BASICS IN BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP LIGHT MICROSCOPY

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1 BASICS IN LIGHT MICROSCOPY

2 OVERVIEW 1. Motivation 2. Basic in optics 3. How microscope works 4. Illumination and resolution 5. Microscope optics 6. Contrasting methods -2-

3 MOTIVATION Why do we need microscopy? Main issues of microscopy -3-

4 -4- The name: Microscopy greek mikros= small skopein= to observe Observation of small objects

-5- HUMAN EYE Normal viewing distance - 250 mm Angular resolution a min 1 Spatial resolution h min 80 mm Nodal distance -17 mm Average retinal cell distance 1.

5 -5- HUMAN EYE Normal viewing distance mm Angular resolution a min 1 Spatial resolution h min 80 mm Nodal distance -17 mm Average retinal cell distance 1.5 mm Spectral range 400 nm nm Can resolve contrast about 5% High dynamic range 10 decades Max sensitivity at 505 nm (night, rods) Max sensitivity at 555 nm (day, cones) More sensitive to color than to intensity Most perfect sensor for light detection up to now

6 MAIN ISSUES OF MICROSCOPY low contrast low resolution low magnification

7 MAIN ISSUES OF MICROSCOPY In order to observe small objects, three preconditions have to be fulfilled 1. Magnification 2. Resolution 3. Contrast Only fulfillment of these three conditions allows translation of information as accurately as possible from object into an image which represents that object.

8 IMAGE FORMATION Light is the messenger and transports the object information from the specimen through the microscope Light translates the object information into a microscopic image of the specimen The observer observes the microscopic image of the specimen not the specimen itself! Only best management of the light allows translation of information as accurately as possible from object into an image which represents that object!

9 MAGNIFYING GLASS a 2 a 1 virtual image object 250 mm f Magnifier increases the angular size of the object M=a 2 /a 1 Magnification is defined by focal distance of lens M=250/f Maximum magnification of magnifying glass is 10x-20x

10 HOW A THIN LENS WORKS Lens focuses collimated beam of light parallel to optical axis into on axis spot Beams in focus are in phase Lens focuses oblique collimated beam into an off axis focal spot

11 HOW MICROSCOPE WORKS Compound microscope Convergent and infinite beam paths Components of microscope

12 COMPOUND MICROSCOPE CONVERGENT BEAM PATH Sample is placed in front of objective focal plane. Intermediate image is formed by objective and is observed through eyepiece.

13 DISADVANTAGE OF A CONVERGENT BEAM PATH Convergent beam Beam is focused differently More aberrations Parallel beam Beam is only shifted Less aberration Presence of parallel light beam is microscope light path is important for modern light microscope (for filters, and other optical elements)

14 COMPOUND MICROSCOPE INFINITY-CORRECTED BEAM PATH The sample is placed in the focal plane of the objective. Parallel light beams are focused by the tube lens. The intermediate image is observed through the eyepiece.

15 OBJECTIVE Objective are constructed of several high quality lenses. For infinity corrected objective the specimen is in the focal plane For not infinity corrected objectives the specimen is in front of the focal plane

16 EYEPIECE The eyepiece acts as a magnifier of the intermediate image

17 CAMERA AS IMAGE DETECTOR When the camera is used, the intermediate image is directly projected on the camera chip (additionally an intermediate magnifier might be used).

18 MICROSCOPE DESIGNS Upright Used in biology mostly for fixed specimens Inverted Widely used in biology for living cell imaging

19 MAIN MICROSCOPE COMPONENTS Hal lamp condenser Hg lamp field diaphragm (t) aperture diaphragm (t) eyepiece objective filter cube turret focus camera field diaphragm (f) aperture diaphragm (f) stage DIC slider

20 ANATOMY OF MICROSCOPE Two independent illumination paths: Transmission Fluorescence Components for contrasting methods: DIC Dark field Phase contrast

21 HOW MICROSCOPE WORKS SUMMARY Magnifying glass has a limited magnification of 10x-20x Compound microscope makes two stage magnification initial magnification with objective further magnification with eyepiece Compound microscope beam path designs finite old microscopes infinity corrected modern microscopes There are several microscope types inverted upright

22 ILLUMINATION AND RESOLUTION Koehler illumination Diffraction of light Numerical aperture Resolution

23 REQUIREMENTS FOR ILLUMINATION Uniform over whole field of view Has all angles accepted by objective Allows optimize image brightness/contrast Allows continuous change of intensity Allows continuous change of field of view Change in illumination and imaging parts do not effect each other Realized in Kohler illumination

CONJUGATED PLANES IN OPTICAL MICROSCOPY Image forming light path (Observed with eyepiece) 1. Variable field diaphragm 2. Specimen plane 3. Intermediate image plane 4.

24 CONJUGATED PLANES IN OPTICAL MICROSCOPY Image forming light path (Observed with eyepiece) 1. Variable field diaphragm 2. Specimen plane 3. Intermediate image plane 4. Image plane (camera, retina) Illumination light path (Observed with Bertrand lens) 1. Lamp (filament, arc) 2. Condenser aperture diaphragm 3. Objective rear (back) focal plane 4. Eyepoint (exit pupil of microscope) Conjugated = imaged onto each other Has one diaphragm in every path If light at given plane is focused in one path, it is parallel in other path

25 COLLECTOR AND CONDENSER Collector gathers light from light source Condenser directs light onto the specimen

26 DIFFRACTION OF LIGHT A parallel beam falls on the screen with pinholes. Secondary spherical waves are formed on each pinhole. Interference results in several plane waves

27 MODULATION/CONTRAST - 27-

28 DIFFRACTION ORDERS d = 2 l 1 st order (d = 5 l) for small enough structures a first diffraction maxima is perpendicular to the direct light d = 1 l 1 st order (d = 1.5 l) 0 +1 d sina ml Direction of diffraction maxima depends on wavelength and period Bigger period results in smaller diffraction angle Bigger wavelength results in bigger diffraction angle

29 NUMERICAL APERTURE OF OBJECTIVE NA nsina 0 a 0 n 1!! n The NA defines how much light (brightness) and how many diffraction orders (resolution) are captured by the objective.

$ROLE OF IMMERSION NA=nsina Refractive indices: Air - 1.003 Water - 1.33 Glycerol - 1.47 Oil - 1.$

30 ROLE OF IMMERSION NA=nsina Refractive indices: Air Water Glycerol Oil Immersion media increase the NA of an objective or a condenser by bringing the beams with higher incidence angle into the light path

31 ROLE OF CONDENSER IN IMAGE FORMATION NA tot =NA obj +NA cond

32 ILLUMINATION AND RESOLUTION Choice of light source depends on application transmission halogen lamp fluorescence HBO, XBO, metal halide Correct illumination is critical for successful imaging always set up Koehler illumination condenser as important as objective Resolution is defined by NA and wavelength higher resolution for higher NA lower resolution for longer wavelength Resolution is much better in lateral direction NA = 1.4, wavelength = 500 nm lateral resolution about 200 nm axial resolution about 800 nm

33 MICROSCOPE OPTICS Aberrations in optics Eyepiece engravings Objective engravings Choice of magnification

34 OPTICAL ABERRATIONS Astigmatism (tangential and meridianal focus are different) Coma (image of dot is not symmetric) Distortion (parallel lines are not parallel in image) Curvature of the field (image of plane is not flat) Chromatic (different focus for different wavelength) Spherical (different focus for on and off axis beams) It is desired to minimize aberrations by proper use of objectives with good aberration correction

35 CHROMATIC ABERRATION Use of lenses with different dispersion Achromat (corrected for two colors) Fluorite (better corrected) Apochromat (corrected at least for three colors)

$17 mm thick or Use objective with correction ring Avoid refraction index$

36 SPHERICAL ABERRATION Use cover slip 0.17 mm thick or Use objective with correction ring Avoid refraction index mismatch of immersion and mounting media

37 EYEPIECE ENGRAVINGS Field number (FN) diameter of view field in mm measured in intermediate image plane. Magnification -10x, 16x, etc. Eyepiece type Pl gives plane image W - wide field of view Also indicated: Diopter correction, use with glasses Field Size = FN/(M obj xm int )

WORKING DISTANCE AND PARFOCAL LENGTH Parfocal distance Distance from objective shoulder till specimen plane 45 mm for most manufactures, 60 mm for Nikon CFI 60 Working

38 WORKING DISTANCE AND PARFOCAL LENGTH Parfocal distance Distance from objective shoulder till specimen plane 45 mm for most manufactures, 60 mm for Nikon CFI 60 Working distance Distance from front edge of objective till cover slip Varies from several mm till several hundreds micrometers. Special long working distance objective are available.

39 ENGRAVINGS ON OBJECTIVES Epi = observation from above (0 = no cover glass) LD = long (working) distance plan = minimal curvature in the image plane APOCHROMAT = especially color corrected HD = hell/dunkel = bright/dark field DIC = differential interference contrast (low strain optics for polarized light)

40 OBJECTIVES WITH CORRECTION COLLARS NEOFLUAR optics is less color corrected than APOCHROMAT Range of cover glass thickness W W Glyc Oil Ph = phase contrast (3 specifies matching condenser) Different immersion media under various cover glass conditions

41 TOTAL MICROSCOPE MAGNIFICATION Defined by magnification of objective, eyepiece and intermediate magnification M tot =M obj x M int x M eyepiece Objective magnification defined by focal lengths of tube lens and objectives M obj =f tl /f obj Tube lens has a standardized value for specific manufacture Zeiss, Leica, Olympus 165 mm, Nikon 200 mm Typical magnification rangies: M obj : 2x 100x M int : 1.5x 2.5x M obj : 10x 25x

42 USEFUL MAGNIFICATION RANGE Microscope resolution is limited by NA and wavelength. Enlargement of image does not necessarily resolve new features. Excessively large magnification is called empty magnification. (The Airy disk on retina/camera should not exceed two cell/pixel sizes). Useful magnification = x NA of objective M obj M eyepiece NA obj M tot M useful Magnification 10x 10x low 40x 10x ok 100x 10x ok 100x 15x empty - 44

43 LIGHT BUDGET IN MICROSCOPE Microscope has a lot of components in light path Microscope optics (T=0.8) Dichroic mirror (T=0.8) Filters (T=0.8) Objective, eyepiece (T=0.9) Objective collects light only within NA (T=0.3) Typically only 10% of light arrives to CCD. Use optics with antireflection coatings Use high quality filters, dichroics Use clean optics Image brightness (transmission) ~ (NA/M) 2 Image brightness (fluorescence) ~ NA 4 /M 2 Use high NA objectives Do not use unnecessary high magnification

44 MICROSCOPE OPTICS SUMMARY Correct choice of microscope optics is the key to successful imaging Pay attention to the engravings on objective and eyepiece Optical aberrations can be minimized use well corrected optics or use green filter use cover slip 0.17 mm thick match refractive index of immersion media and specimen Choose magnification carefully excessive magnification does not reveal new details moreover it deceases the brightness of the image

45 CONTRASTING METHODS Dark field Phase contrast DIC PlasDIC

46 AMPLITUDE AND PHASE SPECIMENS Amplitude specimen changes the intensity of incident light Phase specimen changes the phase of incident light Most unstained biological specimens are phase ones

47 EXAMPLES OF CONTRASTING METHODS Dark field Bone thin section Phase contrast HEK cells DIC Neurons PlasDIC HEK cells

48 DARKFIELD CONTRAST 5 iris diaphragm 4 - objective 3 - sample 2 - condenser 1 - phase stop A - low NA objective B - high NA objective with iris Required: special condenser, sometimes immersion oil Principle: direct light is rejected or blocked, only scattered light is observed Disadvantage: low resolution

49 INTERFERENCE Addition of waves Amplitude of the resulting wave depends on the pahse relation of two waves Extreme cases: destructive interference (res. amplitude =0) positive interference With interference a phase difference can be turned into an amplitude difference Interference is the basic principle of Phase contrast and DIC.

50 PHASE CONTRAST MICROSCOPY

PHASE CONTRAST MICROSCOPY 9 - intermediate image 8 - tube lense 7 - indirect light 6 - direct light 5 - phase ring 4 - objective 3 - sample 2 - condenser 1 - phase stop Required: special objectives

51 PHASE CONTRAST MICROSCOPY 9 - intermediate image 8 - tube lense 7 - indirect light 6 - direct light 5 - phase ring 4 - objective 3 - sample 2 - condenser 1 - phase stop Required: special objectives and special condensers. Principle: direct light is attenuated and its phase is shifted 90. Contrast formed due to interference between direct and scattered light. Disadvantages: relatively low resolution, halos

DIFFERENTIAL INTERFERENCE CONTRAST 9 - intermediate image 8 - tube lens 7 analyzer 7a - λ-plate 6 - Wollaston prism 5 objective 4 sample 3 condenser 2 Wollaston prism 1 - polariser Required: special

52 DIFFERENTIAL INTERFERENCE CONTRAST 9 - intermediate image 8 - tube lens 7 analyzer 7a - λ-plate 6 - Wollaston prism 5 objective 4 sample 3 condenser 2 Wollaston prism 1 - polariser Required: special accessories in light path (prisms, polarizers). Principle: specimen is sensed with two linear polarized slightly shifted (<l) light beams. Difference in optical path of the beams gives a contrast in image. Disadvantages: accessories are relatively expensive.

Beams measure difference in optical path in specimen.

53 DIC IN DETAILS DIC prism split beam into two perpendicularly polarized. Shift between beams less that resolution of microscope. Beams measure difference in optical path in specimen. If retardation is not zero, they are interfere after being recombined on the second DIC prism.

54 DE SERAMONT COMPENSATOR Rotation of polarizer relative to quarter wave plate gives circular polarized light. This results in phase shift between beams after DIC prism. Thus the contrast of the DIC image can be adjusted. This method of contrast change is equivalent to the lateral shift of Wollaston prism but more accurate.

PLASDIC Required: slit diaphragm, prism with polarizer, analyzer. Principle: A slit diaphragm creates a pair of non-polarized light beams that are l/4 out-of-phase.

55 PLASDIC Required: slit diaphragm, prism with polarizer, analyzer. Principle: A slit diaphragm creates a pair of non-polarized light beams that are l/4 out-of-phase. The beams get polarized just before being recombined into a single beam in the DIC-prism. The analyzer (linear) sets a single polarization plane where the components of the beam can interfere.

56 CONTRASTING TECHNIQUES SUMMARY Dark field Fine structural features at, and even below, the resolution limit of a light microscope. Highly suitable for metallographic and crystallographic examinations with reflected light. Phase contrast Used for visualizing very fine structural features in tissues and single cells contained in very thin (< 5 µm), non-stained specimens. DIC Method shows optical path differences in the specimen in a relieflike fashion. The method is excellently suited for thick, non-stained specimens (> 5 µm). Can be used for optical sectioning. PlasDIC The same specimen as conventional DIC but in plastic dishes.

57 IMAGES ARE ARTEFACTS Two images of same object (sample) imaged with the same microscope/objective! Object Image of Object

58 MORE ABOUT LIGHT MICROSCOPY 1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL 2. Books a) Digital microscopy, Sluder, G; Wolf, D.E., eds, Elsevier, 2003 b) Optics, 4th ed., Eugene Hecht, Addison-Wesley, Internet a) b) b) Web sites of microscope manufactures Leica Nikon Olympus Zeiss 4. BIOp EPFL, SV-AI 0241, Sv-AI

59 Acknowledgments These slides are based on a lecture given by Yuri Belyaev (Advanced Light Microscopy Facility, EMBL Heidelberg) during a practical course concerning basics of light microscopy. Thus a big thank to him for providing them and making them available also here at EPFL.

BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP BASICS IN LIGHT MICROSCOPY

BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP BASICS IN LIGHT MICROSCOPY BASICS IN LIGHT MICROSCOPY INTERNAL COURSE 2014 13 TH JANUARY OVERVIEW 1. Motivation 2. Basic in optics 3. How microscope works 4. Illumination and resolution 5. Microscope optics 6. Contrasting methods