Instrumentation of Fluorescence

and Phosphorescence
Presented by Group No.3
• Waqar Rasheed
• Saeed Umar
• Sami UllAllah
• Sania Batool
• Esha Farooq
• Areej Shabir
Table of Contents
• Flourescence and phosphorescence
• Fluorometer
• Instrumentation of Fluorescence
• Instrumentation of Phosphorescence
Fluorescence
• Fluorescence is the ability of certain chemicals to give off visible light after
absorbing radiation which is not normally visible, such as ultraviolet light.
• Fluorescence was first discovered in 1845 by Fredrick W. Herschel. He
discovered that UV light can excite a quinine solution (e.g. tonic water) to emit
blue light.
• Fluorescence is often used to analyze molecules.
Phosphorescence

• Phosphorescence is a process in which energy absorbed by a substance is released

relatively slowly in the form of light.
• Phosphorescence phenomena lifespan is typically from 10-4 -10-2s which
considerably lengthier in comparison to Fluorescence lifespan.
• Examples include glow-in-the-dark stars, some safety signs, and glowing paint.
 Fluorometer
• A fluorometer is an instrument designed to measure the various parameters of
fluorescence, including its intensity and wavelength distribution of the emission
after excitation.
• A fluorometer (and fluorimeter) measures the fluorescence or light emitted by
different fluorescing objects.
Fluorescence instrumentation
• Fluorescence instrumentation refers to the tools and techniques used to measure
and analyze fluorescence, which is the emission of light by a substance that has
absorbed light or other electromagnetic radiation.
• This phenomenon is widely utilized in various scientific fields, including
biochemistry, biotechnology, environmental science, and materials science.
1. Light source
• Fluorescence instrumentation typically begins with a light source. This can be a
xenon lamp, mercury arc lamp, or a more modern and stable option like light-
emitting diodes (LEDs).
• The choice of light source depends on factors such as spectral range, intensity,
stability, and cost.

2. Excitation Monochromator
• The excitation monochromator is used to select a specific wavelength of light to
excite the sample.
• It ensures that only the desired excitation wavelength reaches the sample,
reducing background interference. Monochromators can be prism-based or
grating-based.
3 Sample compartment
• This is where the sample to be analyzed is placed. It can be a cuvette holder for
liquid samples or a sample stage for solid samples.
• The sample compartment is designed to minimize light scattering and ensure
uniform illumination of the sample.
4. Detector
• The detector measures the fluorescence emitted by the sample after excitation.
Photomultiplier tubes (PMTs) are commonly used detectors due to their high
sensitivity and fast response time.
• Other detectors like photodiodes and CCD cameras may also be used, depending
on the application.
5. Emission monochromator
• Similar to the excitation monochromator, the emission monochromator selects a
specific wavelength of emitted fluorescence for detection.
• This helps in separating the fluorescence signal from background noise and
scattered light.
6. Filters and Optics
• Optical filters are used to further refine the excitation and emission wavelengths,
improving the signal-to-noise ratio.
• Lens systems and mirrors are employed to focus and direct light efficiently
through the instrument.
7. Data acquisition system
• Modern fluorescence instruments are equipped with data acquisition systems that
capture, process, and analyze the fluorescence signals.
• This may include software for spectral analysis, fluorescence lifetime measurements,
and data visualization.
8. Calibration Standards
• To ensure accurate and reliable measurements, fluorescence instruments are
calibrated using standard reference materials with known fluorescence properties.
• These standards help validate instrument performance and maintain measurement
accuracy over time.
9. Advanced flouresence technique
• In addition to basic fluorescence measurements, advanced techniques like
fluorescence resonance energy transfer (FRET), fluorescence correlation
spectroscopy (FCS), and fluorescence lifetime imaging microscopy (FLIM) may
require specialized instrumentation with additional components and capabilities.
10. User Interface
• The instrument is typically controlled via a user-friendly interface that allows
researchers to set parameters, acquire data, and perform data analysis. Graphical
interfaces and software packages provide tools for data interpretation and
presentation.
• Overall, fluorescence instrumentation plays a crucial role in research and
analytical laboratories, enabling scientists to study molecular interactions,
characterize materials, monitor biological processes, and conduct various
fluorescence-based assays with high sensitivity and precision.
Instrumentation of Fluorescence
Phosphorescence instrumentation
• Phosphorescence instrumentation involves specialized tools and techniques for
studying phosphorescent phenomena, which is the delayed emission of light by a
substance following exposure to a light source.
• Phosphorescence differs from fluorescence in that it involves a longer-lived
excited state, leading to a delayed emission of light.
1. Light Source
• Similar to fluorescence instrumentation, phosphorescence instrumentation starts
with a suitable light source.
• This can include xenon lamps, mercury arc lamps, or LEDs, depending on the
required spectral range, intensity, and stability.
2.Excitation monochromator
• The excitation monochromator selects a specific wavelength of light to excite the
sample, similar to fluorescence instrumentation.
• This ensures precise excitation and minimizes background interference.
3. Sample Compartment
• The sample compartment is designed to hold the sample to be analyzed. It can
accommodate liquid samples in cuvettes or solid samples on a sample stage.
• The compartment is designed to minimize stray light and ensure uniform
excitation of the sample.
4. Detector
• The detector captures the delayed emission of light (phosphorescence) from the
sample. Photomultiplier tubes (PMTs) are commonly used detectors due to their
sensitivity and ability to detect low-intensity signals over an extended period.
Other detectors like photodiodes and CCD cameras may also be used.
5. Emission Monochromator
• Similar to fluorescence instrumentation, a phosphorescence instrument may
include an emission monochromator to select specific wavelengths of
phosphorescence for detection.
• This helps in separating the phosphorescent signal from background noise.
6. Delayed phosphoresence
Phosphorescence instrumentation includes a delay mechanism to account for
the longer-lived excited state of phosphorescent substances. This delay allows
the instrument to capture the delayed emission accurately.
7. Filters and Optics
Optical filters and lenses are used to further refine the excitation and emission
wavelengths, improve signal-to-noise ratio, and focus light efficiently onto
the detector.
8. Data Acquisition System
• A data acquisition system is essential for capturing and analyzing
phosphorescence signals. It includes hardware and software components for
data acquisition, processing, and visualization. Advanced features may
include time-resolved measurements for studying phosphorescence
lifetimes.
9. Calibration standards
Calibration standards with known phosphorescent properties are used to calibrate
the instrument, ensuring accuracy and reliability in phosphorescence measurements.
10. Application-Specific Features
• Phosphorescence instrumentation may include additional features tailored to
specific applications. For example, in phosphorescence lifetime measurements,
specialized components such as pulsed light sources and time-correlated single
photon counting (TCSPC) detectors are used.
11. User Interface
• The instrument is typically controlled via a user-friendly interface that allows
researchers to set parameters, acquire data, and perform data analysis. Graphical
interfaces and software packages provide tools for data interpretation and
presentation.
• Phosphorescence instrumentation is utilized in various fields, including materials
science, biophysics, environmental monitoring, and chemical analysis, to study
phosphorescent materials, characterize molecular interactions, and perform time-
resolved measurements of phosphorescence lifetimes.
Phosphorescence instrumentation