Lab. 3. Confocal Microscope Imaging Of Single-emitter .
UNIVERSITY OF ROCHESTERTHE INSTITUTE OF OPTICSOPT 453, OPT 253, PHY 434Labs 3-4. SINGLE PHOTON SOURCELab. 3. Confocal microscope imaging ofsingle-emitter fluorescenceLab. 4. Hanbury Brown and Twiss setup.Photon antibunchingInstructor: Dr. Svetlana G. Lukishovasluk@lle.rochester.eduFall 2008
2Summary of these LabsIn these two labs students will learn how to produce single photons obeying the laws of quantummechanics (Lab 3); how to prove that a source of light is a single photon source (Lab4).A single-photon source (SPS) that efficiently produces photonsexhibited antibunching is a pivotal hardware element in photonicquantum information technology. Secure quantum communicationwith single photons will prevent any potential eavesdropper fromintercepting the message without the receiver's noticing. SPS alsoenables quantum computation using linear optical elements andphotodetectors.Students will also get acquainted with a confocal fluorescencemicroscopy of single emitters and photonic bandgap materials. Theywill image the fluorescence of single dye molecules and colloidalsemiconductor quantum dots, will prepare 1-D photonic bandgapchiral liquid crystal samples, and will observe fluorescenceantibunching using a Hanbury Brown and Twiss interferometer.Photograph and schematics of a single photon source setup. Abeamsplitter BS, two single-photon counting avalanche photodiodes APD1and APD2, a time correlated computer card TimeHarp 200 are the mainelements of a Hanbury Brown and Twiss interferometer.
3IMPORTANT SAFETY TIPSSeveral aspects of these Labs are potentially harmful to theexperimenter and to the equipments used. Please review thesewarnings thoroughly before proceeding with this laboratory.LASER SAFETYA diode-pumped solid-state laser is used in this laboratory with 532nm wavelength, 6 ps pulse duration and 76 MHz pulse repetition rate.Laser average output power is near 40 mW, although maximum inputaverage power into microscope is 400 µW. Laser exposure to one’seye can cause permanent damage. One should always be aware ofwhen the laser is operating. In addition, one should always beconscious of the beam path and any reflections which mayoccur in the system. When you are working with laser beamalignment before the spatial filter, use protective goggles.MATERIAL SAFETY When working with powdered dye and with solvents, it is best towork in a chemical hood. Breathing in dye or solvent particles canbe dangerous and should be avoided. It is also necessary to weargloves whenever working with dye and solvents. Contact with skinshould be avoided. If contact does occur, be sure to flush the areawith plenty of water. Always wear gloves when working with liquid crystals. Contactwith skin should be avoided. If contact does occur, wash the areathoroughly with soap and water. Wash your hand after the Lab if you worked with chemicals.
4EQUIPMENT SAFETY(1) Thesingle-photon counting avalanche photodiode modules(APD’s) used in this laboratory are extremely sensitive. Too muchexposure to light can damage them. Whenever the APD’s are inuse, the lights must be turned off. It is also advisable to turn offany extraneous light sources as well (notably computer monitors).Turning on the lights or opening the door while the APD’s are onwill likely ruin the APD’s, which cost about 5,000 a piece. NEVER TURN ON THE ROOM LIGHTS WHILE THE APDs AND EMCCD-CAMERA ARE ON! (1) After turning off the power switch always unplug the APD’s beforeturning on the lights for the long period of time. (2) Also, make sure yourLab View program is turned off before turning on the lights. If APD count rate will exceed 200,000 counts/sec, reduce laserpower or put the screen in front of APD.DON’T SWITCH OFF APD UNDER A HIGH COUNT RATE!!!!(2) The CCD camera used in this lab is internally cooled, and ideallyworks at an internal temperature of -60º C. Never block the venton the camera since doing so can disrupt the coolingmechanism. The temperature inside the camera is displayed atthe bottom left of the screen when the Andor iXon software isopen. If the camera ever reaches a temperature too high tooperate safely, a buzzer will sound from inside the camera. Ifone ever hears this buzzer, it is extremely important to turn off theinternal cooling in the camera and make sure that the internal fanis on and turned to ‘High’. This can be done in the camerasoftware by first click on the ‘Hardware’ drop down menu, andthen choosing ‘Temperature’. Turn the cooling option to off andclick ‘Ok’. Next click on ‘Hardware’ again and choose ‘FanControl’. Make sure that ‘High’ is selected and press ‘Ok’. Makesure to stop any data collection. The camera should then be leftalone to cool off before usage continues.(3) Always follow the directions for turning the laser on and offcarefully. When turning the laser off, it is necessary to press the‘stop’ button on the power supply before turning the key to turn thelaser off. Failing to press the ‘stop’ button before turning thekey on the power supply can cause damage to the laser,which costs about 60,000.
5PREPARATORY QUESTIONS1 What is single photon source?2 What is the difference between laser pulses attenuated to a single-photon level and photons from single photon sources?3 What is fluorescence antibunching?4 How will you produce single photons in this Lab?5 Why do the single emitters emit single photons at a time?6 What is a confocal microscope?7 Make a brief sketch of the main elements of setup for single-emitter fluorescence microscopy.8 What are single-photon counting avalanche photodiode modules?How to work with them without damaging these detectors?9 How will you know if EM-CCD camera is overheated?10 Explain how the image of single-molecule fluorescence is createdin a confocal microscope. Why do you see “the stripes” on someimages?
6References and recommended literature1.H.J. Kimble, M. Dagenais, L. Mandel, Phys. Rev. Lett., 39, 691 (1977).2.T.J. Thom, M.S. Neel, V.W. Donato, G.S. Bergreen, R.E. Davies, M. Beck,Amer. J. Phys., 72, 1210 (2004). See also websitehttp://people.whitman.edu/ beckmk/QM/inter/AAPT 03.pdf3.M. Fox, Quantum Optics: An Introduction, Oxford University Press, 2006.4.Y. Yamamoto., Ch. Santori, J. Vuckovic, D. Fattal, E. Waks, E. Diamanti,5.Progress in Informatics, No 1. 5-37 (2005).6.B. Loonis and M. Orrit, Rep. Progr. Phys., 68, 1129-1179 (2005).7.P. Kumar, P. Kwiat, A. Migdall, A., S.W. Nam, J. Vuckovic, F.N.C. Wong,8.Quantum Information Processing, 3, Nos 1-5, 215-231 (2004).9.New J. Phys., Spec. Issue “Focus on Single Photons on Demand”, 6(2004).10.N. Gisin, G. Ribordy, W. Tittel, H. Zbinden Quantum, Review of ModernPhysics, 74, January 2002, ewMP.pdf11.“Can you keep a secret?”, Nature, df12.http://www.idquantique.com/, logy/information security/quantum cryptography/13.E. Knill, R. Laflamme and G.J. Milburn. Nature 409, 46 (2001).14.L. Novotny and B. Hecht, Principles of Nano-Optics, Cambridge Univ.Press, 2006.15.J.D. Joannopoulos, R.D. Meade, and J.M. Winn, Photonic Crystals,Princeton: Princeton University Press (1995). J.D. Joannopoulos, P.R.Villeneuve, and S. Fan, Nature, 386, 143-149 (1997).16.R.H. Webb, Rep. Prog. Phys., 59, 427 (1996).17.R. Hanbury Brown and R.Q. Twiss, Nature (London), 177, 27 (1956).18.J. Lakowicz, Principles of Fluorescence Spectroscopy. 2nd ed. New York:Kluwer Adademic / Penum Publishers, 1999.271,July2002,
71. INTRODUCTION1.1. Single photon sourcesThe purpose of this laboratory work is to introduce students to singlephoton source (SPS) that efficiently produces photons withantibunching characteristics [1-3]. SPS is a pivotal hardware elementfor quantum communication technology [4–7]. One of thefundamental laws of quantum mechanics, the Heisenberg uncertaintyrelation, tells us that every quantum measurement significantlyinfluences the observed system. Quantum cryptography utilizes thisfeature to guarantee secure communication between Alice(transmitter) and Bob (receiver) (see Figure 1) [8-9]. In contrast toclassical communication, where an eavesdropper (Eve) is able tomeasure the transmitted signals without arousing Alice’s or Bob’sattention, in quantum cryptography eavesdropping can be detectedby Alice or Bob. Quantum communication has a potential largemarket [10], but its practical realization is held back in part because ofthe difficulties in developing robust sources of antibunched photonson demand. In another implementation, a SPS becomes the keyhardware element for quantum computers with linear optical elementsand photodetectors [11].pSinglehotonBobAliceEvaFigure 1 . Schematics of secure communication between Alice and Bobusing single photons.In spite of several solutions for SPSs presented in the literature,significant drawbacks remain. They are the reason for currentquantum communication systems being baud-rate bottlenecked,causing photon numbers from ordinary photon sources to attenuateto the single-photon level ( 0.1 photon per pulse on average). Inaddition to the low efficiency, the drawback of such faint-pulsequantum cryptography is pollution by multiple photons. The pollutionrestriction does not vanish in quantum cryptography based on
8parametric-down-conversion, entangled-photon pairs. A parametricdown-conversion photon source may contain a coherentsuperposition of multiple pairs.An efficient (with an-order-of-magnitude-higher photon number perpulse) and reliable light source that delivers a train of pulsescontaining one, and only one, photon is a very timely challenge. Tomeet this challenge, several issues need to be addressed, fromachieving full control of the quantum properties of the source to easyhandling and integrability of these properties into a practical quantumcomputer and/or communication setup. In addition, in quantuminformation systems it is desirable to deal with single photonssynchronized to an external clock, namely, triggerable single photons(single photons on demand). For practical applications both inquantum cryptography and in quantum computing with qu-bits codedin definite polarization states, well-defined polarization of singlephotons will provide a source efficiency enhancement by a factor oftwo in comparison with producing such polarization from a randompolarization state of photons. Polarization purity is also important forthe coherent properties of the source.The critical issue in producing single photons in another way than bytrivial attenuation of a beam is the very low concentration of photonemitters dispersed in a host, such that within a laser focal spot onlyone emitter becomes excited (Figure 2, left)), emitting only onephoton at a time (because of fluorescence lifetime). In this case allemitted photons will be separated in time (antibunched), see a Figure2, right histogram of the second order correlation function g(2) (t).g2 ( )g(2) (t) is proportional to the measured coincidence count rate(number of the second photons that appeared at a definite timeinterval after a first photon). More details about antibunching and itsmeasurements (Hanbury Brown and Twiss setup) see in Appendix n times (ns)Figure 2. Left: Excitation of a single emitter by a focused laser beam. Right –antibunching histogram showing a dip at zero interphoton time.
9There are various known methods for the production of singlephotons by single-emitter excitation, which are based on a singleatom, a single trapped ion, a single molecule, a single color center indiamond, etc. Tremendous progress has been made in the realizationof SPS’s based on excitonic emission from single heterostructuredsemiconductor quantum dots excited by pulsed laser light. Inheterostructured-quantum-dot SPS’s, microcavities have been usedfor spontaneous emission enhancement in the form of a whisperinggallery-mode resonator (turnstile device), 1-D photonic band-gap,three-dimensional pillar microcavity, and 2-D photonic crystals. Aweakness of heterostructured-quantum-dot SPS’s is that they operateonly at liquid-helium temperatures.To date, three approaches have been suggested for roomtemperature SPS’s: single molecules, colloidal semiconductorquantum dots (nanocrystals), and color centers in diamond. Thecolor-center source suffers from the challenge that it is not easy tocouple out the photons, that the wavelength of this source isrestricted by a specific transition, and random polarization of photons.Both single molecules and colloidal semiconductor nanocrystals(colloidal quantum dots) dissolved in a proper solvent can beembedded in photonic crystals to circumvent the deficiencies thatplague the other system. The primary problems with using fluorescentdyes and colloidal semiconductor nanocrystals in cavities are theemitters’ bleaching and blinking. Using some hosts (e.g., with oxygendepletion) can reduce emitter bleaching. Recently, nonblinkingquantum dots were obtained.In these Labs students will work on a room-temperature SPS basedon single colloidal quantum dot (or dye) fluorescence in photonicbandgap host [12,13] (see Appendix 2). Photonic bandgap hostenhances single-photon emission and provides definite polarization ofsingle photons (in the case of structure asymmetry or chirality).A SPS setup consists of three main elements: (1) Confocalfluorescent microscope [12, 14]; (2) Hanbury Brown and Twiss setup[2, 3, 15]. (3) Sample with single emitters.Confocal fluorescent microscope will be used: to focus laser beam onto a single emitter, to collect and image its fluorescence.
10To prove a single-photon nature of this light source fluorescenceantibunching measurements will be carried out using a HanburyBrown and Twiss setup (see Appendix 1) which is located at the oneof the output ports of a confocal microscope.Students will prepare the samples with single emitters in a photonicbandgap host. Photonic bandgap structure enhances the emissionrate of single photons and can also select a definite polarization ofthem. A planar-aligned cholesteric liquid crystal layer will be used asa 1-D chiral photonic bandgap structure. This structure can produce acircularly polarized light of definite handedness from single emitters.2. BACKGROUND2.1. Fluorescent moleculesFluorescence results from the excitation of electrons into excitedstates [16]. In excited singlet states of fluorescent organic dyes, theexcited electron is paired with a ground-state electron and thereforereturn to the ground-state is spin-allowed. The quick return of theexcited electron to the ground-state orbital results in the emission of aphoton. Excitation of electrons is frequently the result of absorptionfrom a given light source. Fluorescence of an organic molecule isrepresented schematically in the diagram of Figure 3.S2Internal escence210Figure 3. Energy level diagram of an organic molecule. The electronicsinglet states S0, S1 , S2 are complemented by a manifold of vibrationalstates.
11The ground state is denoted S0, with each successive energy levellabeled S1, S2, etc. Notice that each state, including the ground-state,is not one exact energy level. There are vibrational energy levels ateach energy level, denoted 0, 1, 2, etc. Thus each energy level ismore like an energy band than a line. At room temperature, thermalenergy is not adequate to populate excited vibraitonal levels, andabsorption is necessary to populate higher energy levels.A fluorophore is usually excited to a higher vibrational level of S1 orS2, but relaxes to the lowest energy vibrational level before emissionoccurs. This is called internal conversion. Notice also that there is atriplet energy level, denoted T1. It is possible for excited electrons tomove from higher energy levels to this triplet energy level, referred toas intersystem crossing. Because the corresponding electron in theground-state to an electron in the triplet energy level has identicalspin orientation, transmission to the ground-state is forbidden.Remember that this means the transition can still occur, but at sloweremission rates (typically 103 to 10 s-1). The emission of photonsresulting from this transmission from the triplet state is calledphosphorescence.Typical emission rates of dye fluorescence are on the order of 108109 s-1, meaning that the average time between a fluorophore’sexcitation and subsequent return to the ground-state is very small (onthe order of about 10ns). This value is referred to as the lifetime (τ) ofa fluorophore. Keep in mind that a fluorophore’s lifetime is anaverage value which represents the most common lifetime of afluorophore – some lifetimes may be shorter or longer in a givenpopulation.2.2. Confocal Fluorescent MicroscopyToday, confocal microscopy is a technique that applied in manyscientific disciplines, ranging from solid state physics to biology [12,14]. The central idea is to irradiate the sample with focused lightoriginating from a single-mode laser beam and direct the responsefrom the sample into a pinhole.Confocal detection is based on the fact that light not originating fromthe focal area will not be able to pass through the detection pinholeand hence cannot reach the detector. Laterally displaced beams willbe blocked by the detector aperture and beams originating from
12points displaced along the optical axis will not be focused in thedetection plane and therefore will be strongly attenuated by thedetection pinhole (Figure 3).Figure 3. Confocal microscope diagram (from [12]). The detection path ofa scanning confocal optical microscope is shown. Three objects in thesample are depicted. Only the object (circle) on the optical axis lying in theconjugated detection plane in the object space is imaged onto the pinholeand can be detected. The other objects (triangle and squire) are eitherfocused to the side of the pinhole (triangle) or arrive at the pinholeunfocused such that their signals are suppressed.The laser beam spotsize Δx that is achieved at the sample dependson the numerical aperture NA of the objective and the wavelength λused for illumination. It is usually limited by diffraction of the laser lightat the entrance aperture of the objectiveΔx 0.61λ,NAFor NA 1.4 the lateral spotsize (point-spread function width) forgreen light with λ 500 nm is about 220 nm, its length is 750 nm.The lateral resolution of a confocal microscope is not significantlyincreased as compared to a wide-field illumination microscope.However, side lobes are suppressed significantly leading to asignificant increase in the dynamic range of images, meaning thatweak signals may be detected in the proximity of strong ones.The fluorescent light collected by the same objective, has to beseparated from the incoming light using a dichroic mirror.
133. EXPERIMENTAL SETUPSam ple mounted on gla ssmicroscope sl ide and heldon piezo-transl ationsta ge with magnetsAvalanche Photodiodeson adju stable mic rometerstage providing x, y, and zdegrees of freedom532 nm579 nmEyepieceOil able dichroicmirror which ref lectsbut does not tr ansmit532 nmAPDA PDCamer a usedfo r beam ali gnmentNon-polarizing50/50 beam splitterAPD data goes tophoton counting cardin computerNeutral density fil tersRemovableorange glassfilterDiaphragmsLensComputer with dataacqui sition cards andsoftwareMirrorsBlu e filt er for 1064nmfundamental frequencyMic roscope Mi rrorObjective532 nm wavelengthdiode-pumpedSolid state l aserFigure 4. Experimental setup comprising a confocal fluorescentmicroscope and a Hanbury Brown and Twiss setup.One of the important part of the setup (see Figure 4) is a pulsed ( 6ps pulse duration) diode-pumped solid-state laser operating at 76MHz repetition rate. This laser wavelength is 1064 nm, but KTPcrystal placed inside the resonator converts a fundamenta
2 Summary of these Labs In these two labs students will learn how to produce single photons obeying the laws of quantum mechanics (Lab 3); how to prove that a source of light is a single photon source (Lab 4). A single-photon source (SPS) that efficiently produces photons exhibited antibunching is a pivotal hardware element in photonic quantum information technology.
using a confocal microscope to image various sources that exhibit photon antibunching. In order to detect photon antibunching, we imaged 8nm quantum dots in a Hanbury Brown and Twiss Setup. Then, the fluorescence of two different single emitters—quantum dots and color centers in nanodiamonds—were imaged using a confocal microscope.
Introduction - Optical resolution - Optical sectioning with a laser scanning confocal microscope - Confocal fluorescence imaging Stimulated emission depletion (STED) microscopy Fluorescence resonance energy transfer (FRET) Fluorescence lifetime imaging Two photon excitation microscopy Conclusion @Physics, IIT Guwahati Page 3
The Olympus LEXT OLS4000 is a confocal microscope capable of taking high-resolution 3D images. The magnification (Optical and Digital) of this microscope ranges from 108x - 17,280x. It is capable of resolving features 10 nm in size in the z direction (sample height) and 120 nm in the x-y plane. The system is capable of performing a variety of .
microscope focuses a point of light at the in-focus dark blue point by imaging a pinhole aperture placed in front of the light source.[1] Thus, the only regions that are illu-minated are a cone of light above and below the focal (dark blue) point (Fig. 9A). Together the confocal microscope's two pinholes sig-
From routine imaging to live cell research, from super-sensitivity to super-resolution, from multiphoton imaging to CARS - whatever your research, Leica Microsystems has the confocal for your application. SENSITIVITY BY DESIGN The design of a confocal microscope . galvanometric stage insert provides benefits such as real-time xy-scans, beam .
82510 Microscope Lab 2-3 Exercise #1 — Parts of the Microscope Place the microscope on your desk with the oculars (eyepieces) pointing toward you. Plug in the electric cord and turn on the power by pushing the button or turning the switch. In order for you to use the microscope properly, you must know its basic parts. Figure 1
Cells and Systems Section Quiz Unit 2 ANSWER KEY 1. Any microscope that has two or more lenses is a . A. multi-dimensional microscope B. multi-functional microscope C. complex microscope D. compound microscope 2. The part of the microscope the arrow is pointing to is called the A. condenser lens B. diaphragm C. stage D. base 3.
Handling the microscope The microscope accessory is quite heavy. This weight is necessary for stability. There are different ways to lift the microscope accessory. You can choose which way suits you the best. In order to carry the microscope, the microscope is equipped with a h
