Two-Photon Excitation Fluorescence Microscopy
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Absolute Fluorescence Quantum Yields Using the FL 6500 Fluorescence Spectrometer
APPLICATION NOTE Fluorescence Spectroscopy Authors: Kathryn Lawson-Wood Steve Upstone Kieran Evans PerkinElmer, Inc. Seer Green, UK Absolute Fluorescence Quantum Yields Using the FL 6500 Introduction Fluorescence quantum yield Fluorescence Spectrometer (ΦF) is a characteristic property of a fluorescent species and is denoted as the ratio of the number of photons emitted through fluorescence, to the number of photons absorbed by the fluorophore (Equation 1). ΦF ultimately relates to the efficiency of the pathways leading to emission of fluorescence (Figure 1), providing the probability of the excited state being deactivated by fluorescence rather than by other competing relaxation processes. The magnitude of ΦF is directly related to the intensity of the observed fluorescence.1 Number of photons emitted through fluorescence ΦF = (1) Number of photons absorbed Fluorescence quantum yield can be measured using two methods: the relative method and the absolute method. In comparison with relative quantum yield measurements, the absolute method is applicable to both solid and solution phase measurements. The ΦF can be determined in a single measurement without the need for a reference standard. It is especially useful for samples which absorb and emit in wavelength regions for which there are no reliable quantum yield standards available.2 The importance of ΦF has been shown in several industries, standard. It is especially useful for samples which absorb and including the research, development and evaluation of audio emit in wavelength regions for which there are no reliable visual equipment, electroluminescent materials (OLED/LED) and quantum yield standards available. This method requires an fluorescent probes for biological assays.3 This application integrating sphere which allows the instrument to collect all of demonstrates the use of the PerkinElmer FL 6500 Fluorescence the photons emitted from the sample. -
Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections
Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Fall 12-1-2017 Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections Fredrick Michael DeArmond Portland State University Let us know how access to this document benefits ouy . Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Physics Commons Recommended Citation DeArmond, Fredrick Michael, "Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections" (2017). Dissertations and Theses. Paper 4036. 10.15760/etd.5920 This Dissertation is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. For more information, please contact [email protected]. Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections by Fredrick Michael DeArmond A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Applied Physics Dissertation Committee: Erik J. Sánchez, Chair Erik Bodegom Ralf Widenhorn Robert Strongin Portland State University 2017 ABSTRACT As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes increased interest in and demand for robust non-linear fluorescent markers, and characterization of the associated tool set. -
Studies on Fluorescence Efficiency and Photodegradation Of
Laser Chemistry, 2002, Vol. 20(2–4), pp. 99–110 Studies on Fluorescence Efficiency and Photodegradation of Rhodamine 6G Doped PMMA Using a Dual Beam Thermal Lens Technique ACHAMMA KURIAN*, NIBU A GEORGE, BINOY PAUL, V. P. N. NAMPOORI and C. P. G. VALLABHAN International School of Photonics, Cochin University of Science & Technology, Cochin 682022, India (Received11September 2002) In this paper we report the use of the dual beam thermal lens technique as a quantitative method to determine absolute fluorescence quantum efficiency and concentration quenching of fluorescence emission from rhodamine 6G doped Poly(methyl methacrylate) (PMMA), prepared with different concentrations of the dye. A comparison of the present data with that reported in the literature indicates that the observed variation of fluorescence quantum yield with respect to the dye concentration follows a similar profile as in the earlier reported observations on rhodamine 6G in solution. The photodegradation of the dye molecules under cw laser excitation is also studied using the present method. Key words: Thermal lens; Fluorescence quantum yield; Photostability; Dye doped polymer 1 INTRODUCTION From the mid 60s, dye lasers have been attractive sources of coherent tunable radiation because of their unique operational flexibility. The salient features of dye lasers are their tunability, with emission from near ultraviolet to the near infrared. High gain, broad spectral bandwidth enabled pulsed and continuous wave operation. Although dyes have been demonstrated to lase in the solid, liquid or gas phases, liquid solutions of dyes in suitable organic * Corresponding author. Catholicate College, Pathanamthitta 689645, India. E-mail: [email protected] 100 A. KURIAN et al. -
Second Harmonic Imaging Microscopy
170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison. -
Measurement of Fluorescence Quantum Yields on ISS Instrumentation Using Vinci Yevgen Povrozin and Ewald Terpetschnig
TECHNICAL NOTE Measurement of Fluorescence Quantum Yields on ISS Instrumentation Using Vinci Yevgen Povrozin and Ewald Terpetschnig Introduction The quantum yield Q can also be described by the relative rates of the radiative and non-radiative pathways, which deactivate the excited state: kr Q [1 ] kkr nr where and correspond to radiative and non-radiative processes, respectively. In this equation, kr knr knr describes the sum of the rate constants for the various processes that compete with the emission process. These processes include photochemical, and dissociative processes including other, less well characterized changes that result in a return to the ground state with simultaneous dissipation of the energy of the excited state into heat. These latter processes are collectively called non-radiative transitions and two types have been clearly recognized: intersystem crossing and internal conversion. Intersystem crossing related to the radiationless spin inversion of a singlet state (S1) in the excited state into a triplet state (T1). The luminescence quantum yield is also given as the ratio of the number of photons emitted to the number of photons absorbed by the sample: photonsem Q [ 2] photonsabs While measurements of the “absolute” quantum yield do require more sophisticated instrumentation [1], it is easier to determine the “relative” quantum yield of a fluorophore by comparing it to a standard with a known quantum yield. Some of the most common standards are listed in table below: Conditions for Quantum Yield [Q.Y.] Q.Y. Excitation Q.Y. Ref. Standards [nm] [%] Measurements Cy3 4 PBS 540 2 Cy5 27 PBS 620 2 Cresyl Violet 53 Methanol 580 3 1 ISS TECHNICAL NOTE Conditions for Quantum Yield [Q.Y.] Q.Y. -
Fv1000 Fluoview
Confocal Laser Scanning Biological Microscope FV1000 FLUOVIEW FLUOVIEW—Always Evolving FLUOVIEW–—From Olympus is Open FLUOVIEW—More Advanced than Ever The Olympus FLUOVIEW FV1000 confocal laser scanning microscope delivers efficient and reliable performance together with the high resolution required for multi-dimensional observation of cell and tissue morphology, and precise molecular localization. The FV1000 incorporates the industry’s first dedicated laser light stimulation scanner to achieve simultaneous targeted laser stimulation and imaging for real-time visualization of rapid cell responses. The FV1000 also measures diffusion coefficients of intracellular molecules, quantifying molecular kinetics. Quite simply, the FLUOVIEW FV1000 represents a new plateau, bringing “imaging to analysis.” Olympus continues to drive forward the development of FLUOVIEW microscopes, using input from researchers to meet their evolving demands and bringing “imaging to analysis.” Quality Performance with Innovative Design FV10i 1 Imaging to Analysis ing up New Worlds From Imaging to Analysis FV1000 Advanced Deeper Imaging with High Resolution FV1000MPE 2 Advanced FLUOVIEW Systems Enhance the Power of Your Research Superb Optical Systems Set the Standard for Accuracy and Sensitivity. Two types of detectors deliver enhanced accuracy and sensitivity, and are paired with a new objective with low chromatic aberration, to deliver even better precision for colocalization analysis. These optical advances boost the overall system capabilities and raise performance to a new level. Imaging, Stimulation and Measurement— Advanced Analytical Methods for Quantification. Now equipped to measure the diffusion coefficients of intracellular molecules, for quantification of the dynamic interactions of molecules inside live cell. FLUOVIEW opens up new worlds of measurement. Evolving Systems Meet the Demands of Your Application. -
Optical Sectioning in Fluorescence Microscopy by Confocal and 2
Optical Sectioning in Fluorescence achieved with this methodology, Calcium sparks are microscopic calcium release Downloaded from events inside living muscle cells and their properties are giving new insight into Microscopy by Confocal and how excitation leads to contraction (Cannell et al., 1995; Lopez-Lopez et al,, 2-Photon Molecular Excitation 1995; Gomez et al., 1997). Although the wide field microscope had been applied to calcium imaging since about 1985, calcium sparks had not been observed Techniques previously. This is probably because the presence of fluorescence from outside https://www.cambridge.org/core M.B. Cannell & C.Soeller the focal plane results in a marked loss of in-plane contrast for wide field St. George's Hospital Medical School, microscopy. (Note also that fluo-3 was used as the calcium indicator in these Cranmer Terrace, London SW17 ORE experiments as it has low fluorescence in the absence of calcium which also improves image contrast.) The calcium spark illustrates the high sensitivity of Confocal Microscopy current confocal optical methods - the calcium spark finally occupies about 10 fl Fluorescence microscopy has proved to be an invaluable tool for 14 4 (10" l) and represents calcium binding to only -10 indicator molecules, Until biomedical science since it is possible to visualise small quantities of labeled recently, the laser scanning confocal microscope has been the only instrument materials (such as intracellular ions and proteins) in both fixed and living that could measure fluorescence with a spatial resolution of about 0.4 x 0.4 x 0.8 cells, However, the conventional wide field fluorescence microscope suffers . -
Photoremovable Protecting Groups Used for the Caging of Biomolecules
1 1 Photoremovable Protecting Groups Used for the Caging of Biomolecules 1.1 2-Nitrobenzyl and 7-Nitroindoline Derivatives John E.T. Corrie 1.1.1 Introduction 1.1.1.1 Preamble and Scope of the Review This chapter covers developments with 2-nitrobenzyl (and substituted variants) and 7-nitroindoline caging groups over the decade from 1993, when the author last reviewed the topic [1]. Other reviews covered parts of the field at a similar date [2, 3], and more recent coverage is also available [4–6]. This chapter is not an exhaustive review of every instance of the subject cages, and its principal focus is on the chemistry of synthesis and photocleavage. Applications of indi- vidual compounds are only briefly discussed, usually when needed to put the work into context. The balance between the two cage types is heavily slanted to- ward the 2-nitrobenzyls, since work with 7-nitroindoline cages dates essentially from 1999 (see Section 1.1.3.2), while the 2-nitrobenzyl type has been in use for 25 years, from the introduction of caged ATP 1 (Scheme 1.1.1) by Kaplan and co-workers [7] in 1978. Scheme 1.1.1 Overall photolysis reaction of NPE-caged ATP 1. Dynamic Studies in Biology. Edited by M. Goeldner, R. Givens Copyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30783-4 2 1 Photoremovable Protecting Groups Used for the Caging of Biomolecules 1.1.1.2 Historical Perspective The pioneering work of Kaplan et al. [7], although preceded by other examples of 2-nitrobenzyl photolysis in synthetic organic chemistry, was the first to apply this to a biological problem, the erythrocytic Na:K ion pump. -
Innovations of Wide-Field Optical-Sectioning
Innovations of wide-field optical-sectioning fluorescence microscopy: toward high-speed volumetric bio-imaging with simplicity Thesis by Jiun-Yann Yu In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2014 (Defended March 25, 2014) ii c 2014 Jiun-Yann Yu All Rights Reserved iii Acknowledgements Firstly, I would like to thank my thesis advisor, Professor Chin-Lin Guo, for all of his kind advice and generous financial support during these five years. I would also like to thank all of the faculties in my thesis committee: Professor Geoffrey A. Blake, Professor Scott E. Fraser, and Professor Changhuei Yang, for their guidance on my way towards becoming a scientist. I would like to specifically thank Professor Blake, and his graduate student, Dr. Daniel B. Holland, for their endless kindness, enthusiasms and encouragements with our collaborations, without which there would be no more than 10 pages left in this thesis. Dr. Thai Truong of Prof. Fraser's group and Marco A. Allodi of Professor Blake's group are also sincerely acknowledged for contributing to this collaboration. All of the members of Professor Guo's group at Caltech are gratefully acknowledged. I would like to thank our former postdoctoral scholar Dr. Yenyu Chen for generously teaching me all the engineering skills I need, and passing to me his pursuit of wide-field optical-sectioning microscopy. I also thank Dr. Mingxing Ouyang for introducing me the basic concepts of cell biology and showing me the basic techniques of cell-biology experiments. I would like to pay my gratefulness to our administrative assistant, Lilian Porter, not only for her help on administrative procedures, but also for her advice and encouragement on my academic career in the future. -
SPARQ-Ed Risk Assessment Sheet : Immunofluorescence
SPARQ-ed Risk Assessment Sheet : Immunofluorescence Hazard Analyse / Evaluate Risk Overall Risk Category Description of Risk Source Current Controls Event Category Consequences Exposure Probability (see explanation on last page) Exposure PPE worn (gloves, closed Prob footwear, and safety Chemical exposure - VR R U O F C Exposure to Chemical Agents : Risk of Unusual : goggles provided). Use body contact, spills Unusual but eye and skin exposure to chemicals Immuno- AC Low Low Low Low Mod Subs of potentially harmful and splash and Other Contact Minor : General Possible: Less likely (such as fixation / permeabilisation fluorescence QP Low Low Low Low Low Mod Chemical chemicals/substances in inhalation (eg. 4% with Chemical or chemicals used would to occur with the buffer containing 4% staining not a UP Low Low Low Low Low Low fume cupboard/safety paraformaldehyde is Substance be irritants only. control measures but paraformaldehyde) throughout the common cabinet. MSDS available. irritant to eyes and not impossible. RP Low Low Low Low Low Low staining procedure. procedure. Well documented skin). C Low Low Low Low Low Low procedures. PI Low Low Low Low Low Low Personal precaution Exposure procedures to be Prob followed in case of VR R U O F C Sharps : Immunoflourescence is injury. PPE worn Sharps injury (cuts or Unusual : AC Low Low Low Low Mod Subs generally performed with sections on (wearing gloves, closed slicing of fingers) Immuno- Conceivable : Sharps slides or with cells on coverslips. shoes, lab coat and from broken Hitting Objects Minor : May require fluorescence injury is likely to QP Low Low Low Low Low Mod Immunofluorescence staining of cells Mechanical safety goggles are coverslips or pointed with Part of first aid for minor cuts. -
Confocal Microscopy
Confocal microscopy Chapter in Handbook of Comprehensive Biophysics ( in press 2011) Elsevier Brad Amos MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH UK e-mail [email protected] Gail McConnell University of Strathclyde , Centre for Biophotonics 161 Cathedral Street , Glasgow G4 0RE UK [email protected] Tony Wilson Dept. of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK. eMail: [email protected] 1 Introduction A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy. Flying-spot Microscopes A confocal microscope is a special type of ‘flying spot’ microscope. Flying spot systems were developed in the 1950s by combining conventional microscopes with electronics from TV and military equipment. -
Imaging with Second-Harmonic Generation Nanoparticles
1 Imaging with Second-Harmonic Generation Nanoparticles Thesis by Chia-Lung Hsieh In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2011 (Defended March 16, 2011) ii © 2011 Chia-Lung Hsieh All Rights Reserved iii Publications contained within this thesis: 1. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging," Opt. Express 17, 2880–2891 (2009). 2. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes," Biomaterials 31, 2272–2277 (2010). 3. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Second harmonic generation from nanocrystals under linearly and circularly polarized excitations," Opt. Express 18, 11917–11932 (2010). 4. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media," Opt. Express 18, 12283–12290 (2010). 5. C. L. Hsieh, Y. Pu, R. Grange, G. Laporte, and D. Psaltis, "Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle," Opt. Express 18, 20723–20731 (2010). iv Acknowledgements During my five-year Ph.D. studies, I have thought a lot about science and life, but I have never thought of the moment of writing the acknowledgements of my thesis. At this moment, after finishing writing six chapters of my thesis, I realize the acknowledgment is probably one of the most difficult parts for me to complete.