FLUORESCENT DYES TECHNIQUES Shin Hee Yoon, MD Department of Physiology, CUMC

1. FLUORESCENT DYES TECHNIQUESShin Hee Yoon, MDDepartment of Physiology, CUMC Department of Physiology CUMC

2. Fluorescence (형광) fluorescence: an optical phenomenon in which molecular absorption of a photontriggers the emission of another photon with a longer wavelength naturally fluorescent proteins or small molecules: intrinsic fluorescence or autofluorescence. ex, NADH, tryptophan or endogenous chlorophyll, phycoerythrin, green fluorescent protein….. specific or general proteins, nucleic acids, lipids or small moleculescan be "labelled" with an extrinsic fluorophore Fluorescent dyes techniques is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules Department of Physiology CUMC

3. Fluorescence (형광) Primary fluorescence: fluorescence in a sample Secondary (or indirect) fluorescence: fluorescence in a sample stained with a fluorescent dye Fluorescent probes, fluorophores or dyes: The category of molecules capable of undergoing electronic transitions that ultimately result in fluorescence Department of Physiology CUMC

4. Applicationof fluorescent dyes techniques 1. localization of specific molecules: protein, nucleic acid, lipid,… 2. concentration of ions: Ca2+, H+, Zn2+, …. 3. measurement of membrane potential (cell, mitochondrialmembrane)

5. Fluorescence Process 1. Excitation 2. Excited-state: 1-10 nanosecond, conformational change 3. Fluorescence emission Jablonski diagram Department of Physiology CUMC

6. Fluorescence Process 1. Excitation: ground state by photon energy (hvEX) by externallamp or laser → fluorophore adsorb photon → excited electronic state (S1’)cf,  in chemiluminescence: excitated states by chemical reaction 2. Excited-state: excited state for 1-10 nanoseconds Conformational changes a multitude of possible interaction with its molecular environments 1) partial dissipation of S1 ' energy → relaxed singletexcited state (S1) 2) other processes: depopulate S1 collision quenching, fluorescent energy transfer, intersystem crossing Department of Physiology CUMC

7. 3. Emission: relaxed singletexcited state (S1’) → ground state (S0) with emission 1) energy of photon: lower energydue to energy dissipation longer wave length than excitation photon → Stokes shifts: difference in energy or wave length, hex –hvem : fundamental to the sensitivity of fluorescence techniques Entire fluorescence: cyclical Department of Physiology CUMC

8. Characteristics of Fluorescence Spectra 1. The fluorescence excitation spectrum of a single fluorophore species in dilute solution is identical to its absorption spectrum. 2. Under the same conditions, fluorescence emission spectrum is independent of excitation wave length (due to partial dissipation of excitation energy). 3. The intensity ofemission is proportional to the intensity of fluorescence excitation spectrum. Department of Physiology CUMC

9. Color regions of spectrum Ultraviolet rays 200 nm Department of Physiology CUMC

10. Absorption and fluorescence spectral ranges for fluorophores http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook.html Department of Physiology CUMC

11. Essential elements of fluorescence detection system 1. excitation source: laser, Xenon, mercury 2. fluorescent probe or fluorophore 3. wave length filter to isolate emission photons from excitation photons 4. detector Department of Physiology CUMC

12. Fluorescent probe or fluorophore • 1) small molecules: reactive dyes • 2) fluorescent proteins: Green Fluorescent Protein (GFP) from the jellyfish • 3) quantum dots Department of Physiology CUMC

13. Three type of fluorescence instruments 1. spectrofluorometer or microplate reader: average properties of bulk sample (mL to ml) 2. fluorescence microscope: spatial coordination, photobleaching 3. fluorescence scanner including microarray readers: resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms. 4. flow cytometers: fluorescence per cell in a flowing stream, subpopulation in large sample (cell fractionation) Department of Physiology CUMC

14. Essential elements of fluorescence microscope Department of Physiology CUMC

15. Determinant of Fluorescence Signals 1. absorbance of sample 2. fluorescence quantum yield of dye : number of fluorescence photons emitted / number of photos absorbed 3. intensity of excitation source (20 W-200 W) 4. fluorescence collection efficiency of instrument (30%-85%) Department of Physiology CUMC

16. Background Fluorescence Autofluorescence: endogenous fluorescence in sample Reagent background: unbound or nonspecifically bound probes System background: fluorescence in light path → Background correction Department of Physiology CUMC

17. Multicolor Labeling Experiments : monitors different biological functions by using 2 or more dyes application: flow cytometry, DNA sequencing, fluorescence in situ hybridization, fluorescence microscope Strong absorption at a coincident excitation wavelength and well separated emission spectra Department of Physiology CUMC

18. Ratiometric Measurements 1. Ca2+ indicator (fura-2, indo-1), pH indicator (BCECF, SNARF, SNARF) 2. free and bound form of fluorescent ion indicators : different emission and excitation spectrum → ratio of free and bound form → ion concentration 3. eliminate distortion of data caused by photobleaching variation in probe loading and retention instrument instability Department of Physiology CUMC

19. Photobleaching : High-intensity illumination conditions destroy the excited fluorophores → limiting fluorescence detectability Remedy: 1. Maximize detection sensitivity(excitation intensity↓) a. low-light detection device (CCD) b. high-numerical aperture objective in microscope c. wide emission bandpass filter to get higher intensity of fluorescence 2. Use of less photolabile fluorophores Department of Physiology CUMC

20. Signal amplification • increase the number of fluorophores • use effective intracellular concentration of dye • a. marked changes in probe’s chemical and optical characteristics • b. increased labeling of protein or membranes ultimately leads to precipitation of • the protein or gross changes in membrane permeability • c. self quenching • 2. avidin-biotin or antibody-hapten secondary detection techniques • 3. enzymes-labeled secondary detection regents in conjunction with fluorogenic substrates • 4. multiple fluorophores containg probes such as phycobiliproteins and FluoSpheres fluorescent microspheres Department of Physiology CUMC

21. Colocalizationof specific components FITC: ex: 490 nm; em: 518 → synaptophysin TRITC: ex: 550; em: 580 → GABAB receptor merge

22. Multi-color experiment (AJP 297, 930-939) : Caveolin in endothelial NOS activation in sinusoidal endothelial cell confocal microscopy 1. nuclei: DAPI (ex 358; em: 461) 2. Caveolin-1: rabbit caveolin-1 primary Ab + goat anti-rabbit Texas red-conjugated secondary Ab (ex: 595; em: 615) 3. e-NOS: mouse eNOS primary Ab + goat anti-mouse FITC-conjugated secondary Ab (ex: 494; em: 516) Depaartment of Physiology CUMC

23. Ca2+ imaging experiment • cells were loaded with 2 M fura-2 AM HEPES-HBSS containing 0.5% bovine serum albumin for 45 min at 37 C→ loading was terminated by washing with HEPES-HBSS for 15 min (fura-2-AM→ fura-2-Ca2+ + AM) • Fura-2-loaded cells was excited alternately at 340 and 380 nmby a 100 W xenon arc lamp • Excitation light was reflected from a dichroic mirror (400 nm) through a 20 objective (Nikon; N.A. 0.5). Digital fluorescence images (510 nm, 40 nm band-pass) were collected with a cooled CCD camera (Photometrics; 1280  1035 binned to 256  207 pixels). • Ratios were calculated from the two background-subtracted digital images. Department of Physiology CUMC

24. [Ca2+]i imaging and microinjection of ceratin substances in NG108-15 cells by using fura-2 and rhodamin fura-2: imaging for [Ca2+]i excitation: 340nm, 380 nm: emission: 510 nm Dextran-conjugated tretamethyl rhodamin: confirmation of microinjection excitation: 535 nm, emission: 605 nm Department of Physiology CUMC

25. Staining for nucleic acid by Hoechst 33342 (10 μM) Excitation at 535 nm, emission at 617 apoptotic cell death Department of Physiology CUMC

26. pH 7.4 pH 6.6 control - z-VAD-FMK + z-VAD-FMK control - z-VAD-FMK + z-VAD-FMK THAP THAP control - z-VAD-FMK + z-VAD-FMK control - z-VAD-FMK + z-VAD-FMK CPA CPA Department of Physiology CUMC

27. Propidiumiodide: membraneimpermeable nucleic acid intercalater In cultured rat hippocampal neurons 0.1 mM [Mg2+]o: glutamate-induced excitotoxicity cell death: propidiumiodide (ex: 546 nm; em:605) Department of Physiology CUMC

28. Measurement with potentiomteric probes DiBAC4 for membrane potential DiBAC4(3)(bis-(1,3-dibutylbarbituric acid)trimethine oxonol) : Potential sensitive fluorescent dye with an emission at 510 nm and excitation at 490 nm. changes are typically ~1 % per 1 mV. Department of Physiology CUMC

29. After-hyperpolarization : Membrane potential measurement by DiBAC4(3) Department of Physiology CUMC