
Wavelength (nm)
ISSPC1
Correction Factors
400 450 500 550 600
Intensity
(a.u.)
Wavelength (nm)
B
400 450 500 550 600
Intensity
(a.u.)
Wavelength (nm)
C
Correction of Emission Spectra
from Jameson et. Al., Methods in Enzymology, 360:1
Wavelength Wavelength
Fluorescence
Fluorescence
ANS Emission Spectrum, no polarizer ANS Emission Spectrum, parallel polarizer
uncorrected
corrected
Wavelength
29. The Instrument Quantum Counter
Fluorescence
Here we want the inner filter effect!
Optical Filter
Reference
Detector
Quantum Counter
Common Quantum Counters
(optimal range)*
Rhodamine B (220 - 600 nm)
Fluorescein (240 - 400 nm)
Quinine Sulfate (220 - 340 nm)
* Melhuish (1962) J. Opt. Soc. Amer. 52:1256
Wavelength (nm)
200 600
400
1.2
0.8
0.4
0.0
Eppley
Thermopile/
QC
Linearity of Rhodamine
as a quantum counter
30. 250 300 350 400 450
0.0
0.2
0.4
0.6
0.8
1.0
A
Wavelength (nm)
250 300 350 400 450
0.0
0.2
0.4
0.6
0.8
1.0
B
Wavelength (nm)
250 300 350 400 450
0.0
0.2
0.4
0.6
0.8
1.0
C
Wavelength (nm)
Excitation Correction
from Jameson et. Al., Methods in Enzymology, 360:1
Wavelength Wavelength
Wavelength
Fluorescence
Fluorescence
Fluorescence
Absorption (dotted line) and Excitation Spectra (solid line) of ANS in Ethanol
Uncorrected
Ratio Corrected
Lamp
Corrected
31. Two UV selected calcite prisms are
assembled with an intervening air space. The
calcite prism is birefringent and cut so that only
one polarization component continues straight
through the prisms. The spectral range of this
polarizer is from 250 to 2300 nm. At 250 nm
there is approximately 50% transmittance.
The Glan Taylor prism polarizer
Polarizers
Common Types:
Glan Taylor (air gap)
Glan Thompson
Sheet Polarizers
Two Calcite Prisms
0
90
90
0
33. Attenuation of the Excitation Light through Absorbance
Sample concentration
& the inner filter effect
Rhodamine B
from Jameson et. al., Methods in Enzymology (2002), 360:1
34. 3
2
1
x10
6
660
640
620
600
580
560
540
4
3
2
1
x10
6
The second half of the inner filter effect:
attenuation of the emission signal.
Diluted Sample
(1) Spectral Shift
(2) Change in Spectral Shape
1.0
0.8
0.6
0.4
0.2
700
650
600
550
500
450
3
2
1
x10
6
Absorbance Spectrum
Wavelength (nm) Wavelength (nm)
35. How do we handle highly absorbing solutions?
Quartz/Optical Glass/Plastic Cells
Emission
Path Length
Detector
Excitation
Emission
Excitation
Path Length
4 Position Turret
SPEX Fluoromax-2, Jobin-Yvon
36. Front Face Detection
Triangular Cells
Detector
Excitation
Reflected Excitation & Emission
Thin Cells & Special Compartments
Sample
Absorbance
Measurements
Excitation
Emission
[1] Adapted from Gryczynski, Lubkowski, & Bucci Methods of Enz. 278: 538
[1]
IBH, Glasgow G3 8JU
United Kingdom
38. Light Sources for Decay Acquisition:
Frequency and Time Domain Measurements
Pulsed Light Sources (frequency & pulse widths)
Mode-Locked Lasers
ND:YAG (76 MHz) (150 ps)
Pumped Dye Lasers (4 MHz Cavity Dumped, 10-15 ps)
Ti:Sapphire lasers (80 MHz, 150 fs)
Mode-locked Argon Ion lasers
Directly Modulated Light Sources
Diode Lasers (short pulses in ps range, & can be modulated by synthesizer)
LEDs (directly modulated via synthesizer, 1 ns, 20 MHz)
Flash Lamps
Thyratron-gated nanosecond flash lamp (PTI), 25 KHz, 1.6 ns
Coaxial nanosecond flashlamp (IBH), 10Hz-100kHz, 0.6 ns
39. Modulation of CW Light
Use of a Pockel’s Cell
Mirror
Radio Frequency
Input
Pockel’s Cell
Polarizer
Polished on a side
exit plane
CW Light Source
Double Pass Pockel’s Cell
Pulsed Emission
The Pockel’s Cell is an electro-optic device
that uses the birefringment properties of calcite
crystals to alter the beam path of polarized light. In
applying power, the index of refraction is changed
and the beam exiting the side emission port (0
polarized) is enhanced or attenuated. In applying RF
the output becomes modulated.
90
0
Polarizer
40. Time Correlated Single Photon Counting
Pulsed Light Source
PMT
TAC
Multichannel
Analyzer
Constant Fraction
Discriminator
Time
Counts
Sample Compartment
Filter or Monochromator
Time-to-Amplitude
Converter (TAC)
Instrument Considerations
Excitation pulse width
Excitation pulse frequency
Timing accuracy
Detector response time (PMTs 0.2-
0.9 ns; MCP 0.15 to 0.03 ns)
Photon Counting PMT
Timing Electronics
or 2nd PMT Neutral density (reduce to one photon/pulse)
41. 4
6
8
0.01
2
4
6
8
0.1
2
4
6
8
1
300
250
200
150
100
50
0
Channels (50 ps)
Fluorescence
Fluorescence Decay
Instrument Response Function
Histograms built one photon count at a time …
(1) The pulse width and instrument response times determine the time
resolution.
(2) The pulse frequency also influences the time window. An 80 MHz
pulse frequency (Ti:Sapphire laser) would deliver a pulse every 12.5
ns and the pulses would interfere with photons arriving later than the
12.5 ns time.
42. Polarization Correction
There is still a polarization problem in the geometry of our excitation and
collection (even without a monochromator)!!
Will the corrections never end ???
[1]
[2]
[3]
[4]
[5]
[6]
Polarized Excitation
0
0
90
An intuitive argument:
[1] = I0 + I90
[2] = I0 + I90
[3] = I0 + I90
[4] = I0 + I90
[5] = 2 x I90
[6] = 2 x I90
Total = 4 x I0 + 8 x I90
The total Intensity is proportional to:
I0 + 2 x I90
Setting the excitation angle to 0 and the
emission polarizer to 54.7 the proper weighting
of the vectors is achieved.*
*Spencer & Weber (1970) J. Chem Phys. 52:1654
43. Frequency Domain Fluorometry
CW Light Source
Sample Compartment
Filter or Monochromator
PMT
Analog PMTs (can also be done with photon counting)
PMT
S1 = n MHz
S2 = n MHz + 800 Hz
RF
Digital Acquisition
Electronics
Signal
Signal
RF
Locking Signal
S1 S2
Synthesizers
S1 and S2
Computer Driven
Controls
Similar instrument
considerations as
With TCSPC
Reference Turret
Pockel’s Cell