microscopy A great online resource Molecular Expressions, a Microscope Primer Partha Roy

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1 Fundamentals of optical microscopy A great online resource Molecular Expressions, a Microscope Primer Partha Roy 1

2 Why microscopy Topics Functions of a microscope Review of geometric optics Principle of microscopy Types of research microscope Technicalities of microscopy 2

3 Why microscopy? To visualize structural and functional details of cells Structural: cell architecture - visualize cells and organelles - visualize distribution of molecules Actin fibers Lodish 3

4 Functional: study dynamic events (microscopy in a time-lapse fashion) a) Observe cellular processes e.g cell movement, cell division etc.. Movies: moving fish cell, WBC eating yeast, cell division b) Study dynamics of molecules l (e.g transport t of molecules) Movie: melanin-filled granules transported along microtubule track c) protein-protein interaction 4

5 Three major functions of a microscope 1) To magnify an object magnify Empty magnification (makes the image blurry) 5 Lodish 5e

6 2) To provide spatial map of a specimen at a much higher optical resolution than what is perceived by naked eyes. resolution ability to distinguish two closely spaced objects Smaller value higher resolution Limit it for light microscopy µm Human eye ~100 µm (from 250 mm distance) (1 µm =1/1000 mm) 6 Lodish 5e

7 3) To make two different objects optically distinct (Contrast) e.g : specimen from background structures within a specimen Low contrast Enhanced contrast (Phase contrast) 7 Lodish 5e

8 Reflection θ i θ r Review of Geometric optics mirror 45 Refraction Case I: n 1 n 2 Snell s law: θ i = θ r 45 Beam steering at 90 degree θ Case II: i n 1 <n 2 θ n 1 >n 2 i θ r n 1 sin θ i = n 2 sin θ r Bending toward normal n 1 n 2 θ r Bending away from normal n-index of refraction 8

9 Special case of refraction Critical angle θ i =θ n 1 >n c 2 n 1 n 2 θ r =90 n 1 n 2 θ n i >θ 1 >n c 2 θ r Total internal reflection n 1 (glass) = 1.52 n 2 (air) =1.0 n 1 θ 1 c = n 2 Beam steering at 90 degree by a prism 9

10 Lens: two refracting surfaces Refraction at lens surfaces Refracting surface I Refracting surface II Optic axis Principal plane (the plane in the lens where extensions of incident and emergent rays intersect) Reference for distance 10

11 Sets of collimated beam Focal plane Principal plane Focal plane α d=f *tanα Optic axis Focal length (f) Back Front focal plane focal plane F F Focal length Focal length 11

12 Rules of ray tracing 1) A light ray passing through the center of lens remains undeviated object 2) A light ray yparallel to the optic axis passes through the back focal point. object F 3) A ray passing through the front focal point tbecomes parallel lto the optic axis Intersection of any 2 out of 3 traces defines an image point object F 12

13 Real image formation by a convex lens Front focal plane Back focal plane (object) o F F I Optic axis (image) u f v Primary Image plane 1/u+1/v=1/f Magnification (M) = I/O = v/u = f/(u-f) 13

14 Object-image relationship 1) u < f : virtual, magnified image I o F F 2) u=f : no image (@ infinity) F o F 14

15 3) f<u<2f : real, magnified image o F F I 4) u>2f : real, reduced image o F F I 15

16 Modes of flight Microscopy Transmission light microscopy Reflection light microscopy 16

17 Transmission-light microscopy Optical elements Optical elements Light source illumination specimen Image forming light beam altered in property (amplitude/ phase) Detector (eye/camera) *** In reflection microscopy, illumination and imaging paths coincide 17

$V=f(refractive index)$ Bright-field Enhanced

18 Incident light Two methods to generate image contrast Transmitted light specimen (light absorption by sample) amplitude (sample alters velocity) phase V=f(refractive index) Bright-field Enhanced amplitude by using a dye Phase-contrast ast ( phase amplitude) 18

19 Eye Eyepiece lens Primary (intermediate) Image plane Glass coverslip specimen Objective lens Condenser lens Optical elements lamp Basic light path of a microscope 19

20 Upright microscope Recording device Light path Image formation Z specimen X Y illumination 20

2-stage magnification in a microscope Intermediate image plane (within F 2 ) o (object) Back focal plane Eye lens+cornea retina F 1 F 1 F 2 Virtual image objective OTL

21 2-stage magnification in a microscope Intermediate image plane (within F 2 ) o (object) Back focal plane Eye lens+cornea retina F 1 F 1 F 2 Virtual image objective OTL eyepiece F 1 = objective focal point F 2 =eyepiece focal point 250 mm (comfortable viewing distance) Total magnification i = M obj x M eyepiece OTL =optical tube length 21

objective is mounted, to the top edge of the observation tubes where the eyepieces are

22 FINITE vs INFINITY OPTICS eyepiece 160 mm 10 mm eyepieces objective objective condenser Mechanical tube length - Distance from the nosepiece opening, where the objective is mounted, to the top edge of the observation tubes where the eyepieces are inserted (=160 mm: recommended for finite optics) 22

O between F and 2F of the objective O (just outside F) OJ Finite optics F EP EP 150 mm OTL I F Intermediate image OJ Plane (IIP) Shifting of IIP by insertion of additional Optical element δ

23 O between F and 2F of the objective O (just outside F) OJ Finite optics F EP EP 150 mm OTL I F Intermediate image OJ Plane (IIP) Shifting of IIP by insertion of additional Optical element δ IIP (w/o Optical accessory) Glass plate δ OJ-objective EP- eyepiece lateral shifting of ray You need compensatory elements to shift IIP back to its original location not a very flexible system 23

24 O placed at F of objective InFinity optics OJ TL EP O F FEP TL IIP F OJ Infinity space (parallel beam of light) the back focal plane of TL (tube lens) I Infinity space : Between the objective and the tube lens 24

25 O placed at F of objective InFinity optics (continuation) OJ TL EP O F TL F EP IIP F OJ Infinity space (parallel beam of light) IIP the back focal plane of TL (tube lens) I optical accessories inserted into parallel beam of light (infinity space) do not shift the location (either laterally or axially) nor the focal point of the image. 25

26 Upright microscope Recording device Light path Image formation Z specimen X Y illumination Stage moves along X, Y, Z, objective fixed 26

27 Inverted microscope Light source Z X Y condenser Light path objective Stage moves along X,Y objective moves along Z 27

28 Illumination system Light source: Tungsten-halogen bulb Significant intensity in IR Produces heat cut IR by filter (dissipates heat) 28

29 Critical illumination (obsolete) abc filament Specimen plane a b c -image of filament a c b c C Condenser lens b a Specimen Image of light source Condenser lens forms a real image of light source on the specimen plane - creates uneven illumination of specimen. Variation in intensity of the image can result from both the nature of object and non-uniform illumination 29

Kohler illumination (achieves uniform illumination) Enlarged Image of the lamp is formed

CL C background illuminationi F C specimen Each point of lamp illuminates every point in

30 Kohler illumination (achieves uniform illumination) Enlarged Image of the lamp is formed at the front focal plane of condenser (by moving collector lens) sets of parallel rays coming out of the condenser. August Kohler Field diaphragm (FD) Aperture (condenser) diaphragm (AD) Specimen plane Even CL C background illuminationi F C specimen Each point of lamp illuminates every point in the specimen and vice versa. No image of the filament on the specimen plane Variation in intensity of the image can result only from the nature of the object. 30

31 t / i /i d 31

32 Concept of conjugate planes In Kohler illumination Conjugate planes - planes under simultaneous focus SET 1: FD, specimen plane, IIP, retina SET 2: Lamp, AD, objective back focal plane, exit pupil When set 1 planes are in focus, set 2 planes are not and vice-versa collector lens t / i /i d 32

33 Opening or closing of ffd changes the area of illumination (or the field of view), no change in image quality collector lens 33

34 Action of aperture diaphragm (AD) Closing the AD reduces the angle of illumination cone (α) Progressive closing edu/p e / de t 34

35 Numerical aperture (NA)= n.sin (α/2) n-refractive index n α n α condenser (NA defines the illuminating power) Objective (NA defines the light-gathering power) Image brightness (NA/M) 2 M -magnification Resolution (d) 1/NA High NA better resolution, brighter image 35

36 High NA better resolution, brighter image What limits NA? How can you increase NA? objective Air (n=1) α Oil (n=1.53) α Glass (n=1.52) Limited by total internal Reflection at glass-air interface (i>41 o ) specimen Glass (n=1.52) Increased NA by using immersion oil because of no total internal reflection (greater n, α) 36

37 Magnification Medium (n) NA d min (µm) WD (mm) 10x 1 (dry) x 1 (dry) x 1 (dry) x 1.52 (oil) x 1.52 (oil) x 1.52 (oil) Working distance (WD): distance between the surface of the front lens element of the objective and the coverslip (decreases with NA) when the specimen is in focus Higher WD focus through thicker specimen coverslip WD specimen 37

38 Phase Contrast Microscope Examples: Courtesy: Hai Lin

39 Differential Interference Contrast Microscopy Examples: Cultured Hela Cells Phase contrast Courtesy: Hai Lin /Alberts DIC

40 Light paths in a Microscope Image plane The sample is transparent; A point light source (use a diaphragm with a small aperture in the front focal point of the condenser, A beam of parallel rays leaves the condenser passes through the object plane; The beam is focused in the back focal point of the objective; The beam reach the image plane as an extended light beam. This beam is called the direct (undiffracted) beam; Objective focal plane objective sample condenser Courtesy: Hai Lin Light source

41 Light paths in a Microscope Image plane A portion of the light wave is perturbed (diffracted and deflected) by the object; it is called the diffracted beam; The diffracted beam passes through the objective back focal plane in a diffused pattern; The diffracted beam reach the image plane as a focused light beam; Objective focal plane objective sample condenser Courtesy: Hai Lin Light source

42 Interference of the direct and diffracted beams Image plane If the sample refraction index (n) is different from its surroundings, then diffracted beam will have a different phase compared to the direct beam. The interference between the diffracted beam and the direct beam will result in the image of the the sample at the image plane. However, under this unmodified condition, the two beams cannot produce effective constructive or destructive interference to produce a clear image. Objective focal plane objective sample condenser Courtesy: Hai Lin Light source

43 Interference of the direct and diffracted beams The intensity of the diffracted beam is much smaller then the direct beam; The resultant contrast from interference is low; How to increase the contrast? Reduce the intensity (amplitude modification) of the direct beam; Image plane Objective focal plane objective sample condenser Courtesy: Hai Lin Light source

44 Phase Contrast Microscope Image plane ND filter Intensity of the direct beam can be reduced by placing a small neutral density filter at the objective back focal plane, without t affecting the intensity of the diffracted beam; If the intensities of the direct beam and the diffracted beam are equal the image plane, then the two beam will interfere constructively ti when the phase difference is 2kπ (whole wavelengths) and destructively when the phase difference is (2k+1)π (half wavelength). Courtesy: Hai Lin Light source Objective focal plane objective sample condenser

45 Phase Contrast Microscope Image plane Phase shift induced by biological samples are relatively small; n cell = 1.36, n water = 1.335, λ = 550 nm; cell thickness t = 5 µm, Phase shift induce by a cell surrounded by media is ψ =(2π/λ)(n cell -n water )t 0.5π Superposition of two coherent waves: C Courtesy: Hai Lin = A + B + 2 AB cos( ψ ) Light source Objective focal plane objective sample condenser

46 Phase Contrast Microscope Superposition of two coherent waves: C = A + B + 2 AB cos( ψ ) Image plane ND filter & ¼ λ phase shift Objective focal plane Introduction of a ψ=π/2 phase shift (1/4 wavelength) between the direct and diffracted light beams enhanced interference contrast. objective sample condenser Courtesy: Hai Lin Light source

47 Phase Contrast Microscope Real phase contrast microscopes use an annular diaphragm and ring-shaped phase plate; The phase plate is usually built into the objectives; Courtesy: Hai Lin

48 Phase Contrast Microscope Courtesy: Hai Lin

Properties of optical instruments. Visual optical systems part 2: focal visual instruments (microscope type)

Properties of optical instruments Visual optical systems part 2: focal visual instruments (microscope type) Examples of focal visual instruments magnifying glass Eyepieces Measuring microscopes from the