Applications Of Confocal Fluorescence Microscopy In Biological Sciences
Applications of confocal fluorescencemicroscopy in biological sciencesB R BoruahDepartment of PhysicsIIT GuwahatiEmail: brboruah@iitg.ac.in@Physics, IIT GuwahatiPage 1
ContentsIntroduction– Optical resolution– Optical sectioning with a laser scanning confocalmicroscope– Confocal fluorescence imagingStimulated emission depletion (STED) microscopyFluorescence resonance energy transfer (FRET)Fluorescence lifetime imagingTwo photon excitation microscopyConclusion@Physics, IIT GuwahatiPage 2
Reflected lightA simple fraction limitedillumination volumeXBeam splitterZIlluminationlensCollimated beam of wavelength λ is focused by L1 to a diffractionlimited volumeThe illumination volume depends on λ, focal length and diameter ofthe illumination lensA point object is imaged into a diffraction limited volume in the imagespace@Physics, IIT GuwahatiPage 3
Resolution of a microscopeObjectimageZXpoint object and the corresponding imagetwo object points in the lateral direction whoseimages are just resolvedtwo object points are indistinguishable in theimagetwo object points in the axial direction whoseimages are just resolvedResolution: minimum separation between two point objects whoseimages are just resolved– Contributions from diffractions due to the illumination anddetection lensesAxial resolution is worse than lateral resolution@Physics, IIT GuwahatiPage 4
Optical sectioning with a confocalmicroscopedetectordetectorpin holedetection lensSampleplaneLaserbeamFocalpointpin holeSampleplaneLaserbeamIllumination lensConfocal arrangement of focal point and pinhole blocks light from out of focusplanes or points away from the optic axisThe detector receives light mostly from the focal point– Image, free of out of focus blur, of a point object located at the focal point@Physics, IIT GuwahatiPage 5
Optical sectioning with a confocalmicroscopedetectorPCBSWide field imagescanConfocal image(Source :www.olympusfluoview.com)Either the sample holding stage or the illumination spot is scanned– Scanning is controlled by a PCFor each object point at the illumination spot, the detector signal is stored inthe PCResults in an optically sectioned image (image corresponds to a sharplydefined object plane, devoid of out of focus blur) of the sampleMuch better axial and marginally better lateral resolutions than a conventional(wide field) microscopeBest resolution: lateral λ/2, axial λ@Physics, IIT GuwahatiPage 6
A beam scanning confocal setupdetectorpinholeObjectivelensscannerLaserBeam splittertargetScanner unit4f systemX scanmirror@Physics, IIT GuwahatiY scanmirrorPage 7
Confocal fluorescence microscopevibrational nce(λEM)excitationbeamdetectorS1dichroic beam splitter(DBS)fluorescencescanS0Molecules (fluorophores) are excited with a laser beam of wavelength(λEXC), which than undergo a series of spontaneous emissions calledfluorescence at the mean wavelength (λEM)DBS: reflects λEXC and transmits λEMEmission filter : blocks reflected light from the sample at λEXC@Physics, IIT GuwahatiPage 8
Confocal fluorescence imagingThe target molecules are tagged withfluorescent probes or fluorophoresConfocal detection of the fluorescentlight in a beam scanning or stagescanning set upConfocal fluorescence image ofFluorescence image provideshuman T cellsinformation about the physical and(source: PhD thesis, B R Boruah)chemical environment and orientation of the fluorophores andhence of the attached target moleculesBest resolution working in the UV-visible range (lateral 200nm, axial 500 nm)– Not enough for visualising light-matter interaction atnanoscale@Physics, IIT GuwahatiPage 9
Confocal fluorescence imagingHEK293 cells stained withDi-4-ANEPPDHQ, amembrane specific andlipid activated fluorophore,which orients in themembrane,normal to thesurface532nm illumination, 60x1.2NA olympus waterimmersion lensXY(source: PhD thesis, B R Boruah)@Physics, IIT GuwahatiPage 10
Confocal fluorescence imagingZebra fish embryo blood vessels areinjected with fluorescence activequantum dotsHead: 350 µm thick and 71 images,each of 5 µm aparttrunk: 160 µm thick and 80 images,each of 2 µm apart3D animation of zebra fish head and trunkSource: www.helmholtz-muenchen.de@Physics, IIT GuwahatiPage 11
Stimulated emission depletion (STED)vibrational Stimulated emission(λEM)(λSTED)A3A0vibrational relaxationLaser beam (λEXC) excites a molecule to the upper electronic stateAnother laser beam, called STED beam, at (λSTED) shines on the excitedmolecule– Stimulates it to undergo emission at (λSTED)– No emission at (λEM) i.e. No fluorescence from the excited molecule@Physics, IIT GuwahatiPage 12
STED in a confocal fluorescence microscopedetectorλEMSTED beamλSTEDExcitation beamλEXCDBS2:mean emission wavelengthDBS1 : reflects λEXC and transmits wavelengths λEXCDBS2 : reflects λSTED and transmits wavelengths λSTEDDBS1Objective lenssampleBoth excitation and STED beams are pulses following one another,usually derived from the same femto second laserImage is formed by scanning the stage or by scanning the beams@Physics, IIT GuwahatiPage 13
Applications of STED microscopyNanoscale imaging of fluorescent beadsConfocal imageSTED imageXY plane images of 200 nm fluorescent beads(source: PhD thesis, B R Boruah, Imperial College London)@Physics, IIT GuwahatiPage 14
Applications of STED microscopyIn biological scienceConfocal image STED imageReveals nanopattern in the in SNAP-25 protein found in the plasmamembrane of mamalian cells (source: Briefings in functional genomicsand proteomics, Vol 5, No 4, 289-301)@Physics, IIT GuwahatiPage 15
Applications of STED microscopyNanoscale imaging of live cellsConfocal imageSTED imageZLive yeast cellsXLive E-coli bacteria(source: PNAS, 97, 15, 2000, 8206-8210)@Physics, IIT GuwahatiPage 16
Fluorescence resonance energy transferexcitationexcitationDADAUnfolded proteinFolded proteinD : donor fluorescent moleculeA : acceptor fluorescent molecule: fluorescence resonance energy transfer (FRET)Energy transfer from D to A when they are close by– Fluorescence from ANo energy transfer from D to A when they are far apart– No fluorescence from A@Physics, IIT GuwahatiPage 17
Cell protein localizations with confocal FRETIntegrins induce local Rac–effector coupling.Donor (A), uncorrected FRET (B), and corrected FRET (C)Sekar, Periasamy J. Cell Biol. 2008:160:629-633@Physics, IIT GuwahatiPage 18
Fluorescence lifetime eFluorescence (molecule A)τAtimeintensityTime correlatedfluorescencecollectionFluorescence (molecule B)τBA BFluorescencelifetime image (FLIM)Sample plane is excited with short laserpulse (200 ps)Life time (τ) : duration over which thefluorescence decays to 1/e the maximumLifetime is sensitive to local environment:pH, density of oxygen, Ca ion, proximityto other moleculesFLIM– Better contrast than intensity imagingtime– Not effected by scattering@Physics, IIT GuwahatiPage 19
Confocal microscopy with lifetime imagingSource: Photonics group,Imperial College LondonImages of rat’s ear showing two veins, an artery ,and an elasticcartilage. (a) Microscopic image, (b) fluorescence image (c) fastFLIM image, (d) slow FLIM image@Physics, IIT GuwahatiPage 20
Optical trappingmediumLaser beamLensPico newton level forceParticles in a medium (say liquid)Manipulation of trapped micro-beadsA. Jesacher, et al., Optics Express, 2006Laser beam ( 100mW) is focused tightly by a lens into amediumParticle in the medium having contrast in the refractive index willexperience pico newton magnitude force towards the focus pointchanging the direction of the laser beam will change in shift infocal spot along with the trapped particle@Physics, IIT GuwahatiPage 21
Confocal fluorescence microscopy with opticaltrapping(a)(b)RBC cell in (a) isotoinicbuffer (b) hypertonic bufferCells with arrow mark aretrappedConfocal images fromvarious view angles– No change in shape of thetrapped cellK Mohanty, et al., JBO, 2007@Physics, IIT GuwahatiPage 22
Two Photon excitation (TPE)SPEE2νE1TPEfluorescenceνZXTwo photon excitation: E2-E1 2hνExcited molecules in the samplethe time interval between arrivals of the two photons of frequency ν atthe site of the molecule 10-16 secthe molecules sees as if there is a single photon of frequency 2νExcitation probability is proportional to (intensity)2Fluorescence emission is only from a small region near the focus unlikein single photon excitation@Physics, IIT GuwahatiPage 23
Deep tissue imaging using two photonexcitation microscopyIn vivo imaging of brain tissue of an anesthetized rat(up to a depth of 600 µm)M. Oheim et al, Journal of Neuroscience Methods 111 (2001)Excitation wavelength is twice that of single photon excitation– Less scattering (larger the wavelength smaller is the scattering)– Excitation beam enters deep into the sample (upto 1 mm)Less amount of photo damageIn vivo imaging of live tissues@Physics, IIT GuwahatiPage 24
Deep tissue imaging using two photonexcitation microscopyD Kobat, et al., Optics Express, August 2009TPE image of mouse brain shows high contrast blood vessels– Upto a depth of 500 µm when excited with 775 nm– Upto a depth of 1 mm when excited with 1280 nm@Physics, IIT GuwahatiPage 25
Conclusionconfocal fluorescence microscopy is a powerful tool to get ahigh contrast image of a thin slice of the sample in a noninvasive way– Has number of application in biology (and the number isgrowing every day)Confocal fluorescence microscopy using stimulated emissiondepletion provides nanoscale imagingConfocal fluorescence microscopy can be combined with othertechniques such as FRET, FLIM, optical trapping etc. to revealfurther information from the sampleTwo photon excitation instead of single photon excitationprovides high contrast image upto a depth of 1 mm– Useful for imaging in cellular environment@Physics, IIT GuwahatiPage 26
Thank You@Physics, IIT GuwahatiPage 27
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
Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
systems. Examples of real implementations and experimental results will be presented as well. Keywords: confocal microscopy, fluorescence microscopy, laser, beam shaping, flat-top, tophat, homogenizing. 1. INTRODUCTION Lasers are widely used in modern microscopy and make possible realization of various fluorescence techniques and high
FLUORESCENCE MICROSCOPY 177 Overview 177 Applications of Fluorescence Microscopy 178 Physical Basis of Fluorescence 179 . of a microscope under the title “Practical Light Microscopy.” However, the needs of the scientific community for a more comprehensive reference and the furious pace of elec-
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.
\\microscopy-nas1.nri.ucsb.edu Open LASX software –Choose resonant scanner to be on or off . brightfield or fluorescence –Be sure to turn off fluorescence shutter when not observing through eyepiece –Small knob on left side will adjust fluorescence excitation intensity. . mode or manual mode. Manual mode will require some training .
Laser scanning confocal microscopy of BPAE cells stained with DAPI, Alexa 488 phalloidin, Mitotracker Red Sample preparation for fluorescence microscopy
Fritjof Helmchen 1 & Winfried Denk 2 With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional including confocal fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence
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