Nanoscale Resolution. Far-field Fluorescence Microscopy

Size: px
Start display at page:

Download "Nanoscale Resolution. Far-field Fluorescence Microscopy"

Transcription

1 Nanoscale Resolution in Far-field Fluorescence Microscopy Part I Basics and Axial Resolution Improvement Stefan W. Hell Max Planck Institute for Biophysical Chemistry Department of NanoBiophotonics Göttingen, Germany

2 Fluorescence microscopy importance in the life sciences: Electron microscopy, etc. 20 % Ratio 80 % Fluorescence Microscopy

3 Fluorescence excitation and emission of labels: S ns λ exc λem S 0 Synth. organic fuorophore Fluoresc. Proteins (GFP)

4 500 nm Δx = λ 2n sin α α 200 nm λ Wavelength Lens λ S 1 λ fl S 0

5 Sheppard, Kompfner, Appl Opt. (1978) Denk, Strickler, Webb, Science (1990) 500 nm Δx = λ 2n sin α α 200 nm λ Wavelength Lens ~I(r/2) ~I(r) 2 2 ~I(r) Multiphoton processes S 1 2λ λ don t work for resolution improvement! S 0 S 0 2λ S 1 λ fl

6 Z- resolution improvement

7 4Pi- Microscopy: nm r r r (,, ϕ ) = (,, ϕ) + (,, ϕ) 4Pi E r z E1 r z E2 r z Coherent illumination and/or fluorescence detection Hell, Europ Patent OS (1990) Hell, et al JOSA A (1992) Hell, et al, Appl. Phys. Lett., 64, 11 (1994)

8 1997 4Pi type A Detector 2 photon 750 nm Piezo Detector r r E1 + E2

9 ~I(r/2)2 ~I(r) 2-Photon Exc. 1-Photon Exc. Z Z Excitation 800 nm Excitation 488 nm X X Detection 510 nm Detection 510 nm Effective PSF Effective PSF

10

11 Z Confocal 4Pi X 1μm Green and red beads, 100 nm diameter

12 Z Microtubules, mouse fibroblast Immunofluor, Oregon Green 2 µm 2 µm X confocal 4Pi

13 How does the linear lobe filter really work?

14 1-Photon PSF 2-Photon Z Z Excitation 488 nm Excitation 800 nm X. X. Detection 510 nm Detection 510 nm = = Consequence of outer part suppresion is Effective PSF PSF separable in (XY) and (Z) Effective PSF h4 Pi ( x, y, z) c( x, y) h( z) Z Z

15 Separability: h4 Pi ( x, y, z) c( x, y) h( z) -> treat the lobe problem separately, in the z-direction Z X = Z =

16 Real space 1.25 algebraic inversion 1 1 = 0 z z 0 z Frequency domain. = 1 kz kz 0 k z

17 Real space Filtering the PSF = z Frequency domain. = k z

18 Z 4Pi confocal = X z 4Pi 1D-FFT 1D-FFT -1. = k z

19 Non-linear image restoration: - Know the full PSF (not just height and position of lobes) - Positivity of image (and PSF) assumed Examples: Richardson-Lucy Algorithm (RL) Maximum-Likelihood Estimation Algorithm

20 Z Confocal 4Pi X Confocal + Restoration (RL) 4Pi + Restoration (RL) 1μm

21 Z Microtubules / Mouse fibroblast Immunofluor. Oregon Green 100 nm 100 nm X Confocal 4Pi + restor. (RL) Confocal 4Pi + restor. (RL)

22 Higher Speed: Multifocal 4Pi - microscopy - 4Pi-foci in parallel: Second / xy-image - Water immersion Microlens Array Egner,Jakobs & Hell, PNAS, 99, 3370 (2002)

23 Application: Mitochondrial network in S. cerevisiae 3D-reconstruction Electron Microscopy B. J. Stevens & J. G. White, 1975

24 GFP 4Pi microscopy Mitochondrial compartment in live 100 nm 3D-resolution Egner,Jakobs,Hell, PNAS, (2002)

25 Number of nodes increases 4-fold on Glucose on Glycerol 5.8 ± ± 9.3 Quantitative live cell 4Pi microscopy

26 Diameter increases by 20 nm on average on Glucose on Glycerol 339 ±4 nm 359 ±5 nm Quantitative live cell 4Pi microscopy Surface 2.8 x larger

27 4Pi Image Golgi in live Vero Cell UDP-Galactosyltransferase- EGFP z x 2.5 mm Egner & Hell, TiCB (2004)

28 4Pi Image Golgi in live Vero Cell UDP- Galactosyl- transferase- EGFP z 2.5 mm x 3D-resolution: ~100 nm

29 Practical issues

30 Practical issues 1) Alignment of lenses Dark exit of Sagnac interferometer Piezo closed loop alignment (< 50 nm precision)

31 Checking out phase changes and aberrations...induced by live yeast cell Δφ n ( r, z) dz Yeast Cell Cover Slip Water immersion Fluorescent layer

32 Checking out object induced phase changes and aberrations... Water Glass Shift in absolute position < 100 nm Yeast Cell 2 μm.

33 Practical issues 1) Alignment of lenses 2) Need establish PSF (phase, lobes): point or planar objects in sample 3) Refractive index changes: index matching (water lenses) Z erbb1-egfp In Chinese Hamster Ovary (CHO) Cells 18 x 33 x 16 µm (x,y,z)

34 4Pi type C (coherent detection + illumination)

35 1997 4Pi type C Detector r r E1 + E2 2 photon 750 nm Piezo Detector r r E1 + E2

36 Z-Response of a layer (Experiment) Confocal 4 Pi (A) Illumination x z nm Exp. 2-Photon / 840 nm NA = 1.35 Oil Theory 525nm z[µm] nm z[µm] Pi (B) Detection nm z[µm] Pi (C) Illumination + Detection nm z[µm] 1.0

37 Z-Response of a layer (Theory) Confocal z Photon / 840 nm NA = 1.35 Oil 520nm x 500 nm z[µm] Pi (A) Illumination nm z[µm] Pi (B) Detection nm z[µm] Pi (C) Illumination + Detection nm z[µm] 1.0

38 Compact 4Pi-microscope Z- resol < 100 nm (live cells /aqueous cond.) 1 & 2 photon

39 How essential is the use of focused (spherical) wavefronts? 4Pi Isn t it just about making a focal interference standing wave pattern?

40 Standing Wave microscopy Non-Confocal! Bailey et al, Nature (1993) Excitation: flat standing wave Detection: epifluorescence No 4Pi element Does not work for z-resolution improvement! PSF x z exc. det. eff. z-profile k z OTF k x k z -profile

41 I 5 M Non-Confocal! Gustafsson et al, 1997 Conceptually fine, but not very robust Excitation: flat standing wave Detection: 4Pi, spherical waves PSF x z k z exc. det. eff. z-response k z -profile OTF k x

42 Comparison of the effective PSFs z conventional x confocal LUT 0 100% 2ph 4Pi (A) standing wave 1ph 4Pi (C) I 5 M 2ph 4Pi (C)

43 Comparison of the effective OTFs k z conventional k x confocal 2ph 4Pi (A) LUT 0 100% standing wave 1ph 4Pi (C) I 5 M 2ph 4Pi (C)

44 PSFs: Overview Widefield z Confocal SWM I 5 M 4Pi C 2 hν 4Pi A 2 hν 4Pi C r Excitation PSF Detection PSF PSF LUT 0 50% 100% 4% 2% z z z z z z z z z z z z z z

45 Widefield Confocal SWM I 5 M 4Pi C 2 hν 4Pi A 2 hν 4Pi C k z OTFs: Overview k r Excitation OTF Detection OTF OTF k z k z k z k z k z k z k z

46 No. Essential physical element: increase in solid angle ( 4π ) α Isn t it just about making a focal interference standing wave pattern?

47 Two opposing lenses give 3-7 fold increased z-resolution nm..provided that they increase the aperture angle of the microscope

48 Nanoscale Resolution in Far-field Fluorescence Microscopy Part II Breaking the diffraction barrier Stefan W. Hell Max Planck Institute for Biophysical Chemistry Department of NanoBiophotonics Göttingen, Germany

49 500 nm Δx = λ 2n sin α α 200 nm λ Wavelength Lens

50 Jena, Germany

51 "Breaking the diffraction resolution limit by stimulated emission" Hell & Wichmann, Opt. Lett., 19, 11, (1994) axial > 500 nm Δx = λ 2n sin α Z a Airy-Disk X lateral > 200 nm

52 Stimulated Emission Depletion (STED) S1 4 3 τ fl 1ns Fluorescence Excitation Stimulated Emission S0 2 1 τ vib p 1ps Fluoresc. Spectrum Exc Stim. Emission ~ 200 ps ~50 ps 2 Pulses λ [nm] Stim. Em. Excit. t

53 STED microscope y x z focal spot 1 µm Detector 50 ps 200 ps Excitation Sync Depletion (STED) Fluorescence time [s] S 1 Absorption S 0 Fluorescence τ 1ns fl Stimulated Emission τ vib p 1ps Fluorescence ON OFF I STED [GW/cm 2 ]

54 STED microscope y x z x 200 nm Detector PhaseMod 2π 0 y 50 ps 200 ps Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

55 STED microscope y x z x 200 nm Detector PhaseMod 2π 0 y 50 ps 200 ps Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

56 Fluorescence I I s I STED [GW/cm 2 ] Focal spot... probed with 1 molecule STED 48 nm 200nm Confocal 254 nm x [nm] λ STED = 770 nm Westphal, Hell, PRL (2005)

57 40 nm beads (Molecular Probes) Confocal STED... just physics! 10 counts/0,3ms counts/0,3ms 89 1µm X Y

58 Biological imaging. (Immunofluorescence)

59 SNAP- 25 / plasma membrane...just physics! Confocal: STED: K. Willig et al, NJP (2006)

60 Syntaxin 1A/ plasma membrane...just physics! c d Confocal STED 9 x 9 µm e f g Sample: T. Lang, J. Sieber, R. Jahn

61 Neuronal communication Presynaptic Synaptic bouton Synaptotagmin I antibody labeled Postsynaptic Confocal STED New biology: Synaptic vesicle protein synaptotagmin I is clustered after exocytosis Y X Willig, Rizzoli, Jahn, Hell, Nature (2006)

62 Nonbiological imaging.

63 65 nm Colloids: Silica beads with fluorescent core 130 nm Confocal STED X Y 0 counts/0,2ms nm 5 counts/0,2ms 80 Sample by A van Blaaderen Willig et al, New J Phys (2006)

64 65 nm Colloids: Silica beads with fluorescent core 130 nm Confocal + Lin Deconv. STED + Lin Deconv maths! X Y 500nm Willig et al, New J Phys (2006)

65 Confocal + Lin. Deconv. STED + Lin. Deconv maths! Field: (6x6) µm; Pitch 120 nm; Line width: 60 nm E-beam lithography on dyed PMMA Westphal et al, J Phys B (2005)

66 STED microscope y x z x 200 nm Detector y PhaseMod 2π 0 Sync 1.0 Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

67 STED microscope y x z x 200 nm Detector y PhaseMod 2π 0 Sync 1.0 Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

68 STED microscope y x z x 200 nm Detector y PhaseMod 2π 0 Sync 1.0 Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

69 STED microscope y x z x 200 nm Detector y PhaseMod 2π 0 Sync 1.0 Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

70 STED microscope y x z x 200 nm Detector y PhaseMod 2π 0 Sync 1.0 Excitation Depletion (STED) Fluorescence I STED [GW/cm 2 ]

71 In far-field field fluorescence microscopy: Diffraction resolution barrier does no longer exist! Molecular scale resolution is possible with visible light and regular lenses. With a rectangular depletion curve, the resolution could be increased to infinity. Hell & Wichmann, Opt. Lett. 19 (1994)

72 STED Microscopy y x z x 200 nm Detector y λ /2 Phaseplate Grating SHG Delay OPO Ti:Sa Depletion (STED) Excitation Westphal & Hell, PRL 94 (2005)

73 I I I I s sat x below Abbe Fluorescence I STED λ STED = 770 nm STED 16 nm λ STED / 46 STED 200nm Confocal x [nm] Westphal & Hell, PRL 94 (2005)

74 Triplet Relaxation STED microscopy..provides <20 nm resolution (x,y) Donnert et al, PNAS (2006)

75 Heavy subunit of neurofilaments in neuroblastoma Donnert et al, PNAS (2006)

76 Axial resolution improvement by STED - 4Pi microscopy

77 S.W. Hell, in Topics Fluoresc Spectr V, Plenum Press (1997)

78 Microtubules / HEK cell Immunofluorescence XY-Overview Confocal STED-4Pi x improved 53 nm Monolayer M. Dyba &SWH, Nature Biotech 21, (2003)

79 How does resolution scale with intensity?

80 Resolution gain with intensity I/ I S nm Resolution gain ~ I I s 10 FWHM r / [nm] 118nm 95nm 60nm 44nm 34nm 27nm 22nm Resolution gain Intensity (STED) / [GW/cm²] 0 I S ~30 MW /cm 2

81 The basic principle...

82 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

83 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

84 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

85 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

86 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

87 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A I s = k BA σ

88 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A Δ x π n λ I I s I k BA s = = σ 1 στ BA

89 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis N B / N A B r / λ I( r) A Δ x π n λ Iστ BA I k BA s = = σ 1 στ BA

90 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis STED microscopy S 1 A Absorption Fluorescence τ ~ 1 ns k AB = σ I( r) S 0 B Is = 10 cm 10 στ 9 s I s 30 MW/cm 2

91 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis There must also be Other Molecular States & transitions to break the diffraction barrier

92 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis Ground State Depletion Microscopy Confocal 40 nm beads Ground State Depletion Micr. t~20ns t ~ 1 ns t~1µs A Singlet B Triplet 500nm S. Bretschneider, C. Eggeling, SWH Phys Rev Lett (2007) Hell & Kroug, Appl. Phys. B 60 (1995) I s ~ kw/cm 2

93 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis Optical Bistability (Synthetic compounds) A fluorescent dark B Δ x π n λ Iστ BA trans - O 3 S cis - - O 3 S SO 3 N O O N O O N N + O N O O O O O N O O + N - SO 3 O O N O O I s ~ W/cm 2 Photoisomerisation Hell, in Topics Fluoresc Spectr V, Plenum Press (1997) Dyba & Hell, Phys. Rev. Lett. 88 (2002) Hell, Jakobs, Kastrup, Appl. Phys. B 77 (2003)

94 BRIGHT fluorescent absorbing trans A k AB = σ I( r) k BA = optical, thermal... B DARK Non-fluorescent Non-absorbing cis Optical Bistability (Fluorescent proteins) A fluor dark B Δ x π n λ Iστ BA I s ~ W/cm 2 Photoswitchable fluorescent proteins Hell, Jakobs, Kastrup, Appl. Phys. B 77 (2003) Hofmann, et al PNAS (2005)

95 Fluor. On Fluor. Off Object 0.6 λfl nsinα Read CCD-camera Out Image

96 Fluor. On Fluor. Off Object 0.6 λfl nsinα S 1 A Absorption Fluorescence S 0 B Read CCD-camera Camera detection possible. Zeros & scanning are required! Out Image MGL Gustafsson, PNAS (2005)

97 Sequential read-out with at least one Intensity Zero + Reversible Saturable/Switchable Optical Linear (Fluorescence) Transition = IZ RESOLFT

98 RESOLFT Switching molecule ensembles (confinement by zeros) STORM / PALM Switching molecules individually! Object 0.6 λfl nsinα A B 0.6 λfl nsinα SW Hell Nature Biotech (2003) Betzig, Hess et al Science Rust, et al Nature Meth Hess et al Biophys J (2006) Read CCD-camera Read CCD-camera Out Image Out Center Gravity

99 RESOLFT Switching molecule ensembles (confinement by zeros) STORM / PALM Switching molecules individually! Object 0.6 λfl nsinα A B 0.6 λfl nsinα SW Hell Nature Biotech (2003) Δx λ 2nsinα N CCD-camera CCD-camera Read Out Image Read Out

100 What is the common enabling physical element behind all these approaches?

101 Establishing a state A contrasted with B in a sub-λ/2 region moved through the object and detected sequentially A A B B B B B B B B B B Δr<<λ/2 BB B B B B B A B B B B

102 Energy state: S 0, S 1, T 1,.. S 1 Fluorescence T 1 (Photophysics) Absorption A B Conformational states: cis, trans, binding S 0 (Photochemistry) A need not be fluorescent, but just detectable (e.g. heat). Long lifetimes of states τ are advantageous Key is the states, not the density of photons!

103 m-photon absorption does not really work for resolution! S 1 2- photons 2 λ 2 λ Fluorescence α ~ I 2 S 0 STED S 1 A 1.0 Absorption Fluorescence Stimulated emission 0.5 ~ exp (-Iτσ) S 0 B I STED [GW/cm 2 ]

104 ... Abbe s equation? 1.0 0Signal 2 Intensity 4 6 Concepts using zeros...expanded. SWH, Nature Biotech 21, 1347 (2003) SWH, Phys. Lett. A 326, 140 (2004) Westphal, SWH, Phys. Rev. Lett. (2005)

105 Acknowledgements References Pictures/Movies SW Hell (Review article) Science, May 25, (2007) Physics: G. Donnert et al PNAS, July 24 (2006) K Willig, J Keller, M Bossi, SWH New J Phys, (2006) V Westphal, SWH Phys Rev Lett, 94 (2005) L Kastrup, H Blom, C Eggeling, SWH Phys Rev Lett, 94 (2005) Applications: K Willig, S Rizzoli, R Jahn, SWH Nature, April 13, (2006) R Kittel, et al Science, May 19, (2006)

Applications of confocal fluorescence microscopy in biological sciences

Applications of confocal fluorescence microscopy in biological sciences Applications of confocal fluorescence microscopy in biological sciences B R Boruah Department of Physics IIT Guwahati Email: brboruah@iitg.ac.in Page 1 Contents Introduction Optical resolution Optical

More information

Optics and Image formation

Optics and Image formation Optics and Image formation Pascal Chartrand chercheur-agrégé Département de Biochimie email: p.chartrand@umontreal.ca The Light Microscope Four centuries of history Vibrant current development One of the

More information

Super Resolution Optical Microscopy. Bo Huang

Super Resolution Optical Microscopy. Bo Huang Super Resolution Optical Microscopy Bo Huang Mar 30, 2012 0.1mm 10µm 1µm 100nm Naked eye: ~ 50 100 μm 1595, Zaccharias and Hans Janssen First microscope, 9x magnification Antony Van Leeuwenhoek (1632 1723),

More information

Measuring the Point Spread Function of a Fluorescence Microscope

Measuring the Point Spread Function of a Fluorescence Microscope Frederick National Laboratory Measuring the Point Spread Function of a Fluorescence Microscope Stephen J Lockett, PhD Principal Scientist, Optical Microscopy and Analysis Laboratory Frederick National

More information

Confocal Microscopy and Atomic Force Microscopy (AFM) A very brief primer...

Confocal Microscopy and Atomic Force Microscopy (AFM) A very brief primer... Confocal Microscopy and Atomic Force Microscopy (AFM) of biofilms A very brief primer... Fundamentals of Confocal Microscopy Based on a conventional fluorescence microscope Fluorescent Microscope Confocal

More information

Fluorescence Microscopy for an NMR- Biosensor Project

Fluorescence Microscopy for an NMR- Biosensor Project Fluorescence Microscopy for an NMR- Biosensor Project Ole Hirsch Physikalisch-Technische Bundesanstalt Medical Optics Abbestr. -1, 10587 Berlin, Germany Overview NMR Sensor Project Dimensions in biological

More information

Contents. Fluorescence Microscopy Confocal Microscopy. Nearfield Scanning Optical Microscopy. Electron Microscopy. Atomic Force Microscopy

Contents. Fluorescence Microscopy Confocal Microscopy. Nearfield Scanning Optical Microscopy. Electron Microscopy. Atomic Force Microscopy Material Chemistry Contents Optical Microscopy Historical development Lens aberrations Transmission / Reflection Microscopy Dark-field Microscopy Differential Interference Contrast Microscopy 4 -Microscopy

More information

12. CONFOCAL MICROSCOPY. Confocal microscopy can render depth-resolved slices through a 3D object by

12. CONFOCAL MICROSCOPY. Confocal microscopy can render depth-resolved slices through a 3D object by 12. CONFOCAL MICROSCOPY Confocal microscopy can render depth-resolved slices through a 3D object by rejecting much of the out of focus light via a pinhole. The image is reconstructed serially, i.e. point

More information

Microscopy Optical Sectioning. Stephen Ross Ph.D. UCSF Principles and Practice Of Light Microscopy May 3 rd, 2010

Microscopy Optical Sectioning. Stephen Ross Ph.D. UCSF Principles and Practice Of Light Microscopy May 3 rd, 2010 Microscopy Optical Sectioning Stephen Ross Ph.D. UCSF Principles and Practice Of Light Microscopy May 3 rd, 2010 Confocal Microscopy In Principle Point Scanning Confocal Spinning Disk Confocal Swept Field

More information

The Basic Concept of Light Microscope. Taiwan Instrument Co, Ltd Eva Lin

The Basic Concept of Light Microscope. Taiwan Instrument Co, Ltd Eva Lin The Basic Concept of Light Microscope Taiwan Instrument Co, Ltd Eva Lin 1 Today's Outline Different types of light microscopes Upright microscope Inverted microscope Stereomicroscope Basic concepts of

More information

Neuro imaging: looking with lasers in the brain

Neuro imaging: looking with lasers in the brain Neuro imaging: looking with lasers in the brain Aim: To image life cells, label free, with cellular resolution in deep tissue Marloes Groot Vrije Universiteit Amsterdam Faculteit Exacte Wetenschappen Natuurkunde

More information

Principles in Light Microscopy. Lecture I. Technical Introduction Lecture II. Applications

Principles in Light Microscopy. Lecture I. Technical Introduction Lecture II. Applications Principles in Light Microscopy Lecture I. Technical Introduction Lecture II. Applications Microscopy Techniques http://www.mcb.ucdavis.edu/faculty-labs/kaplan/ Visible Light Microscopy: http://www.micro.magnet.fsu.edu/index.html

More information

A new high-aperture glycerol immersion objective lens and its

A new high-aperture glycerol immersion objective lens and its Journal of Microscopy, Vol. 206, Pt 2 May 2002, pp. 146 151 Received 11 September 2001; accepted 15 January 2002 A new high-aperture glycerol immersion objective lens and its Blackwell Science Ltd application

More information

Spotlight. The Basic Concept of Light Microscope. The incident angle determines the size that we see

Spotlight. The Basic Concept of Light Microscope. The incident angle determines the size that we see Spotlight 理 The Basic Concept of Light Microscope 行 Basic Introduction of Light Microscopy What is the difference between the light microscope and the magnifier? Two kinds of beam path: reflected light

More information

Overview 5/11/2015 MICROSCOPIC TECHNIQUES 1 LIGHT MICROSCOPY FLUORESCENCE MICROSCOPY. Microscopy, light microscopy

Overview 5/11/2015 MICROSCOPIC TECHNIQUES 1 LIGHT MICROSCOPY FLUORESCENCE MICROSCOPY. Microscopy, light microscopy courtesy: seelastslide UNIVERSITY OF PÉCS MEDICAL SCHOOL www.medschool.pte.hu MICROSCOPIC TECHNIQUES 1 LIGHT MICROSCOPY FLUORESCENCE MICROSCOPY BIOPHYSICS 2. 2015 18th March Dr. Beáta Bugyi Department

More information

Bio 321 Lightmicroscopy Electronmicrosopy Image Processing

Bio 321 Lightmicroscopy Electronmicrosopy Image Processing Bio 321 Lightmicroscopy Electronmicrosopy Image Processing Urs Ziegler Center for Microscopy and Image Analysis Light microscopy (Confocal Laser Scanning Microscopy) Light microscopy (Confocal Laser Scanning

More information

Chapter 1 High-Resolution Optical and Confocal Microscopy

Chapter 1 High-Resolution Optical and Confocal Microscopy Chapter 1 High-Resolution Optical and Confocal Microscopy Olaf Hollricher and Wolfram Ibach Abstract In this chapter, the theory of optical image formation in an optical microscope is described, and the

More information

Fluorescence Workshop UMN Physics June 8-10, Fluorescence Microscopy and Fluorescence Correlation Spectroscopy Joachim Mueller

Fluorescence Workshop UMN Physics June 8-10, Fluorescence Microscopy and Fluorescence Correlation Spectroscopy Joachim Mueller Fluorescence Workshop UMN Physics June 8-10, 2006 Fluorescence Microscopy and Fluorescence Correlation Spectroscopy Joachim Mueller Fluorescence Microscopy Use a microscope as a fluorometer Advantages:

More information

5. Scanning Near-Field Optical Microscopy 5.1. Resolution of conventional optical microscopy

5. Scanning Near-Field Optical Microscopy 5.1. Resolution of conventional optical microscopy 5. Scanning Near-Field Optical Microscopy 5.1. Resolution of conventional optical microscopy Resolution of optical microscope is limited by diffraction. Light going through an aperture makes diffraction

More information

Revision problem. Chapter 18 problem 37 page 612. Suppose you point a pinhole camera at a 15m tall tree that is 75m away.

Revision problem. Chapter 18 problem 37 page 612. Suppose you point a pinhole camera at a 15m tall tree that is 75m away. Revision problem Chapter 18 problem 37 page 612 Suppose you point a pinhole camera at a 15m tall tree that is 75m away. 1 Optical Instruments Thin lens equation Refractive power Cameras The human eye Combining

More information

Introduction to Light Microscopy

Introduction to Light Microscopy Introduction to Light Microscopy Paul S. Maddox " You can observe a lot by watching -Yogi Berra Reference materials Fundamentals of light microscopy and electronic imaging Douglas B. Murphy, 2001, Wiley-Liss

More information

Near-field scanning optical microscopy (SNOM)

Near-field scanning optical microscopy (SNOM) Adviser: dr. Maja Remškar Institut Jožef Stefan January 2010 1 2 3 4 5 6 Fluorescence Raman and surface enhanced Raman 7 Conventional optical microscopy-limited resolution Two broad classes of techniques

More information

Lab instrumentation. Polarized light microscopy

Lab instrumentation. Polarized light microscopy Polarized light microscopy Polarized light microscopy uses plane-polarized light to analyze structures that are birefringent; unisotropic structures that have two different refractive indices, and capable

More information

Scanning Near-Field Optical Microscopy for Measuring Materials Properties at the Nanoscale

Scanning Near-Field Optical Microscopy for Measuring Materials Properties at the Nanoscale Scanning Near-Field Optical Microscopy for Measuring Materials Properties at the Nanoscale Outline Background Research Design Detection of Near-Field Signal Submonolayer Chemical Sensitivity Conclusions

More information

Lecture 20: Scanning Confocal Microscopy (SCM) Rationale for SCM. Principles and major components of SCM. Advantages and major applications of SCM.

Lecture 20: Scanning Confocal Microscopy (SCM) Rationale for SCM. Principles and major components of SCM. Advantages and major applications of SCM. Lecture 20: Scanning Confocal Microscopy (SCM) Rationale for SCM. Principles and major components of SCM. Advantages and major applications of SCM. Some limitations (disadvantages) of NSOM A trade-off

More information

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND

DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND DOING PHYSICS WITH MATLAB COMPUTATIONAL OPTICS RAYLEIGH-SOMMERFELD DIFFRACTION INTEGRAL OF THE FIRST KIND THE THREE-DIMENSIONAL DISTRIBUTION OF THE RADIANT FLUX DENSITY AT THE FOCUS OF A CONVERGENCE BEAM

More information

Physics 1653 Final Exam - Review Questions

Physics 1653 Final Exam - Review Questions Chapter 22 Reflection & Refraction Physics 1653 Final Exam - Review Questions 1. The photon energy for light of wavelength 500 nm is approximately A) 1.77 ev B) 3.10 ev C) 6.20 ev D) 2.48 ev E) 5.46 ev

More information

Chapter 4. Microscopy, Staining, and Classification. Lecture prepared by Mindy Miller-Kittrell North Carolina State University

Chapter 4. Microscopy, Staining, and Classification. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 4 Microscopy, Staining, and Classification 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Microscopy and Staining 2012 Pearson Education Inc.

More information

Holographically corrected telescope for high bandwidth optical communications (as appears in Applied Optics Vol. 38, No. 33, , 20 Nov.

Holographically corrected telescope for high bandwidth optical communications (as appears in Applied Optics Vol. 38, No. 33, , 20 Nov. Holographically corrected telescope for high bandwidth optical communications (as appears in Applied Optics Vol. 38, No. 33, 6833-6835, 20 Nov. 1999) Geoff Andersen and R. J. Knize Laser and Optics Research

More information

Biomedical & X-ray Physics Kjell Carlsson. Light Microscopy. Compendium compiled for course SK2500, Physics of Biomedical Microscopy.

Biomedical & X-ray Physics Kjell Carlsson. Light Microscopy. Compendium compiled for course SK2500, Physics of Biomedical Microscopy. Biomedical & X-ray Physics Kjell Carlsson Light Microscopy Compendium compiled for course SK2500, Physics of Biomedical Microscopy by Kjell Carlsson Applied Physics Dept., KTH, Stockholm, 2007 No part

More information

Super-Resolution Fluorescence Microscopy: Practical Approaches Using 3D-SIM

Super-Resolution Fluorescence Microscopy: Practical Approaches Using 3D-SIM Super-Resolution Fluorescence Microscopy: Practical Approaches Using 3D-SIM Claire Walczak IUB Light Microscopy Imaging Center Super-Resolution Fluorescence Microscopy Using Structured Illumination Applied

More information

Study of the Human Eye Working Principle: An impressive high angular resolution system with simple array detectors

Study of the Human Eye Working Principle: An impressive high angular resolution system with simple array detectors Study of the Human Eye Working Principle: An impressive high angular resolution system with simple array detectors Diego Betancourt and Carlos del Río Antenna Group, Public University of Navarra, Campus

More information

Generating contrast in light microscopy Orion Weiner Principles & Practice of Light Microscopy 2013

Generating contrast in light microscopy Orion Weiner Principles & Practice of Light Microscopy 2013 Generating contrast in light microscopy Orion Weiner Principles & Practice of Light Microscopy 2013 Absorption is not the only way samples interact with light. (polarization, phase shift) Brightfield Phase

More information

Microscopy. MICROSCOPY Light Electron Tunnelling Atomic Force RESOLVE: => INCREASE CONTRAST BIODIVERSITY I BIOL1051 MAJOR FUNCTIONS OF MICROSCOPES

Microscopy. MICROSCOPY Light Electron Tunnelling Atomic Force RESOLVE: => INCREASE CONTRAST BIODIVERSITY I BIOL1051 MAJOR FUNCTIONS OF MICROSCOPES BIODIVERSITY I BIOL1051 Microscopy Professor Marc C. Lavoie marc.lavoie@cavehill.uwi.edu MAJOR FUNCTIONS OF MICROSCOPES MAGNIFY RESOLVE: => INCREASE CONTRAST Microscopy 1. Eyepieces 2. Diopter adjustment

More information

Optical laser beam scanner lens relay system

Optical laser beam scanner lens relay system 1. Introduction Optical laser beam scanner lens relay system Laser beam scanning is used most often by far in confocal microscopes. There are many ways by which a laser beam can be scanned across the back

More information

4Pi-Confocal Imaging in Fixed Biological Specimens

4Pi-Confocal Imaging in Fixed Biological Specimens Biophysical Journal Volume 75 October 1998 1659 1668 1659 4Pi-Confocal Imaging in Fixed Biological Specimens Martin Schrader,* Karsten Bahlmann,* Günter Giese, # and Stefan W. Hell* *High Resolution Optical

More information

Analytical Technologies in Biotechnology Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee

Analytical Technologies in Biotechnology Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Analytical Technologies in Biotechnology Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Module 1 Microscopy Lecture - 2 Basic concepts in microscopy 2 In this

More information

What is Laser Ablation? Mass removal by coupling laser energy to a target material

What is Laser Ablation? Mass removal by coupling laser energy to a target material Laser Ablation Fundamentals & Applications Samuel S. Mao Department of Mechanical Engineering University of California at Berkeley Advanced Energy Technology Department March 1, 25 Laser Ablation What

More information

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question.

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. Exam Name MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) A single slit forms a diffraction pattern, with the first minimum at an angle of 40 from

More information

Experiment #5: Qualitative Absorption Spectroscopy

Experiment #5: Qualitative Absorption Spectroscopy Experiment #5: Qualitative Absorption Spectroscopy One of the most important areas in the field of analytical chemistry is that of spectroscopy. In general terms, spectroscopy deals with the interactions

More information

Z-polarized confocal microscopy

Z-polarized confocal microscopy Journal of Biomedical Optics 6(4), 48 484 (October 21) Publisher s note: This paper originally appeared in Journal of Biomedical Optics 6(3), 273 276 (July 21). Figures 2 and 3 were printed in black and

More information

Info Note UV-VIS Nomenclature and Units

Info Note UV-VIS Nomenclature and Units Info Note 804: Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV/VIS) involves the spectroscopy of photons in the UV-visible region. It uses light in the visible and adjacent

More information

Triple Stage Raman spectrograph/spectrometer Raman system with scanning microscopy attachment: QTY: One

Triple Stage Raman spectrograph/spectrometer Raman system with scanning microscopy attachment: QTY: One Specifications: Triple Stage Raman spectrograph/spectrometer Raman system with scanning microscopy attachment: QTY: One A. Triple Stage Raman spectrograph/spectrometer: 1. Spectral range : UV_Vis_NIR :

More information

Principles in Light Microscopy. Lecture I. Technical Introduction Lecture II. Applications

Principles in Light Microscopy. Lecture I. Technical Introduction Lecture II. Applications Principles in Light Microscopy Lecture I. Technical Introduction Lecture II. Applications Microscopy Techniques http://www.mcb.ucdavis.edu/faculty-labs/kaplan/ Visible Light Microscopy: http://www.micro.magnet.fsu.edu/index.html

More information

Observing a nanomachine at work: Single-molecule imaging or spectroscopy (SMI or SMS)

Observing a nanomachine at work: Single-molecule imaging or spectroscopy (SMI or SMS) Observing a nanomachine at work: Single-molecule imaging or spectroscopy (SMI or SMS) Principle: SMS allows one to observe the function and the motion of nano-objects in realtime in living systems. Usually,

More information

Microscopy: Principles and Advances

Microscopy: Principles and Advances Microscopy: Principles and Advances Chandrashekhar V. Kulkarni University of Central Lancashire, Preston, United kingdom May, 2014 University of Ljubljana Academic Background 2005-2008: PhD-Chemical Biology

More information

Basic principles and mechanisms of NSOM; Different scanning modes and systems of NSOM; General applications and advantages of NSOM.

Basic principles and mechanisms of NSOM; Different scanning modes and systems of NSOM; General applications and advantages of NSOM. Lecture 16: Near-field Scanning Optical Microscopy (NSOM) Background of NSOM; Basic principles and mechanisms of NSOM; Basic components of a NSOM; Different scanning modes and systems of NSOM; General

More information

AP Physics B Ch. 23 and Ch. 24 Geometric Optics and Wave Nature of Light

AP Physics B Ch. 23 and Ch. 24 Geometric Optics and Wave Nature of Light AP Physics B Ch. 23 and Ch. 24 Geometric Optics and Wave Nature of Light Name: Period: Date: MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) Reflection,

More information

Single-Pinhole Confocal Imaging of Sub-Resolution Sparse Objects Using Experimental Point Spread Function and Image Restoration

Single-Pinhole Confocal Imaging of Sub-Resolution Sparse Objects Using Experimental Point Spread Function and Image Restoration MICROSCOPY RESEARCH AND TECHNIQUE 51:464 468 (2000) Single-Pinhole Confocal Imaging of Sub-Resolution Sparse Objects Using Experimental Point Spread Function and Image Restoration A. DIASPRO,* S. ANNUNZIATA,

More information

Pump-probe experiments with ultra-short temporal resolution

Pump-probe experiments with ultra-short temporal resolution Pump-probe experiments with ultra-short temporal resolution PhD candidate: Ferrante Carino Advisor:Tullio Scopigno Università di Roma ƒla Sapienza 22 February 2012 1 Pump-probe experiments: generalities

More information

Laser-induced surface phonons and their excitation of nanostructures

Laser-induced surface phonons and their excitation of nanostructures CHINESE JOURNAL OF PHYSICS VOL. 49, NO. 1 FEBRUARY 2011 Laser-induced surface phonons and their excitation of nanostructures Markus Schmotz, 1, Dominik Gollmer, 1 Florian Habel, 1 Stephen Riedel, 1 and

More information

Imaging Methods: Breath Patterns

Imaging Methods: Breath Patterns Imaging Methods: Breath Patterns Breath / condensation pattern: By cooling a substrate below the condensation temperature H 2 O will condense in different rates on the substrate with the nucleation rate

More information

Electromagnetic Radiation

Electromagnetic Radiation Electromagnetic Radiation Wave - a traveling disturbance, e.g., displacement of water surface (water waves), string (waves on a string), or position of air molecules (sound waves). [ π λ ] h = h sin (

More information

Time resolved photoluminescence studies of blue light-emitting diodes (LED)

Time resolved photoluminescence studies of blue light-emitting diodes (LED) APPLICATION NOTE Time resolved photoluminescence studies of blue light-emitting diodes (LED) WITec GmbH, Lise-Meitner-Str. 6, 89081 Ulm, Germany fon +49 (0) 731 140 700, fax +49 (0) 731 140 70 200 info@witec.de,

More information

Lecture PowerPoints. Chapter 24 Physics: Principles with Applications, 7th edition Giancoli

Lecture PowerPoints. Chapter 24 Physics: Principles with Applications, 7th edition Giancoli Lecture PowerPoints Chapter 24 Physics: Principles with Applications, 7th edition Giancoli This work is protected by United States copyright laws and is provided solely for the use of instructors in teaching

More information

Microscopic Techniques

Microscopic Techniques Microscopic Techniques Outline 1. Optical microscopy Conventional light microscopy, Fluorescence microscopy, confocal/multiphoton microscopy and Stimulated emission depletion microscopy 2. Scanning probe

More information

Exam 3--S12--PHYS April 2012

Exam 3--S12--PHYS April 2012 ame: Exam 3--S12--PHYS102 30 April 2012 Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Which of the following statements is true? a. Newton believed light

More information

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question.

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. Exam Name MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) Two radio antennas are 120 m apart on a north-south line. The two antennas radiate in

More information

CHEM 343: Problem Set #4 (Spectroscopy)

CHEM 343: Problem Set #4 (Spectroscopy) CHEM 343: Problem Set #4 (Spectroscopy) 1) What is the energy, in ev, of UV radiation at 250 nm? What about Visible radiation at 550 nm? hc a) Use the expression E = = hv. Where c is the speed of light,

More information

Christian Huygen Light is a wave, not merely a ray As waves propagate each point on the wavefront produces new wavelets. Wave Nature of Light

Christian Huygen Light is a wave, not merely a ray As waves propagate each point on the wavefront produces new wavelets. Wave Nature of Light Wave Nature of Light Christian Huygen Light is a wave, not merely a ray As waves propagate each point on the wavefront produces new wavelets Chapter 24 Wavelength Changes Wavelength of light changes in

More information

Optical Microscopy Beyond the Diffraction Limit: Imaging Guided and Propagating Fields

Optical Microscopy Beyond the Diffraction Limit: Imaging Guided and Propagating Fields Optical Microscopy Beyond the Diffraction Limit: Imaging Guided and Propagating Fields M. Selim Ünlü, Bennett B. Goldberg, and Stephen B. Ippolito Boston University Department of Electrical and Computer

More information

Arrayoptik für ultraflache Datensichtbrillen

Arrayoptik für ultraflache Datensichtbrillen Arrayoptik für ultraflache Datensichtbrillen Peter Schreiber, Fraunhofer Institut für Angewandte Optik und Feinmechanik IOF, Jena 1. Introduction 2. Conventional near-to-eye (N2E) projection optics - first

More information

Appendix. Cell biologists often need to examine the structure of cells. Principles and Techniques of Microscopy. Optical Principles of Microscopy

Appendix. Cell biologists often need to examine the structure of cells. Principles and Techniques of Microscopy. Optical Principles of Microscopy Appendix Principles and Techniques of Microscopy Cell biologists often need to examine the structure of cells and their components. The microscope is an indispensable tool for this purpose because most

More information

Nederland België / Belgique

Nederland België / Belgique Cronus Brochure 2 Introduction Cronus: The first spectrometer and colorimeter in one The Cronus is world s first spectrocolorimeter combining a high en VIS spectrometer with a high speed XYZ colorimeter.

More information

DIFFRACTION GRATINGS AND SPECTROSCOPY

DIFFRACTION GRATINGS AND SPECTROSCOPY Experiment 8 Name: S.N.: SECTION: PARTNER: DATE: DIFFRACTION GRATINGS AND SPECTROSCOPY Objectives To introduce and calibrate a diffraction grating, and use it to examine several types of spectra. To learn

More information

VECTORIAL ELECTRIC FIELD MONTE CARO SIMULA- TIONS FOR FOCUSED LASER BEAMS (800 nm 2220 nm) IN A BIOLOGICAL SAMPLE

VECTORIAL ELECTRIC FIELD MONTE CARO SIMULA- TIONS FOR FOCUSED LASER BEAMS (800 nm 2220 nm) IN A BIOLOGICAL SAMPLE Progress In Electromagnetics Research, Vol. 142, 667 681, 2013 VECTORIAL ELECTRIC FIELD MONTE CARO SIMULA- TIONS FOR FOCUSED LASER BEAMS (800 nm 2220 nm) IN A BIOLOGICAL SAMPLE Fuhong Cai 1, Jiaxin Yu

More information

Ultrashort pulse laser processing current industrial applications and beyond

Ultrashort pulse laser processing current industrial applications and beyond Ultrashort pulse laser processing current industrial applications and beyond Stefan Nolte Institute of Applied Physics, Abbe Center of Photonics, Friedrich Schiller University Jena, Albert-Einstein-Str.

More information

A Brief History of the Microscope and its Significance in the Advancement of Biology and Medicine

A Brief History of the Microscope and its Significance in the Advancement of Biology and Medicine Chapter 1 A Brief History of the Microscope and its Significance in the Advancement of Biology and Medicine This chapter provides a historical foundation of the field of microscopy and outlines the significant

More information

It has long been a goal to achieve higher spatial resolution in optical imaging and

It has long been a goal to achieve higher spatial resolution in optical imaging and Nano-optical Imaging using Scattering Scanning Near-field Optical Microscopy Fehmi Yasin, Advisor: Dr. Markus Raschke, Post-doc: Dr. Gregory Andreev, Graduate Student: Benjamin Pollard Department of Physics,

More information

CH 35. Interference. A. Interference of light waves, applied in many branches of science.

CH 35. Interference. A. Interference of light waves, applied in many branches of science. CH 35 Interference [SHIVOK SP212] March 17, 2016 I. Optical Interference: A. Interference of light waves, applied in many branches of science. B. The blue of the top surface of a Morpho butterfly wing

More information

A Decade of Super-Resolution Innovation

A Decade of Super-Resolution Innovation www.leica-microsystems.com A Decade of Super-Resolution Innovation Working with passion for the global research community: The TCS SP8 STED 3X marks one decade of innovative super-resolution technology

More information

Guide for the Measurements of Absolute Quantum Yields of Liquid Samples

Guide for the Measurements of Absolute Quantum Yields of Liquid Samples ` T e c h n i c a l N o t e Page 1 Guide for the Measurements of Absolute Quantum Yields of Liquid Samples This Guide is designed to help you to determine the Fluorescence Quantum Yields (QY) of liquid

More information

Laue lens for Nuclear Medicine

Laue lens for Nuclear Medicine Laue lens for Nuclear Medicine PhD in Physics Gianfranco Paternò Ferrara, 6-11-013 Supervisor: prof. Vincenzo Guidi Sensors and Semiconductors Lab, Department of Physics and Earth Science, University of

More information

Application Note AN03 3D Imaging and Analysis

Application Note AN03 3D Imaging and Analysis Application Note AN03 3D Imaging and Analysis Summary This Application Note illustrates the use of Spatial Light Interference Microscopy (SLIM) for tomographic imaging and 3D analysis of live cells. Tomography

More information

PREVIOUS 8 YEARS QUESTIONS (1 mark & 2 marks) 1 mark questions

PREVIOUS 8 YEARS QUESTIONS (1 mark & 2 marks) 1 mark questions 230 PREVIOUS 8 YEARS QUESTIONS (1 mark & 2 marks) 1 mark questions 1. An object is held at the principal focus of a concave lens of focal length f. Where is the image formed? (AISSCE 2008) Ans: That is

More information

Nanoscale Resolution Options for Optical Localization Techniques. C. Boit TU Berlin Chair of Semiconductor Devices

Nanoscale Resolution Options for Optical Localization Techniques. C. Boit TU Berlin Chair of Semiconductor Devices berlin Nanoscale Resolution Options for Optical Localization Techniques C. Boit TU Berlin Chair of Semiconductor Devices EUFANET Workshop on Optical Localization Techniques Toulouse, Jan 26, 2009 Jan 26,

More information

Physics 441/2: Transmission Electron Microscope

Physics 441/2: Transmission Electron Microscope Physics 441/2: Transmission Electron Microscope Introduction In this experiment we will explore the use of transmission electron microscopy (TEM) to take us into the world of ultrasmall structures. This

More information

Organic nanofibers. From fundmental optics to devices

Organic nanofibers. From fundmental optics to devices Organic nanofibers From fundmental optics to devices Horst-Günter Rubahn Fysisk Institut, Syddansk Universitet, Danmark Topographic background H.C.Andersen Egeskov Goal New devices on the basis of a new

More information

Magneto-optical microscopy (incl. time-resolved)

Magneto-optical microscopy (incl. time-resolved) Magneto-optical microscopy (incl. time-resolved) J. McCord (abstract in cooperation with R. Schäfer) IFW Dresden - Institute for Metallic Materials Helmholtzstr. 20 01169 Dresden j.mccord@ifw-dresden.de

More information

Image Acquisition. Fluorescent Microscope. Imaging Device. Four Types of Images. Benefits of Electronic Detectors. The Imaging Triangle 5.12.

Image Acquisition. Fluorescent Microscope. Imaging Device. Four Types of Images. Benefits of Electronic Detectors. The Imaging Triangle 5.12. Fluorescent Microscope Image Acquisition Lightsource Optics Detector Fluorescence Microscopy and Quantitative Imaging Image acquired can not be better than image generated by microscope (but it can be

More information

BA410. Microscope. Clinical&Lab Platform

BA410. Microscope. Clinical&Lab Platform BA410 Microscope Clinical&Lab Platform BA410 Clinical&Lab Microscope Platform Motic s premier BA410 is the flagship model of its BA Upright Compound Series, and exhibits Motic s advancing optical development

More information

Microlenses immersed in nematic liquid crystal with electrically. controllable focal length

Microlenses immersed in nematic liquid crystal with electrically. controllable focal length Microlenses immersed in nematic liquid crystal with electrically controllable focal length L.G.Commander, S.E. Day, C.H. Chia and D.R.Selviah Dept of Electronic and Electrical Engineering, University College

More information

CREOL, College of Optics & Photonics, University of Central Florida

CREOL, College of Optics & Photonics, University of Central Florida OSE6650 - Optical Properties of Nanostructured Materials Optical Properties of Nanostructured Materials Fall 2013 Class 3 slide 1 Challenge: excite and detect the near field Thus far: Nanostructured materials

More information

20.309: Biological Instrumentation and Measurement. Heejin Choi Rumi Chunara Yuri Matsumoto

20.309: Biological Instrumentation and Measurement. Heejin Choi Rumi Chunara Yuri Matsumoto 20.309: Biological Instrumentation and Measurement Instructors: Laboratory Instructor: Teaching Assistants: Scott Manalis and Peter So Steve Wasserman Jaewon Cha Heejin Choi Rumi Chunara Yuri Matsumoto

More information

Raman spectroscopy Lecture

Raman spectroscopy Lecture Raman spectroscopy Lecture Licentiate course in measurement science and technology Spring 2008 10.04.2008 Antti Kivioja Contents - Introduction - What is Raman spectroscopy? - The theory of Raman spectroscopy

More information

Diffraction of a Circular Aperture

Diffraction of a Circular Aperture Diffraction of a Circular Aperture Diffraction can be understood by considering the wave nature of light. Huygen's principle, illustrated in the image below, states that each point on a propagating wavefront

More information

Two-photon FCS Tutorial. Berland Lab Department of Physics Emory University

Two-photon FCS Tutorial. Berland Lab Department of Physics Emory University Two-photon FCS Tutorial Berland Lab Department of Physics Emory University What is FCS? FCS : Fluorescence Correlation Spectroscopy FCS is a technique for acquiring dynamical information from spontaneous

More information

CONFOCAL LASER SCANNING MICROSCOPY TUTORIAL

CONFOCAL LASER SCANNING MICROSCOPY TUTORIAL CONFOCAL LASER SCANNING MICROSCOPY TUTORIAL Robert Bagnell 2006 This tutorial covers the following CLSM topics: 1) What is the optical principal behind CLSM? 2) What is the spatial resolution in X, Y,

More information

When selecting a sensor for an application the following parameters should be considered:

When selecting a sensor for an application the following parameters should be considered: Memo Title: CCD Sensor Selection Guide Memo Number: 0007 Revision No. / Date: 1.0 Date: 8-Sep-2009 The CCDsp Scientific camera is available for a variety of different sensors that cover a broad spectrum

More information

Immersion Oil and the Microscope FAQs

Immersion Oil and the Microscope FAQs Immersion Oil and the Microscope FAQs Aren t all immersion oils for microscope objectives basically the same? No, there actually are many different types of oils with different refractive indices, viscosities,

More information

Amplification Atomic (or molecular, or semiconductor) system has energy levels Some higher energy states are stable for a short time (ps to ms)

Amplification Atomic (or molecular, or semiconductor) system has energy levels Some higher energy states are stable for a short time (ps to ms) Part 5: Lasers Amplification Atomic (or molecular, or semiconductor) system has energy levels Some higher energy states are stable for a short time (ps to ms) Incident photon can trigger emission of an

More information

Ch. 27. Interference and the Wave Nature of Light

Ch. 27. Interference and the Wave Nature of Light Ch. 27. Interference and the Wave Nature of Light Up to now, we have been studying geometrical ti optics, where the wavelength of the light is much smaller than the size of our mirrors and lenses and the

More information

Ch 24 Wave Optics. concept questions #8, 11 problems #1, 3, 9, 15, 19, 31, 45, 48, 53

Ch 24 Wave Optics. concept questions #8, 11 problems #1, 3, 9, 15, 19, 31, 45, 48, 53 Ch 24 Wave Optics concept questions #8, 11 problems #1, 3, 9, 15, 19, 31, 45, 48, 53 Light is a wave so interference can occur. Interference effects for light are not easy to observe because of the short

More information

Biophotonics. Basic Microscopy. NPTEL Biophotonics 1

Biophotonics. Basic Microscopy. NPTEL Biophotonics 1 Biophotonics Basic Microscopy NPTEL Biophotonics 1 Overview In this lecture you will learn Elements of a basic microscope Some imaging techniques Keywords: optical microscopy, microscope construction,

More information

TopHat StableTop Beamshaper

TopHat StableTop Beamshaper TopHat StableTop Beamshaper The top-hat beam shaper is a diffractive optical element (DOE) used to transform a near-gaussian incident laser beam into a uniform-intensity spot of either round, rectangular,

More information

Physics 111 Fall 2007 Wave Optics Solutions

Physics 111 Fall 2007 Wave Optics Solutions Physics 111 Fall 2007 Wave Optics Solutions 1. The pupil of a cat s eye narrows to a vertical slit of width 0.500 mm in daylight. What is the angular resolution for horizontally separated mice? Assume

More information

Taking the Confusion out of Confocal Microscopy

Taking the Confusion out of Confocal Microscopy KEYWORDS: confocal microscopy, fluorescence imaging, three dimensional Special section on techniques: Taking the Confusion out of Confocal Microscopy Nana Rezai Pathology, University of British Columbia

More information

Time out states and transitions

Time out states and transitions Time out states and transitions Spectroscopy transitions between energy states of a molecule excited by absorption or emission of a photon hn = DE = E i - E f Energy levels due to interactions between

More information

Fluorescence spectroscopy

Fluorescence spectroscopy Spectral characteristics A Jablonski diagram represents absorption and emission in a fluorescent molecule A typical molecule absorbs from the ground state (S 0 ) into the vibrational manifold above the

More information