Invitation for a walk through microscopy. Sebastian Schuchmann Jörg Rösner

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1 Invitation for a walk through microscopy Sebastian Schuchmann Jörg Rösner joerg.roesner@charite.de

2 Techniques in microscopy Conventional (light) microscopy bright & dark field, phase & interference contrast Fluorescence microscopy light sources, fluorescence detectors, digital image, objectives Single- & two-photon confocal microscopy basic idea & differences, advantages & disadvantages What is the optimal technique (for my question)?

3 ( Conventional (light) microscopy

4 Light microscope: general structure I Illumination Magnification Specimen (with modification

5 Light microscope: general structure II 2f imaginary image f (magnifying glass) object Illumination Magnification specimen ocular objective condensor light source object F 2f F f real image

6 A light microscope is a combination of a slide projector with a magnifying glass Intermediate (real) image (on the projector screen) ocular (magnifying glass) Final (imaginary) image objective (slide projector) light Total magnification = M objective x M ocular

7 Bright and dark field illumination bright field with specimen objective object plane specimen condensor

8 Bright and dark field illumination dark field without specimen dark field with specimen objective object plane specimen condensor - ring diaphragm (usually) - dark field condensor

$high contrast Objects with a sharp rise in refraction index (with modification http://mikroskopie.$

9 Bright and dark field illumination Bright field total illumination of the specimen direct light collection by objective dark/colored object on bright background Dark field part-illumination of the specimen scattered light collected by objective bright object on dark background Objects with high contrast Objects with a sharp rise in refraction index (with modification

Köhler (1866-1948) retina optical elements (eye) objective specimen condensor

10 Microscopy illumination after Köhler (or the mystery condensor adjustment) conjugate planes in the image path A. Köhler ( ) retina optical elements (eye) objective specimen condensor diaphragm intermediate image plane focused specimen field diaphragm ocular objective condensor aperture diaphragm light source

How to do adjust the Köhler illumination 1.Light source on 2.

Choose prefered objective (at least 10x), focus specimen 4.

sharply onto the already focused specimen 5.

11 How to do adjust the Köhler illumination 1.Light source on 2.Open up fully field diaphragm and aperture diaphragm 3. Choose prefered objective (at least 10x), focus specimen 4.Close down field diaphragm; focus the image of the field diaphragm sharply onto the already focused specimen 5.If neccessary center the condensor; then open the field diaphragm until it just disappears from view 6.Take out one of the eyepieces, look down the tube and adjust the aperture diaphragm Diaphragm should be 2/3 to 3/4 open (compromise between resolution & contrast)

12 Amplitude and phase objects influence light waves: Basic principle for phase & interference contrast amplitude object amplitude Reduction in amplitude is equal with a reduction in light intensity (used in bright field microscopy!) phase object amplitude phase Slow down of light wave passing the phase object

13 intermediate image plane Phase contrast scattered light slowed down, unscattered light phase ring objective focussed specimen condensor and light source phase diaphragm

difference Analysator unpolarized light linear polarized light two

14 Differential interference contrast (DIC) Prisma (Nomarski) Specimen (inhomogen phase object) DIC prisma (Nomarski) Polarisator Phase difference Analysator unpolarized light linear polarized light two vertical polarized waves linear polarized light (analysator vertical vs. polarisator)

15 Phase contrast vs. DIC Buccal epithelial cell (monolayer) Kidney tissue (tubule with some cells > 100 µm thick section) Phase contrast DIC (with modification

16 Light microscopy: illumination & contrast techniques Illumination Try to optimise your illumination (condensor adjustment after Köhler) Bright field illumination: standard technique for most specimen Dark field illumination: specific technique for monolayer specimen with distinct differences in the refraction index Contrast Check and improve all contrast techniques available at your microscope Phase contrast: standard technique for low-contrast monolayer specimen DIC: standard technique for low-contrast specimen, in particularily for thick (non-monolayer) preparations

17 Fluorescence microscopy

18 Basic idea of fluorescence microscopy: Stokes shift excitation level 1 E1 E = hν E1 E2 excitation level 2 E2 c = λν λ 1 λ 2 E ~ 1 / λ Stokes shift base level E0

19 The use of the Stokes shift in fluorescence microscopy Detection system (eye, conventional camera, CCD, photo diode, PMT) (filter) emission wavelenght λ em > λ ex light source (arc lamp, laser) dichroic mirror excitation wavelenght λ ex fluorescence object/dye

20 Fluorescence microscopy requires... Fluorochrome (or autofluorescence) Light source

21 Light source Arc lamps Xenon Mercury Laser types UV IR Argon Blue diode Helium-Cadmium Krypton-Argon Green Helium-Neon 543 Yellow Helium-Neon 594 Orange Helium-Neon 612 Red Helium-Neon 633 Red diode Ti:Sapphire

22 Fluorescence microscopy requires... Fluorochrome (or autofluorescence) see Molecular Probes ( Light source Fluorescence detection

23 Fluorescence detector systems... Temporal resolution photo diode PMT CCD conventional photography Spartial resolution

24 Fluorescence detector systems produce digital images Analog Image Digital Sampling Pixel Quantization - observer eye - conventional photography - CCD - PMT (in combination with scan technique) (with modification

25 Fluorescence detector systems produce digital images pixel counts normal contrast low contrast high contrast grey level grey level grey level (with modification

26 Mainly fluorescence detector systems are color-blind! (Colors are based on a [pseudo-]color look-up table)

27 Fluorescence microscopy requires... Fluorochrome (or autofluorescence) see Molecular Probes ( Light source Fluorescence detection (prefers) immersion objectives

$Immersion objectives: Remember the refraction index!$

28 Immersion objectives: Remember the refraction index! α α β β α β = α n 1 n 2 sin α = n 2 n 1 < n 2 sin β n 1 total reflection

29 Immersion objectives: Remember the refraction index! water or oil immersion objective medium (water or oil) specimen Emission Light source Excitation DM refraction index (n) air 1.00 water = 1.37 oil = 1.5 glass = 1.5 Immersion objective with specimen

30 Conventional fluorescence microscopy Advantage low cost uncomplicated handling fast imaging technique Disadvantage no 3-dimentional imaging possible low depth of light penetration bleaching

31 (Schmitz et al., 2001) Confocal microscopy

32 Basic idea of confocal microscopy I Conventional fluorescence microscope Laser scanning microscope full field detection point scan detection specimen full field illumination Arc lamp (Hg, Xe) + excitation filter point scan illumination laser light source

33 Laser: light source for confocal microscopy Laser (Light Amplification by Stimulated Emission of Radiation) = highly precise light source in direction, frequency, phase, polarisation - monochromatic = light has the same wavelength (continuous-wave lasers) - coherent = light is oscilating in the same phase - linear polarized = light is oscilating in the same direction - can be focussed to a very high density power (compared to arc lamps) Different wavelengths require different laser, for example... visible spectrum ultra violet infra red Argon Green Helium-Neon 543 Red Helium-Neon 633

34 Basic idea of confocal microscopy II laser y z x y x point scan illumination (fluorescence excitation) point scan detection (fluorescence emission)

35 Confocal microscope: general structure PMT excitation emission pinhole filter laser source x/y-scanning device and dichroic mirror objective focal plane specimen z y x

36 Confocal microscope: the power of the pinhole PMT PMT pinhole pinhole objective objective specimen focal plane specimen

37 Confocal microscope: excitation profil in z-direction focal plane A(x) ~ 1/I(x)

38 Confocal microscope: depth of light penetration visible light depth of light penetration (µm) UV IR wavelenght (µm)

39 Confocal fluorescence microscopy Advantage improved spartial resolution 3-dimentional scanning Disadvantage more complicated imaging control low depth of light penetration bleaching

40 Two-photon microscopy A B 5 µm 100 ms

41 Basic idea of two-photon microscopy single-photon excitation two-photon excitation Absorbtion hν Emission hν Absorbtion hν* hν* Emission hν E = hν c = λν E ~ 1 / λ E* = 1/2 E E* ~ 1 / 2λ Two photons at the same time and at the same place with doubled wavelenght photons from the infra red spectrum (> 750 nm) high photon density

Light source for two-photon microscopy: Ti/Sa-laser Pump laser: solid-state cw laser, 532 nm, 5 W (Millennia, Spectra Physics) Mode-locked Titan-Sapphire laser

42 Light source for two-photon microscopy: Ti/Sa-laser Pump laser: solid-state cw laser, 532 nm, 5 W (Millennia, Spectra Physics) Mode-locked Titan-Sapphire laser (Tsunami, Spectra Physics) avarage power > 0.7 W at 800 nm pulsewidth < 100 fs nominal repetition rate 80 MHz turning range nm Titan-Sapphire spectra Excitation Emission

43 Two-photon excitation (with modification Piston, 1999) laser pulse focal plane photon non-excited dye molecule 2p-excited dye molecule the required photon density for two-photon excitation can be established only in the focal plan and within a laser puls

44 Single vs. two-photon microscope: general structure emission excitation PMT PMT emission excitation pinhole IR laser x/y-scanning device and dichroic mirror z y x

45 Fluorescence detection using 2-photon excitation descanned detection Non descanned detection (NDD) point scan detection full field detection specimen point scan illumination pulsed Ti:Sa laser point scan illumination pulsed Ti:Sa laser

46 Two-photon microscopy with descanned and NDD-PMT excitation beam x/y-scanning device & dichroic mirror (DM) prisma for spectral analyse DM DM descanned PMT 1 & 2 non-descanned (NDD) PMT 3 & 4 objective specimen condensor DM trans-non-descanned (NDD) PMT 5 (with modification Oertner, 2002)

47 Single vs. two-photon excitation: excitation profile single-photon excitation two-photon excitation focal plane A(x) ~ 1/I(x) A(x) ~ 1/I 2 (x)

48 Two-photon microscope: depth of light penetration visible light depth of ligh penetration (µm) UV IR wavelenght (µm)

49 Single vs. two-photon microscopy: bleaching (3D-FITC-dextran gel; irradiated area ~ 10 x 20 µm) single-photon absorbtion (488 nm; Ar) focal plane y x x z two-photon absorbtion (760 nm; Ti:Sa) focal plane 20 µm 10 µm (with modification Kubitscheck et al., 1996)

50 Two-photon microscope: excitation spectra Simply doubling the excitation wavelenght? Absorbtion hν hν Emission hν Two-photon cross section Calcium green (506/533) Fluo-3 (505/526) Lucifer yellow (428/533) Cascade blue (400/420) Excitation wavelenght (nm) (with modification

51 Two-photon microscopy Advantage optimized z-resolution reduced bleaching higher efficiency (removed pinhole) higher depth of light penetration Disadvantage complicate combination of laser and imaging control cost reduced temporal resolution

52 Limitations of fluorescence microscopy Eternal triangle of compromise (Shotton, 1995) light source & fluorescence dye Intensity and spectral resolution (dynamic range, signal-to-noise-ratio) Spatial resolution fluorescence detection Temporal resolution

53 What is the proper technique for my question? Fluorescence microscopy conventional confocal (single-photon) two-photon spatial resolution depth of penetration bleaching temporal resolution available dyes ++ + (+) cost increasing

54 What is the proper technique for my question? conventional fluorescence imaging - fast and full frame imaging - dual-wavelenght functional imaging (Fura-2, BCECF, etc.) confocal (single-photon) fluorescence imaging - thin preparation (< 100 µm): cell culture (monolayer), fixed preparations - multilabling using different dyes (require of different wavelenght) two-photon fluorescence imaging - thick preparation: acute and cultured brain slice - in vivo imaging with interest on deeper structures

55 Spinning Disk Microscope Advantage -fast scanning (300 frames/s) -low phototoxicity Disadvantage -fixated pinhole -no FRAP source Duke University

56 TIRF Microscope TIRF Total Internal Reflection Fluorescence Advantage -improved axial resolution (50-150nm) -signal to noise ratio Disadvantage -very low depth of light penetration (< 150nm) Nikon sourcenikon

57 STED Microscope STED Stimulated Emission Depletion Advantage -improved lateral resolution (50-70nm) as compared with a fluorescence- or confocal microscope Disadvantage -cost -bleaching Grafik: Max-Planck-Institut für biophysikalische Chemie"

58 PALM Microscope PALM Photoactivated Localization Microscopy Advantage -high lateral resolution ( 20nm) -multi channel recording Disadvantage -cost -higher background fluorescence than STED source Science 2006

59 Notes on confocal resolution Lateral resolution FWHM = 0.4 * λ / NA Axial resolution FWHM = 0.45 * λ / n (1-cosα) NA = n*sinα FWHM: full width half maximum (or spatial resolution) NA: numeral aperture of the chosen objective n: refraction index of the sample medium (for air: n = 1, for immersion oil: n = 1.5) λ: laser wavelength

60 Notes on confocal resolution The improvement in spatial resolution corresponds lateral to 1.4x and axial to 6x compaired with the conventional fluorescence microscopy. You can adjust the spatial resolution in the LCS software using Zoom and Format. As a simple rule you can use: Lateral: resolution/3 = optimal size of the voxel Axial: resolution/3 = optimal choice of the z-scan Please note: the spatial resolution depends on the used wavelength.

Bio 407. Applied microscopy. Introduction into light microscopy. José María Mateos. Center for Microscopy and Image Analysis

Bio 407. Applied microscopy. Introduction into light microscopy. José María Mateos. Center for Microscopy and Image Analysis Center for Microscopy and Image Analysis Bio 407 Applied Introduction into light José María Mateos Fundamentals of light Compound microscope Microscope composed of an objective and an additional lens (eyepiece,