Instrumental Analytics Part II

Instrumental Analytics Part II

• The course is titled Instrumental Analytics Part II, taught by Harald Hundsberger at the University of Applied Sciences, Krems, during the WS 23/24 semester.

INSA Overview

• Fluorescence Spectroscopy & Applications
  • Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, FACS).
  • Time-resolved fluorescence
  • Fluorescence polarization
  • Fluorescence Lifetime
  • Luminescence
  • Label-Free Detection
  • Plasmon Surface Resonance Spectroscopy

Fluorescence

• Fluorescence became a crucial detection method in biomedical research, enabling parallel analysis of thousands of genes (e.g., microarrays).
• Fluorescence is essential for various applications, including tissue engineering (antibody labeling), drug discovery (high-throughput screening), and food safety testing (e.g., BSE).
• Key advantages of fluorescence include high sensitivity, specificity, and multiplexing capability.

Fluorescence: 3-Step Process

• Absorption of light by a fluorophore, exciting it to a higher energy level.
• The excited state exists for a finite period.
• Emission of light as the fluorophore returns to its ground state.

Excitation and Emission

• Excitation peak at 485 nm, emission peak at 535 nm in fluorescein.
• The distance between excitation and emission peaks is the Stokes shift.
• Fluorescence detection tools must separate excitation and emission light to prevent interference.

Excitation and Emission (Fluorophore)

• The excitation of fluorophores at three wavelengths, EX1, EX2, and EX3, do not change the emission profile; however, the variations in fluorescence emission intensity (EM1, EM2, EM3) correspond to the amplitude of the excitation spectrum.

Fluorescence and Multiplexing

• Detect multiple substances simultaneously in mixed samples.
• Identify substances with overlapping excitation spectra, using different filters to isolate specific emission.

Fluorescence Dyes

• Many dyes can be coupled with biomolecules (proteins, antibodies).
• These dyes have varying spectral properties useful for specific applications, like calcium sensing or pH measurement.

Photobleaching

• Fluorescence signals diminish from continued exposure to light or high-power laser radiation.
• Cyanine dyes tend to be more photobleaching resistant than other fluorescence dyes.

Fluorescence Quenching

• Energy transfer from an excited dye molecule to another molecule (quencher) interferes with fluorescence processes.
• Increased dye concentration or labeling density can lead to quenching.
• Added quenchers can reduce fluorescence output drastically.

Hardware for Fluorescence Detection

• Simple fluorescence detectors use excitation light sources, bandpass filters, dichroic mirrors/beamsplitters, lenses, and detectors like photomultipliers (or CCDs, photodiodes).

Detectors for Fluorescence Spectroscopy

• Photodiode, spectrophotometer, photomultiplier tube are sensitive and have a wide dynamic range.
• Devices like cuvette fluorometers, microarray scanners, microplate readers, flow cytometers, and CCD-cameras are specialized for the task.

Light Sources in Fluorescence

• LEDs: Popular, cost-effective, and efficient, particularly in pulsed applications (e.g., PCR).
• Halogen lamps: Provide a broad wavelength range, suitable for various instruments.
• Xenon Flash lamps: Offer tunability for different wavelengths, often used in pulsed mode.
• Lasers: Offer high intensity with a narrow spectral bandwidth, used for many fluorescence applications.

Fluorescence Detection Methods

• Fluorescence Intensity & FRET
• Time-Resolved Fluorescence
• Fluorescence Polarization
• Fluorescence Lifetime
• Fluorescence Imaging (Luminescence)

Fluorescence Intensity

• Continuous light source used.
• PMT detector for emission light through a defined period.
• Used for measurements in microplates (e.g., 96, 384, or 1536 wells).

Fluorescence Intensity Applications

• High-throughput screening assays
• Quantification of nucleic acids and proteins
• Cell-based assays
• Microarray analysis
• Flow cytometry
• Quantitative PCR
• Confocal Laser Scanning Microscopy

Flow Cytometry

• Essential technology for cell characterization.
• Enables analysis of complex samples.
• Cells are labeled with antibodies or via intrinsic fluorescence.
• Provides data about cell populations, including ratio of subtypes.

Cytograms

• Visual representations of data from flow cytometry experiments; used to display expression levels of surface markers.
• Provides clinical insights (e.g., infection detection).
• Allows analysis of cells not readily identified using light microscopy.

Fluorescence Activated Cell Sorting (FACS)

• Separates cells based on fluorescence, sorting into different tubes or compartments.
• The computer determines how cells will be sorted by applying an electrical charge to the drop stream.
• Provides pure cell populations.

FRET – Fluorescence Resonance Energy Transfer

• The excited donor molecule transfers energy to the acceptor molecule without photon emission.
• Donor and acceptor molecules must be in close proximity.
• Used in high-throughput screening (HTS) assays and imaging.
• Useful for measuring molecular interactions, including drug interactions.

Dual Color Optics for FRET

• Separates FRET signals from other signals through optical filters in different wavelength ranges.
• Facilitates simultaneous measurement of donor and acceptor fluorescence in multicolor analyses.

FRET-Imaging

• Used for spatial localization of interactions in cells
• Useful for drug discovery

Real-Time PCR (Quantitative PCR)

• Monitors PCR amplification in real-time to quantify target molecules.
• The exponential phase of amplification is tracked continuously.
• Enables high precision and throughput
• Important for RNA and DNA quantification

Hardware and Application RT-PCR

• LED based detectors are increasingly used for efficiency in real-time PCR.
• Enables crucial applications such as RNA quantitation (viral load), gene expression studies.

Nucleic Acid Quantification with Fluorescence

• Fluorescence methods offer advantages for DNA and RNA quantification and protein measurement compared to absorbance methods.
• Dyes such as PicoGreen, Hoechst, and Ethidium bromide are used.
• NanoOrange™ is a commonly available dye for protein measurement.

Microarray & Scanners

• Analyzing large numbers of genes in parallel.
• Green/red spots indicate increased/decreased gene expression.
• Important in drug discovery and research.

Microarray Drug Discovery

• This technology analyses the expression of various genes to investigate drug effects and identify genes with co-expression patterns.
• Screens drug candidate potential effects and toxicity and to monitor their responses over time.

Confocal Optics

• Confocal microscopes utilize a spatial filtering technique to eliminate out-of-focus light, offering enhanced image quality.
• This is achieved through a pinhole in the imaging path.
• Used for detailed imaging of cells and samples.

Laser Scanning Microscopy

• Used to image fast cellular events like calcium influx, central second messenger activity, or receptor activation.
• Used for studies of different processes in cells and processes in organisms at the cellular level.

Calcium Imaging

• Ratiometric measurement of calcium fluorescence.
• Serves as internal control.
• Important for screening applications.

Special Measurement Modes (FRAP)

• Fluorescence Recovery After Photobleaching (FRAP) studies the mobilities of various fluorescent molecules—often proteins—within cells.
• Measures the rate of fluorescence restoration after an area of fluorescent molecules is bleached.
• Useful for studying protein movement, diffusion, and transport.

Multiparameter Fluorescence Spectral Detector

• Multiparameter fluorescence microscopy enables the measurement of multiple spectral signals simultaneously.
• This approach utilizes a spectral detector to capture signals from different dyes or fluorophores that may be present in a substance or sample.

Drug Molecules

• Overview of different drug targets relevant in biological systems.

Time Resolved Fluorescence (TRF)

• Highly sensitive methodology.
• Uses sensitive labels.
• Uses europium chelates and cryptates
• Background fluorescence is short lived.

TRF-FRET Assay

• Ratiometric read-out for internal normalization.
• A standard HTS assay for drug discovery.
• Used to study interactions between kinases and phosphatases.
• Important for studying enzyme interactions.

Luminescence

• Chemiluminescence: Light emission from a chemical reaction.
• Bioluminescence: Visible light emission from living organisms (often through protein-mediated reactions).

Bioluminescence-based Assays–ELISA

• A sensitive and versatile technique for detecting and measuring enzymes by using enzyme reactions that elicit measurable fluorescence or color change.

Firefly Luciferase

• ATP dependent enzyme that produces bioluminescent light.
• Light emission at specific wavelengths in the visible range.
• Widely used assay for reporter genes.

Luciferase Assays

• Measuring the reaction of luciferase substrates to generate light.
• Used in variety of assays and studies.

Reporter Plasmids

• Plasmids containing genes and control elements to monitor expression or strength of different genetic sequences in different cells and conditions.
• Important tool to investigate genetic mechanisms.

Calcium Imaging (Aequorin)

• Used for measuring intracellular calcium levels by using the bioluminescence resonance energy transfer (BRET) method.
• Aequorin is a calcium-sensitive protein.

Luminescence Detectors

• PMT-based detectors, cuvettes, microplates, imaging systems are used depending on the specific needs of the experiments.

Reporter Systems

• Enzyme fluorescence detection, time-resolved fluorescence, radioactivity, chemiluminescence using luminol, and peroxidase-based detection and alkaline phosphatase-based detection.
• Used to measure the expression and activity of different molecules.

This quiz covers key concepts from Instrumental Analytics Part II, focusing on fluorescence spectroscopy and its applications in biomedical research. Topics include fluorescence intensity, time-resolved fluorescence, and luminescence, among others. Explore how these techniques are vital for high-throughput screening and other applications.