Introduction to light microscopy

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1 Center for Microscopy and Image Anaylsis Introduction to light microscopy Basic concepts of imaging with light Urs Ziegler

2 Light interacting with matter Absorbtion Refraction Diffraction Scattering

$Refraction$

3 Light interacting with matter Absorbtion Refraction Diffraction Scattering

4 Light interacting with matter Light emitted from fluorochromes How is an image formed? Why are there limits in resolution?

5 Basic concepts of light microscopical imaging - General setup of microscopes Introduction to light microscopy - Image formation by diffraction and interference - Resolution limits - Light emission from molecules and fluorescent imaging Methods and techniques in microscopy - Summary of microscopical techniques

6 Fundamental Setup of Light Microscopes

7 F objective F eyepiece Fundamentals of light microscopy Object Objective Primary image Eyepiece The objective forms a magnified image of the object near (or in) the eyepiece Virtual image seen by eye The intermediate image is examined by the eyepiece and eye forming a real image on the retina

Magnification: M final = M objecive x M occular What is the maximal

8 Fundamentals of light microscopy Compound microscope: Microscope composed of an objective and an additional lens (eyepiece, occular, tube lens) Magnification: M final = M objecive x M occular What is the maximal magnification? Is there a limit in useful magnification? => Why is there a limit in resolution?

9 Properties of light

$Diffraction and interference Diffraction and interference: the key principles to understand how a microscope forms an image Object Diffraction and interference are$

10 Diffraction and interference Diffraction and interference: the key principles to understand how a microscope forms an image Object Diffraction and interference are phenomena of wave optics Light carries information about the object and creates the image in the focal plane of a lens Diffraction of light after passing object (grating)

11 Diffraction at an aperture or substrate Disturbance of the electric field of a planar wavefront by diffraction upon passage through an aperture A mixture of particles diffracts an incident planar wavefront inversely proportional to the size of particles

Image formation in the microscope Ernst Abbe (1840 1905)

$microscope Diffracted light from a periodic specimen$

12 Image formation in the microscope Ernst Abbe ( ) developed the theory for image formation in the light microscope Diffracted light from a periodic specimen produces a diffraction pattern of the object in the back focal plane

13 Image formation in the microscope

$Image formation in the microscope Diffracted light from a periodic specimen$ $illuminated image plane Diffraction spots in the back focal plane correspond$ to constructive interference of waves differing in 1, 2,. wavelengths.

to constructive interference of waves differing in 1, 2,. wavelengths.

14 Image formation in the microscope Diffracted light from a periodic specimen produces a diffraction pattern of the object in the back focal plane Not interacting incident light is transmitted undeviated and produce the evenly illuminated image plane Diffraction spots in the back focal plane correspond to constructive interference of waves differing in 1, 2,. wavelengths. Image formation in the image plane is by interference of undeviated and deviated waves

$Diffraction patterns in the back focal plane Generation of an image by interference requires collection of two adjacent orders of$

15 Diffraction patterns in the back focal plane Generation of an image by interference requires collection of two adjacent orders of diffracted light! a,b: no image is formed c: image is formed d: image with high definition due to multiple diffracted orders collected

$Diffraction image of a point source of light The image of a self-luminous point in a$ central bright spot surrounded by a series of rings The central spot contains 84% of

central bright spot surrounded by a series of rings The central spot contains 84% of

16 Diffraction image of a point source of light The image of a self-luminous point in a microscope is a pattern created by interference in the image plane The pattern is a central bright spot surrounded by a series of rings The central spot contains 84% of light The image is called: Airy disk (after Sir George Airy ( ))

17 Theory 0.1 µm bead focal plane Spatial resolution in x,y and z Implications: Reality Objects smaller than the resolution limit of the chosen objective will always be 1Airy disk Objects larger than the resolution limit of the chosen objective will always be the size of the object convolved with the optical transfer function 1 µm Crossection Note: the optical transfer function is a function describing how the imaging is occurring in the microscope

18 Resolution and aperture angle Concept: Object is approximated with self luminous points Image of each individual point is not influenced by any other points

19 Resolution and aperture angle The image of a self luminous point is an airy disk The self luminous point generates a spherical wavefront Ideally the path length between object and corresponding conjugate image is preserved Smaller apertures increase the size of airy disks

20 α α Resolution and aperture angle The objective aperture must capture light from a wide angle for maximum resolution (diffracted or emitted light) NA = n sin α α: half angle of the cone of specimen light accepted by the objective n: refractive index of medium between lens and specimen

21 Resolution and aperture angle Aperture of objective determines the resolution, not the magnification! Objective with high aperture (NA 1.25) Objective with low aperture (NA 0.3)

$61 λ NA a) Single diffraction pattern b) Two Airy disks with maximum of one overlapping first minimum of the other objects just resolved c) Two Airy disks with maximum of one overlapping the second$

22 Resolution and Rayleigh criterion Resolving power of microscope: d = 0.61 λ NA a) Single diffraction pattern b) Two Airy disks with maximum of one overlapping first minimum of the other objects just resolved c) Two Airy disks with maximum of one overlapping the second minimum objects well resolved Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks).

$Resolution and size of Airy disk Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the$

23 Resolution and size of Airy disk Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks). Note: this theme of diffraction limited spots and their separation in space and time will again be used and taken up in superresolution microscopy.

24 Theory Reality 0.1 µm bead focal plane Spatial resolution in x,y and z Objects are (always) 3 dimensional The resulting image will also be a 3D image in the image space Again: an image of an extended object consists of a pattern of overlapping diffraction spots 1 µm Crossection

25 In focus Resolution and size of Airy disk Objects are (always) 3 dimensional The resulting image will also be a 3D image in the image space Out of focus Again: an image of an extended object consists of a pattern of overlapping diffraction spots Take home: In widefield microscopy the out of focus information is increasing the background and results in low contrast images

26 Resolution and size of Airy disk Objects are (always) 3 dimensional The resulting image will also be a 3D image in the image space Again: an image of an extended object consists of a pattern of overlapping diffraction spots Take home: In widefield microscopy the out of focus information is increasing the background and results in low contrast images

27 Resolution limits d xy = 0.61 λ NA d z = n λ NA 2 These formula are used for the calculation of resolution in widefield microscopy. In other techniques like confocal laser scanning, multiphoton microscopy, etc other formula are used.

28 Fluorescence in microscopy DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C

29 Fluorescence in microscopy Advantages: Very high contrast resulting in high sensitivity Tagging of specific entities possible Excitation / emission allows for various variants of microscopy techniques Jablonski scheme

30 Fluorescent microscopy

$Confocal laser scanning microscopy Sample is excited by a diffraction limited point of a focused laser spot Emitted fluorescent light from focus is$

31 Confocal laser scanning microscopy Sample is excited by a diffraction limited point of a focused laser spot Emitted fluorescent light from focus is focused at pinhole and reaches detector Emitted fluorescent light from outof-focus is also out-of- focus at pinhole and largely excluded from detector

Comparison of widefield and confocal microscopy d z = n λ

microscopy has a very high signal to noise ratio

well resolved 3D imaging (without any image processing) dz

32 Comparison of widefield and confocal microscopy d z = n λ NA 2 Image acquired with a widefield microscope Confocal microscopy has a very high signal to noise ratio (prominent in thick samples) Confocal microscopy allows well resolved 3D imaging (without any image processing) dz n 0.88 n 2 NA 2 n em 2 2 NA PH 2 Image acquired with a confocal microscope

33 Spinning disk microscopy Increase acquisition speed

Fluorescence recovery after photobleaching Image sample using widefield microscopy Bleach defined region using intense illumination Measure

34 Fluorescence recovery after photobleaching Image sample using widefield microscopy Bleach defined region using intense illumination Measure fluorescence intensity over time in the photobleached region Time for recovery of fluorescence is an indication for: Diffusion Mobility Binding

35 Multiphoton microscopy Imaging deep into tissue

36 Multiphoton microscopy Imaging in scattering tissue All fluorescent photons provide useful signals. Helmchen and Denk, Nature Methods 2005

37 Multiphoton microscopy Deep tissue two-photon microscopy Helmchen and Denk, Nature Methods 2005

38 Selective Plane Illumination Microscopy SPIM 4D imaging Light-sheet-imaging technique Better signal-to-noise ratio Low phototoxicity Wu, Y. et al. Proc. Natl. Acad. Sci. USA 108, (2011).

Total internal reflection fluorescence microscopy TIRF Laser

slide at a specific, oblique angle (critical angle) Most of the

tissue sample (total internal reflection) Induction of an

39 Total internal reflection fluorescence microscopy TIRF Laser excitation light is directed at a tissue sample through a glass slide at a specific, oblique angle (critical angle) Most of the light is reflected at the interface between glass and the tissue sample (total internal reflection) Induction of an evanescent wave parallel to the slide Decay of the evanescent wave over 200 nm

$Superresolution microscopy Beyond the diffraction limit d = 0.$

40 Superresolution microscopy Beyond the diffraction limit d = 0.61 λ / NA Confocal Imaging EGFP in living cells has a resolution of approximately 200 (XY) and 500 nanometers (Z) STED Sample courtesy Martin Engelke, Urs Greber, Institute of Zoology, University of Zurich

41 Super resolution microscopy

42 Super resolution microscopy Enhanced PSF microscopy SSIM Saturated structured illumination microscopy Statistical microscopy STORM Stochastic optical reconstruction microscopy STED Stimulated emission depletion PALM Photoactivated localization microscopy GSD Ground state depletion microscopy

the body of the donut to emit (this is the emission depletion part of STED).

43 Stimulated emission depletion microscopy STED In STED, an initial excitation pulse is focused on a spot. The spot is narrowed by a second, donut-shaped pulse that prompts all excited fluorophores in the body of the donut to emit (this is the emission depletion part of STED). This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence.

44 Saturated structured illumination microscopy SSIM Sample structure Illumination pattern Image (Moiré) Algorithm (calculation of sample structure)

45 Position of a single molecule can be localized to 1 nm accuracy or better if enough photons are collected and there are no other similarly emitting molecules within ~200 nm (Heisenberg 1930, Bobroff 1980). Statistical microscopy Imaging single molecules GSD Ground state depletion microscopy PALM Photoactivated localization microscopy STORM Stochastic optical reconstruction microscopy

Position of a single molecule can be localized to 1 nm accuracy or better if enough photons are collected and there are no other similarly emitting molecules within ~200 nm (Heisenberg 1930, Bobroff

46 Position of a single molecule can be localized to 1 nm accuracy or better if enough photons are collected and there are no other similarly emitting molecules within ~200 nm (Heisenberg 1930, Bobroff 1980). Statistical microscopy Imaging single molecules GSD Ground state depletion microscopy PALM Photoactivated localization microscopy Price and Davidson Florida State University STORM Stochastic optical reconstruction microscopy

47 Literatur Thank you Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley-Liss, 2001 ISBN X (Sehr verständliches Buch mit allem nötigen Grundlagenwissen zu Lichtmikroskopie) Light Microscopy in Biology A practical approach, A. J. Lacey; Oxford University Press, 2004 (Einfache Beschreibung der Lichtmikroskopie mit praktischen Übungen und Anleitungen) Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992 (Detailierte und oft mathematische Beschreibung der Licht und Elektronenmikroskopie. Gutes Referenzwerk) (Ausführliche und vorzügliche Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)

Introduction to light microscopy

Introduction to light microscopy Center for Microscopy and Image Anaylsis Introduction to light Basic concepts of imaging with light Urs Ziegler ziegler@zmb.uzh.ch Microscopy with light 1 Light interacting with matter Absorbtion Refraction