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The Light Microscope
• Four centuries of history • Vibrant current development • One of the most widely used research tools
(Image: T. Wittman, Scripps)
Major Imaging Functions of the Microscope • Magnify • Resolve features
A. Khodjakov et al.
• Generate Contrast • Capture and Display Images
Photons vs. n v = c/n n=1 n>1 n=1 2 . Rays • Quantum wave-particle duality • Rays: photon trajectories • Rays: propagation direction of waves Rays are perpendicular to wavefronts Light travels more slowly in matter The speed ratio is the Index of Refraction.An Upright Epifluorescence Microscope Waves vs.
38 ? 1.5–1.46 • Optical glasses 1.417 Refractive index n2 Speed = c/n λ/n θ2 Refracted wave ⇒ Snell’s law: Mirror law: Depends on wavelength and temperature n1 Sin(θ1) = n2 Sin(θ2) θr = θ1 Which Direction? Lenses work by refraction n1 Incident light focus n2 > n1 Focal length f Refraction goes towards the normal in the higher-index medium 3 .0003 1.Refractive Index Examples • Vacuum • Air • • • • Water Cytoplasm Glycerol Immersion oil 1 1.35–1.9 • Diamond 2.333 1.515 Refraction by an Interface Incident wave θr θ1 Refractive index n1 = 1 Speed = c Reﬂected wave λ • Fused silica 1.475 (anhydrous) 1.
Ray Tracing Rules of Thumb (for thin ideal lenses) Imaging f Object d1 Image Parallel rays converge at the focal plane Rays that cross in the focal plane end up parallel d2 f f L1 L2 Rays through the lens center are unaffected The lens law: Magniﬁcation: Finite vs. Inﬁnite Conjugate Imaging • Finite conjugate imaging (older objectives) Object f0 Image f0 Object >f0 f0 Back focal plane Back focal plane • Inﬁnite conjugate imaging (modern objectives). f1 Object f0 Image at inﬁnity ⇒ Need a tube lens Image f0 f0 f0 f0 (uncritical) f1 Rays that leave the object with the same angle meet in the objective’s back focal plane Magniﬁcation: 4 .
The Compound Microscope The Compound Microscope Final image Eye Exit pupil Eyepiece Primary or intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Object plane Objective Sample Tube lens Eyepiece Exit pupil Intermediate image plane Back focal plane (pupil) Object plane The Compound Microscope Final image Eye Exit pupil Eyepiece Intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Object plane The Compound Microscope Final image Eye Exit pupil Eyepiece Intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Object plane 5 .
The Compound Microscope Final image Eye Exit pupil Eyepiece Intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Object plane The Compound Microscope Camera Final image Secondary pupil Projection Eyepiece Intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Object plane Eyepieces (Oculars) Trans-illumination Microscope Camera Final image plane Features • Magniﬁcation (10x typical) • “High eye point” (exit pupil high enough to allow eyeglasses) • Diopter adjust (at least one must have this) • Reticle or ﬁtting for one • Eye cups Secondary pupil plane Imaging path Projection Eyepiece Intermediate image plane Tube lens Back focal plane (pupil) Objective Sample Condenser lens Aperture iris Illumination path Field lens Field iris Collector Light source (pupil plane) (image plane) Object plane (pupil plane) The aperture iris controls the range of illumination angles The ﬁeld iris controls the illuminated ﬁeld of view 6 .
Köhler Illumination Sample Object plane Critical Illumination Conjugate Planes in A Research Microscope Aperture iris (pupil plane) Field iris (image plane) Light source (pupil plane) • Each light source point produces a parallel beam • The source is imaged onto the of light at the sample sample • Uniform light intensity at the sample even if the • Usable only if the light source light source is “ugly” (e. a ﬁlament) is perfectly uniform How view the pupil planes? By far the most important part: the Objective Lens Two ways: • “Eyepiece telescope” • “Bertrand lens” Each major manufacturer sells 20-30 different categories of objectives. What are the important distinctions? 7 .g.
75 WD = 1. high NA lenses have short working distances However.Working Distance The focal length of a lens depends on the refractive index… Refractive index n In general. extra-long working distance objectives do exist Some examples: 10x/0.2mm 20x/0.0mm 100x/1.3 WD = 15.13mm Focal length f f ∝ 1/(n-1) … and the refractive index depends on the wavelength (“dispersion”) ⇒ Chromatic aberration • Different colors get focused to different planes • Not good… Glass types 8 .4 WD = 0.
monochromatic) aberrations also improves in the same order 9 .Dispersion vs. refractive index of different glass types Refractive index Achromatic Lenses • Use a weak negative ﬂint glass element to compensate the dispersion of a positive crown glass element Abbe dispersion number (Higher dispersion→) Achromats and Apochromats Focal length error Correction classes of objectives Wavelength Achromat (cheap) Fluor “semi-apo” (good correction.e. high UV transmission) Apochromat (best correction) Apochromat (≥3 glass types) Achromat (2 glass types) Simple lens Correction for other (i.
Curvature of Field Focal plane Focal surface Plan objectives • Corrected for ﬁeld curvature • More complex design • Needed for most photomicrography Tube lens objective sample Focal surface • Plan-Apochromats have the highest performance (and highest complexity and price) Interference In phase constructive interference Diffraction by a periodic structure (grating) + = Opposite phase destructive interference + = 10 .
75% Tube lens …or can be formed at a ﬁnite distance by a lens… Intermediate image Objective pupil “Airy disk” diameter d = 2.44 λ f/d (for small angles d/f) d f …as happens in a microscope 11 .Diffraction by a periodic structure (grating) Diffraction by an aperture drawn as waves Light spreads to new angles θ d θ Larger aperture ⇔ weaker diffraction dS in(θ ) In phase if: d Sin(θ) = m λ for some integer m Diffraction by an aperture drawn as rays The Airy Pattern = the far-ﬁeld diffraction pattern from a round aperture The pure. “far-ﬁeld” diffraction pattern is formed at ∞ distance… Height of ﬁrst ring ≈ 1.
Aperture and Resolution Diffraction spot on image plane = Point Spread Function Objective Tube lens Intermediate image plane Objective Aperture and Resolution Diffraction spot on image plane = Point Spread Function Tube lens Intermediate image plane Sample Sample Back focal plane aperture Back focal plane aperture Aperture and Resolution Diffraction spot on image plane = Point Spread Function Objective Tube lens Intermediate image plane Objective Aperture and Resolution Intermediate image plane Diffraction spot on image plane (resolution) Tube lens Sample Sample α Back focal plane aperture Back focal plane aperture • Image resolution improves with aperture size Numerical Aperture (NA) NA = n sin(α) where: α = light gathering angle n = refractive index of sample 12 .
33 max NA ≈ 1.515 max NA ≈ 1.5° Oil immersion: n ≈ 1. but two spots get through → interference → image formed! β Diffracted beams Sample Condenser Light source Consider ﬁrst a point light source If β > α.20 NA α = 11. Resolution (smallest resolvable d) with incoherent illumination (all possible illumination directions): Resolution (smallest resolvable d): dmin = λ sample/sin(α) = λ/n sin(α) = λ/NA λ/2 NA if dmin = λ/(NAobj + NAcondenser ) NAcondenser ≥ NAobj (“Filling the back focal plane”) 13 .45 (85%) max NA ≈ 1. only one spot makes it through ⇒ no interference ⇒ no image formed d d sin(β) = λ Smaller d → larger β d βout βin d [sin(βin) + sin(βout) ] = λ Two spots get through if βout < α and βin < α. continued) Now consider oblique illumination (an off-axis source point): Objective lens One spot hopelessly lost.4 (1.45–1.2 Ernst Abbe’s argument (1873) Consider a striped sample ≈ a diffraction grating Back focal plane Resolution (Abbe’s argument.49 for TIRF) NA can approach the index of the immersion ﬂuid Glycerol immersion: n ≈ 1.95 NA α = 71.8° ⇒ NA cannot exceed the lowest n between the sample and the objective lens ⇒ NA >1 requires ﬂuid immersion 4X / 0.Numerical Aperture Immersion Objectives 100X / 0.35 (Leica) Water immersion: n ≈ 1.
3rd ed.Aperture.magnet.edu Douglas B. Resolution & Contrast Can adjust the condenser NA with the aperture iris Alternate Deﬁnitions of Resolution Imaging path Intermed. contrast FWHM ≈ 0. Ed. image Tube lens Back aperture Objective Sample Condenser lens Aperture iris As the Full Width at Half Max (FWHM) of the PSF Q: Don’t we always want it full open?? A: No Why? Tradeoff: resolution vs. “Handbook of Biological Confocal Microscopy.com micro.61 λ /NA Illumination path Field lens Field iris Collector Light source Objective Types • Plan or not Further reading Field ﬂatness www.microscopyu.fsu. Murphy “Fundamentals of Light Microscopy and Electronic Imaging” James Pawley.353 λ /NA As the diameter of the Airy disk (ﬁrst dark ring of the PSF) = “Rayleigh criterion” (Probably most common deﬁnition) Airy disk diameter ≈ 0.” • • • • • Magniﬁcation Numerical Aperture (NA) Inﬁnite or ﬁnite conjugate Cover slip thickness if any Immersion ﬂuid if any Basic properties • Positive or negative • Diameter of ring (number) Phase rings for phase contrast • Strain free for Polarization or DIC Special Properties • Achromat • Fluor • Apochromat Correction class • • • • Correction collar for spherical aberration Iris Spring-loaded front end Lockable front end Features Acknowledgements Ron Vale / Mats Gustafsson 14 .
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