Instrumental Analytics Part II PDF

Induction Week Instrumental Analytics Part II Harald Hundsberger, Krems WS 23/24 INSA Overview Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, FACS) Time resolved fluorescence Fluorescence Polarization...

Induction Week Instrumental Analytics Part II Harald Hundsberger, Krems WS 23/24 INSA Overview Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, FACS) Time resolved fluorescence Fluorescence Polarization Fluorescence Life Time Luminescence Label Free Detection Plasmon Surface Resonsance Spectroscopy Fluorescence Fluorescence became to one of the most important detection technologies in the biomedical continuum Gene expression analysis of 40,000 genes in paralell ! Only with fluorescence detection (Microarray !) Instruments in labs using FI for detection and quantitation of molecules Important tool in tissue engineering (fluorescence labeled antibodies) Key technology in drug discovery (HTS- High throughput screening assays), Cell bases assays Fluorescence is moving more and more into the speciality testing market (BSE-testing, detection of genetically modified food…….) Key advantages of fluorescence: Sensitivity Specifity Multiplexing Part I: What is Fluorescence ? Fluorescence 1. 3 step process 2. Fluorophore is excited by/ absorbs light 3. The excited state exists for a finite time 4. Energy(light) is emitted returning the fluorophore to ground state Excitation and Emission Spectrum of fluorescein: Excitation at 485nm (peak) Emission at 535nm (peak) Distance between excitation and emission peak is called stoke´s shift Fluorescence detection devices have to separate excitation from emission light Excitation and Emission Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum.  Fluorescence and Multiplexing Fluorescence detection of mixed species. Excitation (EX) in overlapping absorption bands A1 and A2 produces two fluorescent species with spectra E1 and E2. Optical filters isolate quantitative emission signals S1 and S2.  Fluorescence Dyes A lot of fluorescence dyes are available Can be coupled to biomolecules (proteins and antibodies) Different spectral properties Calcium sensing pH-sensing  Photobleaching Fluorescence process is rapid (10-8 seconds) and cyclical. The excited state is more chemical reactive than the ground state. When high power light sources are used (lasers), occurs also with lower energy light sources. Cyanine dyes are more resistant to photobleaching  Fluorescence Quenching Quenching causes decrease or loss of signals: – Energy of an excited dye is transmitted to another dye molecule – when samples are to labeled too densely, or if dye is too concentrated – Alternatively a quencher can be added – Fluorescence output dramatically decreased Part II: Hardware for Fluorscence Detection A Simple Fluorescence Detector Example: Microplate Reader (optics only), electronics and temperature control not shown ! Excitation light source Bandpass filters Alternative (double monochromator based Dichroic Mirror or Beamsplitter Lenses 1 light source 2+3+4 lenses Photomultiplier (CCD camera, photodiode 5 excitation/emission filters 6 beamsplitter 7 detector (Photomultiplier) 8 well of a microplate  Detectors for Fluorescence Photodiode Spectroscopy Spectrophotometer Photomultiplier Tube sensitive and high dynamic range Cuvette Fluorometers Microarray scanners Microplate readers FACS Flow Cytometer CCD - Cameras  Light Sources in Fluorescence LED`s light emitting diodes cheap but efficiancy can be high when high power LED´s are used (Light cycler from Roche, and DTX 880 from Beckman), can be pulsed ! Halogen lamp lower priced instruments Xenon flash lamp flexible because of wavelength range (200-> 1000nm) can be pulsed ! Lasers (argon, he/ne, nitrogen, laser diodes) Laser scanning microscopy FACS-fluorescence activated cell sorting Flow Cytometer High resolution scanners (microarray scanners) Part 3: Fluorescence Detection Methods and Applications  Fluorescence Detection Methods Fluorescence Intensity & FRET Time Resolved Fluorescence Fluorescence Polarization Fluorescence Lifetime Fluorescence Imgaging (Luminescence) Fluorescence Intensity Continuous light source (not pulsed) PMT collects emission light for a defined time period (integration time) Optical path for microplate reader fast measurements for 96, 384 or 1536 samples Fluorescence Intensity Applications High Throughput Screening assays Nucleic Acid and Protein quantitation Cell based assay Microarray FACS/Flow cytometry Quantitative PCR Confocal Laser Scanning Microscopy Flow Cytometry Flow cytometry Essential technology for cell characterization Expensive but a lot of informationfrom complex samples can be gained Cell sare characterized via fluorescence Cell are labled with antibodies or via intrinsic expression of fluorescence (GFP) Flow Cytometry Cytograms Expression of surface markers recognized by fluorescence labeled antibodies Clinical relevance: infected or not ? Detection of cells which could not be recognized by normal light microscopy methods Ratio calculation of subtypes of cells  Fluorescence Activated Cell Sorting If you want to separate a subpopulation of cells, you could do so by tagging those of interest with an antibody linked to a fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells you want to separate. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected. The final step is sorting the cells which is accomplished by electrical charge. The computer determines how the cells will be sorted before the drop forms at the end of the stream. As the drop forms, an electrical charge is applied to the stream and the newly formed drop will form with a charge. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes. Drops that contain no cells are sent into the waste tube. The end result is three tubes with pure subpopulations of cells. The number of cells is each tube is known and the level of fluorescence is also recorded for each cell.  FRET – Fluorescence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Primary Conditions for FRET – Donor and acceptor molecules must be in close proximity (typically 10–100 Å). – The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (see Figure). – Donor and acceptor transition dipole orientations must be approximately parallel.  Dual Color Optics for FRET Detection For multicolor analysis or FRET Both signals are separated simultanously FRET main application are HTS assays and imaging(microscopes) FRET-Imaging Localization of a protein interaction inside cells FRET assays are used for drug discovery see later: Time Resolved Fluorescence Assays  Real Time PCR (Quantitative PCR) Principle of quantitative PCR  Hardware and Application RT-PCR Most successful instruments in the life science market during the last 10 years ! Size decreased rapidly Today: LED based detectors Applications: RNA quantitation (Viral titer), Gene expression studies DNA quantitation from genetically modified food Nuleic Acid Quantitation with Fluorescence Superior performance over absorbance dsDNA ssDNA RNA Dyes also for protein available NanoOrange TM Cuvette based or microplate based Microarray & Scanners Analysis of 20,000-40,000 spots in parallel Each spot represents a single gene of an organism. Green means increase in expression, red means decrease in expression Very successful technology for research and pharma industry-drug discovery Focus Microarray in 5th semester- Biochemical Analysis Microarray Drug Discovery: – MA technology can provide useful info throughout the process of drug discovery. – Toxic properties of a drug can be monitored by analyzing expression profile induced by a drug candidate. – Looking for co expressed genes – https://www.youtube.com/watch?v=VN sThMNjKhM Confocal Optics Optics of confocal scanning microscope Difference Confocal non Confocal Drosophila embryo Stained with FITC (Fluoresceine) A non confocal B confocal Laser Scanning Microscopy Imaging of fast cellular events: Calcium influx Central second messenger Activity of mucles Fertilization Synaptic activity Apoptosis Differentitation Evolutionary conserved signal transduction pathway Calcium Imaging Ratiometric measurement Serves as internal control Important for screenings Special Measurement Modes FRAP-detection (Fluorescence Recovery After Photobleaching) Definition of the cell region to be bleached Brief illumination of the region with very high laser intensity Recording the progress of fluorescence recovery in the bleached area with high temporal resolution Changes in intensity in the bleached region represent the sum of all movements of the fluorescent molecules, whether passive (e.g., diffusion) or active (e.g., transport). The regeneration time (half-recovery period) is a measure of the speed of protein movement. Laser Scanning Microscopy Multiparameter Fluorescence: Spectral Detector of our microscope Time Resolved Fluorescence (TRF) TRF-Highly Specific & Sensitive Used Labels: Europium Chelates Europium Cryptates Samarium Dysprosium Terbium Hardware Requirements for TRF Pulsed Light Source Laser LED Xenon flash lamp High sensitivity Detection in the attomole range High specificity Background fluorescence is short lived TRF-FRET Assay TRF-FRET Assay Ratiometric read out for internal normalization Standard HTS assay in drug discovery Kinase, Phosphatase Molecular Interaction Luminescence Chemiluminescence:...is luminescence as the result of a chemical reaction. Bioluminescence:...is visible chemiluminescence from living organisms Source of Bioluminescence are Photoproteins Luminescencent ELISA Firefly Luciferase Photinus pyralis ATP dependent Light emission at 565 nm Luciferase + Luciferin + ATP + O2 ⎯⎯ ⎯⎯→ Oxyluciferin + AMP + PPi + CO2 + Light Luciferase  Luciferase Assays Firefly luciferase is by far the most commonly used bioluminescent reporter. This monomeric enzyme of 61kDa catalyzes a two-step oxidation reaction to yield light, usually in the green to yellow region, typically 550– 570nm Upon mixing with substrates, firefly luciferase produces an initial burst of light that decays over about 15 seconds to a low level of sustained luminescence Dual Luciferase Assay Reporter Plasmids Checking Promotors for strength or when they are induced Indentification of enhancer elements Aequorine For monitoring intarcellular Calcium BRET Bioluminescence resonance energy transfer Luminescence Detectors PMT based Injectors Cuvettes Microplates Imaging Systems Reporter Systems