Introduction to light microscopy

Size: px

Start display at page:

Download "Introduction to light microscopy"

Merryl Jackson
5 years ago
Views:

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

$Refraction Diffraction$ emitted from

2 Light interacting with matter Absorbtion Refraction Diffraction Scattering Light interacting with matter Light emitted from fluorochromes How is an image formed? Why are there limits in resolution? 2

$Basic concepts of light microscopical imaging - General setup of microscopes Introduction to light - Image formation by diffraction and interference - Resolution limits - Light emission from$

3 Basic concepts of light microscopical imaging - General setup of microscopes Introduction to light - Image formation by diffraction and interference - Resolution limits - Light emission from molecules and fluorescent imaging Methods and techniques in - Summary of microscopical techniques Fundamentals of light 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? 3

image of the object near (or in) the eyepiece Virtual image seen by eye The

4 Fundamental Setup of Light Microscopes F objective F eyepiece Fundamentals of light 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 4

5 F objective F eyepiece Fundamentals of light detector Object Objective Primary image The objective forms a magnified image of the object after the objective The intermediate image is detected with a CCD camera or any other suitable detector 5

$Diffraction and interference Diffraction and interference: the key$ $Diffraction and interference are phenomena of wave optics Light carries$ $a lens Diffraction of light after passing object (grating) Diffraction at$ $wavefront by diffraction upon passage through an aperture A mixture of$

6 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) 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 6

$the theory for image formation in the light microscope Diffracted$ $light from a periodic specimen produces a diffraction pattern of$

7 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 Image formation in the microscope 7

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

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

$waves Diffraction image of a point source of light The image of a self-luminous$ pattern is a central bright spot surrounded by a series of rings The central spot

pattern is a central bright spot surrounded by a series of rings The central spot

8 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 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 ( )) 8

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

9 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 Resolution and aperture angle Concept: Object is approximated with self luminous points Image of each individual point is not influenced by any other points 9

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

10 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 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 10

$61 λ a) Single diffraction pattern b) Two Airy disks with maximum of one overlapping first minimum of the$ $resolved Concept: an image of an extended object consists of a pattern of overlapping diffraction spots$

11 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) Resolution and Rayleigh criterion Resolving power of microscope: 0.61 λ 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). 11

$overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction$ $Note: this theme of diffraction limited spots and their separation in space and time will again be used$ and taken up in superresolution. Theory Reality 0.

and taken up in superresolution. Theory Reality 0.

12 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. 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 12

13 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 the out of focus information is increasing the background and results in low contrast images 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 the out of focus information is increasing the background and results in low contrast images 13

14 Resolution limits 0.61 λ λ These formula are used for the calculation of resolution in widefield. In other techniques like confocal laser scanning, multiphoton, etc other formula are used. 16th / 17th Century 18th Century 19th Century 14

15 20th Century Fluorescence in DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C 15

16 Fluorescence in Advantages: Very high contrast resulting in high sensitivity Tagging of specific entities possible Excitation / emission allows for various variants of techniques Jablonski scheme Fluorescent 16

$Confocal laser scanning Sample is excited by a diffraction$ light from focus is focused at pinhole and reaches detector

focus at pinhole and largely excluded from detector

a widefield microscope Confocal has a very high signal to

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

17 Confocal laser scanning 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 λ Image acquired with a widefield microscope Confocal has a very high signal to noise ratio (prominent in thick samples) Confocal allows well resolved 3D imaging (without any image processing) dz n n NA 2 em 2 2 n 2 PH NA Image acquired with a confocal microscope 17

intense illumination Measure fluorescence intensity over time in the

18 Spinning disk Increase acquisition speed Fluorescence recovery after photobleaching Image sample using widefield 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 18

19 Multiphoton Imaging deep into tissue Multiphoton Imaging in scattering tissue All fluorescent photons provide useful signals. Helmchen and Denk, Nature Methods

20 Multiphoton Deep tissue two-photon Helmchen and Denk, Nature Methods 2005 Light sheet Huisken J, Stainier D Y R Development 2009;136:

Light sheet Development, September 1, 2012vol. 139 no. 17 3242-3247 TIRF Microscopy 1.

21 Light sheet Development, September 1, 2012vol. 139 no TIRF Microscopy 1. Laser excitation light is directed at a tissue sample through a glass slide at a specific, oblique angle (critical angle) 2. Most of the light is reflected at the interface between glass and the tissue sample (total internal reflection) 3. Induction of a evanescent wave parallel to the slide 4. Decay of the evanescent wave over 200 nm

22 TIRF Microscopy The penetration depth of the incident wave is in the range of nm. Animations and detailed explanation see: micro.magnet.fsu.edu TIRF Microscopy Image: micro.magnet.fsu.edu 22

$resolution Beyond the diffraction limit The common feature:$ $the signals can be recorded consecutively beneath the diffraction$

23 Superresolution Structured illumination José María Mateos Super resolution Beyond the diffraction limit The common feature: switching fluorophores on and off sequentially in time so that the signals can be recorded consecutively beneath the diffraction limit 23

Super resolution Enhanced PSF STED Stimulated emission depletion SIM Structured illumination Statistical STORM Stochastic optical reconstruction PALM Photoactivated localization GSD Ground state

24 Super resolution Enhanced PSF STED Stimulated emission depletion SIM Structured illumination Statistical STORM Stochastic optical reconstruction PALM Photoactivated localization GSD Ground state depletion Structured Illumination Microscopy A grid pattern is projected on sample Grid pattern is translated over the specimen Principle 1: only structures emit light if grid lines excite 2: grid pattern in the size of the resolution limit allow superresolution because of interference (moiré fringes with lower frequency) 24

Stimulated emission depletion STED Configuration of microscope: Confocal laser

$edu/tutorials Superresolution Beyond the diffraction limit d = 0.$ 61 λ / NA Confocal Imaging EGFP in living cells has a resolution of approximately

25 Stimulated emission depletion STED Configuration of microscope: Confocal laser scanning microscope (single point not spinning disk) Note and reminder: Stimulated emission is the core principle of a laser! Superresolution 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 25

26 Statistical Microscopy (STORM) stochastic photoswitching of fluorescent proteins where most of the molecules remain dark ->PALM Price and Davidson, Florida State University Statistical Microscopy (STORM) Ground state depletion with stochastic individual molecule return of fluorescent dyes where most of the molecules remain dark ->GSD or GSDIM 26

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

27 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 Imaging single molecules GSD Ground state depletion PALM Photoactivated localization STORM Stochastic optical reconstruction Literatur Thank you Fundamentals of light 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) 27

Introduction to light microscopy

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