1. Fluorescence microscopy I Basic concepts of optical microscopy Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING

2. Further reading: Introduction to Confocal Microscopy and Image Analysis, J. P. Robinson, http://tinyurl.com/2dr5p Molecular Expressions Microscopy Primer http://micro.magnet.fsu.edu/primer/index.html Nikon Microscopy Tutorials, http://www.microscopyu.com/ Zeiss Microscopy Tutorials, http://zeiss-campus.magnet.fsu.edu/index.html Olympus Microscopy Tutorials, http://www.olympusmicro.com /, http://www.olympusfluoview.com/index.html/ Stowers Institute Tutorials (especially FCS) http://research.stowers- institute.org/microscopy/external/Technology/index.htm

3. Sources of image contrast: Absorption (bright field – the “basic” optical microscopy) Refractive index (refraction, scattering, phase shift) Emission (fluorescence) Raman scattering Others (birefringence, reflection, …) Why do we see the objects? Because they differ in optical properties from the background:

4. Bright field microscopy: light form the condenser passes through the sample, where it is attenuated by absorbing objects

5. Bright field microscopy: ocular light objective light form the condenser passes through the sample, where it is attenuated by absorbing objects Magnification = M(objective) x M(eyepiece) the image formed by the objective in its back focal plane (the intermediate image plane) contains all information accessible by the microscope. Further magnification of the image by eyepiece or lenses of a camera only change it size for easier observation or to fit to the chip of the camera, but do not add any information. We will forget about the eyepiece and magnification. The objective and the resolution and contrast it can achieve are essential

6. Köhler illumination – conjugated planes: A. Köhler (1866-1948) optimal adjustment of the illumination pathway uses the concept of two sets of conjugated planes (planes in which the beam is simultaneously focused) to ensure even illumination of the sample

7. Objectives – infinity system: Inserted optical components (filters, polarizers, …) do not disturb the optical path tube lens

8. Objectives – aberrations and corrections: Chromatic aberration is corrected by combination of lenses of different refractive index (Achromat – 2 different wavelength focused to 1 point, Apochromat – 3 different wavelength focused to 1 point Flat-Field correction ensures planarity of the image – important for its projection on a chip of a camera

9. Objectives – numerical aperture: NA = n sin  TR = 41 ° Dry objective Immersion objective the width of the acceptance cone of the objective determines how much light contributes to the image formation and it is important for the resolution and contrast of the image Why refractive index n??? Refraction occurring of the interface of glass (cover glass of the sample) and air Immersion liquid reduces the refractive index mismatch

10. Objectives – immersion liquids: immersion oils – chosen to match closely the refractive index of glass n G = 1.52 oilvs.water water – n W = 1.33, worse match, however, biological samples consist mainly of water and water immersion is better for imaging thick biological samples objectives have corrections for aberrations introduced by the cover glass of given thickness and refractive index.

11. Sources of image contrast: Bright field microscopy is based on absorption of light in the sample. Most biological objects, however, absorb only weakly in the visible spectrum. This lead to: Development of specific staining (nowadays almost entirely replaced by fluorescent labeling) Development of UV microscopy (Köhler) facing technical difficulties due to absorption of UV light by glass Use of difference in refractive index between the object and medium manifested by:  refraction (scattering) of light  introduction of phase shift to the passing light

12. Dark field microscopy: Objects with a sharp rise in refraction index part-illumination of the specimen scattered light collected by objective bright object on dark background

13. Phase contrast microscopy: annular diaphragm image plane positive phase contrast objective back focal plane & phase plate specimen (phase object) condenser front focal plane  condenser aperture  pinhole positive phase contrast: object of higher optical path appears darker uncertainty in image interpretation arises when objects induce larger phase shift than /2 or when absorption appears simultaneously to phase shift Frits Zernike (1888-1966)

14. the lateral profile of the object optical thickness object-induced phase shift a prism-induced phase differential between the two perpendicularly polarised wavefronts individual phase profiles in the polarised components of the doubled image local phase differences in the overlapping images revealed by the analyser brightness profile in the differential image WP C - compensator (eyepiece) analyser (- 45  ) doubled image Wollaston prisms WP O and WP C WP O - beamsplitter objective specimen condenser iris diaphragm polariser (+45  ) A’ B’ A’’ B’’ 00 A 00 A’’ A’ Differential interference contrast: Objects appear as if illuminated from one side

15. Phase contrast vs. DIC: Kidney tissue (tubule with some cells > 100 µm thick section) Phase contrast Buccal epithelial cell (monolayer) DIC (with modification http://mikroskopie.de) Images suffer from a halo of bright light surrounding some objects – caused by a fraction of diffracted light which has passed the phase ring Can resolve differences in thickness down to about 2 nm Small gradients of thickness give little contrast

16. Fluorescence Microscopy: Possibility of molecule-specific labeling – chemical sensitivity Example: Cytoskeleton (tubulin antibody-Alexa647) Mitochondria (streptavidin-Alexa488) Nucleus (Hoechst-DNA intercalator) High sensitivity – single molecule observation possible Fluorescence is sensitive to environment – provides information on polarity, pH, …

17. Fluorescence microscope: Epi-Fluorescence setup: excitation light passes through the same objective which collects the fluorescence objective sample camera sets of filters and dichroics are available for every common fluorophore

18. Fluorescence microscope: Typically the inverted setup – objective below the sample camera Many cell strands tend to adhere to the bottom of the chamber Sample chamber can be open – we can add something during the measurement

19. Photobleaching in fluorescence microscopy: source of artefacts and irreproducibility, low excitation intensity to avoid photobleaching and saturation microscopy.duke.edu/gallery.html E1 It can be however used to investigate molecular diffusion: Fluorescence recovery after photobleaching (FRAP) – how fast are fluorophores, which had been photobleached by a pulse of high intensity, replaced by new ones lipid bilayer adsorbed to solid surface – mobile lipids lipid monolayer adsorbed to immobilized alkyl chains – immobile lipids D found by fitting the recovery curve with a model accounting for the size and shape of the bleached area Fraction of immobile fluorophores I∞I∞ I0I0 IBIB

20. Microscope resolution – Rayleigh criterion: 240 200 160 120 80 40 0 50100150200250 240 200 160 120 80 40 0 50100150200 250300 Light from a point source is diffracted by the objective forming an Airy disc, the size of which depends on and NA of the objective Airy disc Corresponding intensity profile Rayleigh criterion: points are resolvable if the maximum of one Airy disc corresponds with the first minimum of the adjacent Airy pattern Digital contrast enhancement of images may help resolution of closer points. The improvement may be, however, overestimated due to smaller distance between the maxima than between the centers of Airy discs

21. R ’’ a’  Y Y’ Diffraction sin = 0.61'/R Rayleigh criterion Y' = a' tan Simple geometry yields: R/a’ = tan’ Y' = 0.61'/tan’ a Abbe Sine Condition: Y n sin = Y' n' sin‘  Y' n' tan‘ Y min = 0.61 / n sin considering that ‘ = / n’ NA Microscope resolution – Rayleigh criterion:

22. f e dcba Light passing through a periodic structure in the sample (a diffraction grating) results in a characteristic diffraction pattern in the objective back focal plane. The observable number of diffraction maxima is determined by NA of the objective Microscope resolution – Abbe’s theory: Ideal image diffraction pattern & mask image brightness profile image appearance Description by Fourier optics: Wavefront in the back focal plane W is a Fourier transform of the object transmission function O. The image I is the inverse Fourier transform of W W = F (O)I = F -1 (W) = F -1 (F(O))

23. Microscope resolution – Abbe’s theory: Description by Fourier optics: Wavefront in the back focal plane W is a Fourier transform of the object transmission function O. The image I is the inverse Fourier transform of W W = F (O)I = F -1 (W) = F -1 (F(O)) The objective aperture filters out higher order diffraction maxima from W and, thus, filters out high spatial frequencies from I Light Microscopy in Biology. A practical Approach. A.J.Lacey (ed.), IRL Press, Oxford, 1989, p.33. Any aperiodic object O can be theoretically described as an infinite series of periodic functions (Fourier series)

24. Abbe’s theory and oblique illumination: With oblique illumination higher orders of diffraction maxima can enter the objective of the same NA than with axial illumination  Improved resolution However, less light enters the objective  worse contrast

25. Microscope resolution – Elastic scattering: The shape of polar scattering diagrams for small spherical particles depends on the size of the particle r and. The smaller r, the more symmetric is the scattering diagram. The size of the central scattering lobe corresponds to the acceptance angle of the microscope when r ≈ 3 d r ≈ d r ≈ d/3

26. Microscope resolution – Summary: The lateral resolution of an optical microscope d: The axial resolution (in the direction of optical axis) d z : Sufficient contrast is necessary for full utilization of the available resolution

27. Acknowledgement The course was inspired by courses of: Prof. David M. Jameson, Ph.D. Prof. RNDr. Jaromír Plášek, Csc. Prof. William Reusch Financial support from the grant: FRVŠ 33/119970