RECENT ADVANCES IN FLUORESCENCE EMISSION SPECTROSCOPY

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RECENT ADVANCES IN FLUORESCENCE EMISSION SPECTROSCOPY:

RECENT ADVANCES IN FLUORESCENCE EMISSION SPECTROSCOPY Presented by ROHITH.T M.PHARM 2 nd sem Pharmaceutical Analysis

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CONTENTS INTRODUCTION INSTRUMENTATION TECHNIQUES APPLICATIONS CONCLUSION REFERENCES

INTRODUCTION :

INTRODUCTION Emission spectroscopy Emission spectroscopy is a spectroscopic technique which examines the wavelengths of photons emitted by atoms or molecules during their transition from an excited state to a lower energy state.

Luminescence :

Luminescence Luminescence is the emission of light from any substance, and occurs from electronically excited states. Luminescence is divided into two categories- Photo Luminescence & chemi Luminescence. fluorescence & phosphorescence comes under Photo Luminescence fluorescence emission rates are typically 10 8 s –1 and lifetime is near 10 ns. Phosphorescence emission rates are slow (10 3 to 100 s –1 ), lifetimes are milliseconds to seconds. Fluorescence is much more widely used for chemical analysis than phosphorescence.

Theory of molecular fluorescence :

Theory of molecular fluorescence Molecular fluorescence is measured by exciting the sample at the absorption wavelength, also called the excitation wavelength, and measuring the emission at a longer wavelength called the emission or fluorescence wavelength. For example, the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH) can absorb radiation at 340 nm. The molecule exhibits fluorescence with an emission maximum at 465 nm.

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In a non- flurescent molecule when an electron is excited to the electronic excited state, it return back to the ground state by losing the energy it has acquired through conversion of the excess electronic energy into vibrational energy. The energy emitted is of lower energy than the energy absorbed because the excited electron moves to the lowest energy vibrational state in the excited state in the excited state before returning to the ground state.

Thus fluorescence emission is typically shifted by 50- 150 nm (Stokes shift) to the longer wavelength in comparison to the wavelength of the radiation used to produce excitation. :

Thus fluorescence emission is typically shifted by 50- 150 nm (Stokes shift) to the longer wavelength in comparison to the wavelength of the radiation used to produce excitation. Relaxation processes Once the molecule is excited to E 1 or E 2 several processes can occur that cause the molecule to lose its excess energy. Two of the most important of these mechanisms, nonradiative relaxation and fluorescence emission are illustrated in Figure b and c. The two most important nonradiative relaxation methods that compete with fluorescence are illustrated in Figure b.

INSTRUMENTATION :

INSTRUMENTATION CCD Cameras are also used for detection

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APPLICATIONS Raman Laser Induced Breakdown spectroscopy (LIBS) Luminescence and Fluorescence Near IR Absorption, Reflection and Transmission Raman Luminescence and Fluorescence Near IR Absorption, Reflection and Transmission Frequency Domain / Time Domain FluorescenceTime -resolved imaging & spectroscopy Plasma diagnostics Combustion Planar laser-induced fluorescence (PLIF) Particle imaging velocimetry (PIV) Nanotechnology

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Ideal spectrofluorometer Light source must yield constant photon output at all wavelengths Monochromator must pass photons of all wavelengths with equal efficiency The PMT must detect photons of all wavelengths with equal efficiency

Quenching and photobleaching:

Quenching and photobleaching Photobleaching (fading): permanent loss of fluorescence due to photo-induced chemical modification of molecule Quenching: Competing processes that induce non- radiative relaxation of excited-state electrons to the ground state Quenching as a tool to probe fluorophore environment Requires contact between fluorophore and quencher Typical quenchers: iodide, acrylamide and O 2

TECHNIQUES:

TECHNIQUES Fluorescence Recovery after Photo-bleaching (FRAP) Fluorescence Resonance Energy Transfer (FRET) Fluorescence fluctuation spectroscopy a)Fluorescence correlation spectroscopy b)Fluorescence cross correlation spectroscopy Time-resolved Fluorescence Spectroscopy

Fluorescence Recovery after Photo-bleaching (FRAP):

Fluorescence Recovery after Photo-bleaching (FRAP) Useful technique for studying transport properties within a cell, especially transmembrane protein diffusion Label the molecule with a fluorophore Bleach (destroy) the fluorophore is a well defined area with a high intensity laser Use a weaker beam to examine the recovery of fluorescence as a function of time FRAP can be used to estimate the rate of diffusion, and the fraction of molecules that are mobile/immobile Can also be used to distinguish between active transport and diffusion

Fluorescence Resonance Energy Transfer (FRET):

Fluorescence Resonance Energy Transfer (FRET) Non- radiative transfer of excited state energy from donor molecule to acceptor molecule, i.e., a form of quenching! Donor and acceptor molecules must be in close proximity (<10nm) Molecular dipoles must be somewhat oriented Absorption spectra of acceptor molecule must overlap to some extent with the fluorescence spectra of donor molecule

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FRET is very sensitive to the distance between donor an acceptor and is therefore an extremely useful tool for studying molecular dynamics Efficiency  R -6

Fluorescence correlation spectroscopy:

Fluorescence correlation spectroscopy Method for observing fluorescence from single molecules Relies on a low concentration, a small illuminated volume and a fast detector Using the auto-correlation function simplifies averaging over many measurements Useful for molecular binding and aggregation studies

Fluorescence cross correlation spectroscopy:

Fluorescence cross correlation spectroscopy Fluorescence cross-correlation spectroscopy (FCCS) was introduced by Eigen and Rigler in 1994 and experimentally realized by Schwille in 1997 FCCS provides a highly sensitive measurement of molecular interactions independent of diffusion rate. FCCS utilizes two species which are independently labeled with two differently colored fluorescent probes FCCS is primarily utilized for measurements of bio-molecular interactions both in living cells and in vitro

Time-resolved Fluorescence Spectroscopy:

Time-resolved Fluorescence Spectroscopy Short pulse excitation followed by an interval during which the resulting fluorescence is measured as a function of time Lifetimes can be very sensitive to local environment Possibly linked to the refractive index of the environment Lifetime measurements are robust against variation in intensity, i.e., an absolute measurement

APPLICATIONS:

APPLICATIONS

Diagnostic Application of Fluorescence Spectroscopy in Oncology Field :

Diagnostic Application of Fluorescence Spectroscopy in Oncology Field Cancer is one of the big killers of world population. The majority of cancers are diagnosed at a late stage, making a cure almost impossible. Fluorescence spectroscopy is an emerging diagnostic tool for various medical diseases including premalignant and malignant lesions. Fluorescence spectroscopy is a noninvasive technique and has been applied successfully for the diagnosis of multisystem cancers with high sensitivity and specificity. Fluorescence spectroscopy minimizes the need for repetitive biopsy, which is routine practice for cancer patient follow-up.

Determine compounds at low concentrations :

Determine compounds at low concentrations In Agrochemicals (pesticides, fungicides, insecticides, etc) The potential increase in fluorescence of a benzimidazole -type fungicide ( carbendazim ) due to complexation with cucurbit[6] uril was reported . The fluorescence enhancement of the fungicide carbendazim by cucurbit[6] uril has been observed in solution due to formation of a host–guest inclusion complex.

Analysis of Fumonisins(A Corn based product):

Analysis of Fumonisins (A Corn based product) Fumonisins (FBs) are worldwide distributed and produced by Fusarium verticillioides and Fusarium proliferatum . Although several other fumonisin analogues have been characterized, fumonisin B1 (FB1) remains the most abundant in naturally contaminated Corn-based foods followed by fumonisin B2 (FB2).The problems and risks associated with fumonisin contamination lead to development of new sensitive methods(LC-Fluorescent detector)

Accurate determination of glucose:

Accurate determination of glucose Glucose is considered as a major component of animal and plant carbohydrates in biological systems. Furthermore, blood glucose levels are also an indicator of human health conditions: the abnormal amount of glucose provides significant information of many diseases such as diabetes or hypoglycemia. Fluorophotometry was used widely owing to its operational simplicity and high sensitivity. Recently biomolecule -stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose

SOME MORE APPLICATIONS:

SOME MORE APPLICATIONS Characterization of Tooth decay Analysis of water quality Detection of fluorescent pigments in living corals Selective excitation of tryptophan fluorescence decay in proteins Determination of Vitamin B1. Elucidating and visualising local viscosity Food composition Detection of explosives In-vivo determination of sunscreen UV-A detection factors Fluorescent dyes are extensively used in industry

CONCLUSION:

CONCLUSION Fluorescence spectroscopy is more useful in the field of pharmacy & as well as in other fields. Till today, so many scientists are continuing their researches to strengthen it. More & more further Researches should be conducted for the betterment of fourth coming generations of the society.

REFERENCES:

REFERENCES GURDEEP R .CHATWAL, Instrumental methods of chemical analysis,2.399-2.416. Axelrod, D.L., Koppel, D.E., Schelessinger , J., Elson, E. and Webb, W.W. Mobility measuremtns by analysis of photobleaching recovery kinetics. Biophysics journal, 16: 1055-1069. 1976. Spector , D.L., Goldman, R.D. and Leinward , L.A. Cells, a laboratory manual, Volume 2. Light microscopy and cell structure. Cold spring harbour press. 1997.

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Bacskai , B.J., Skoch , J., Hickey, G.A., Allen, R. and Hyman, B.T. Fluorescence resonance energy transfer determination using multiphoton fluorescence lifetime in microscopy to characterize amyloid -beta plaques. Journal of Biomedical Optics 8 (3) 368-375. 2003 Time‐Resolved Fluorescence Application Note TRFA‐4, www.horbia.com/scientific Dessertation work on Advanced Fluorescence Fluctuation Spectroscopy, Hoeller Matthias,201

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