basics in light microscopy - biop - epfl - biopbiop.epfl.ch/pdf/basics in microscopy.pdf · pt-biop...

Dr. Arne SeitzPT-BIOP Course, Basics in Light Microscopy 2010, EPFL

BioImaging &Optics Platform

Dr. Arne Seitz

Swiss Institute of Technology (EPFL) Faculty of Life Sciences

Head of BIOIMAGING AND OPTICS – [email protected]

Basics in

light microscopy



Overview

1. Motivation2. Basic in optics3. How microscope works4. Illumination and resolution5. Microscope optics6. Contrasting methods

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1.Motivation

• Why do we need microscopy?• Main issues of microscopy

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The name:Microscopy

greekmikros= small

skopein= to observe

“Observation of small objects”

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Human eye

Normal viewing distance - 250 mm Angular resolution αmin ≈ 1’Spatial resolution hmin ≈ 80 µmNodal distance -17 mmAverage retinal cell distance 1.5 µmSpectral range 400 nm - 800 nmCan resolve contrast about 5%High dynamic range – 10 decadesMax 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

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Main issues of Microscopy

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In order to observe “small objects”, three preconditions have tobe fulfilled

1. Magnification2. Resolution3. Contrast

Only fulfillment of these three conditions allows translation ofinformation as accurately as possible from object into an imagewhich represents that object.



Image formation

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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 asaccurately as possible from object into an image which represents that object!



2.Basics in Optics

• What is light?• Geometrical optics• Thin lenses

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What is light?

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Light can be described as an electromagnetic wave (=electromagnetic radiation).Light can be described as a particle (photon)

Wave-particle duality

Main properties of light are:

• Intensity• Frequency or wavelength• Polarization• Phase

Study of light in known as optics



Geometrical optics

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• also known as Gaussian optics•light propagation is explained in terms of “rays”•an optical axis can be defined and all rays are almost parallel to it(= paraxial approximation)•does practically an excellent job(even under conditions where the paraxial condition is not fulfilled!)• no wavelength (fails to explain resolution!)

Optical axis

sin Θ ~ Θ tan Θ ~ Θ cos Θ ~ 1



Basics of geometrical optics

h - object height; h’’ - image heights - object distance; s’’ - image distance

f - effective focal lengthLens formula: 1/f = 1/s’+1/s’’,

m - magnificationm = s”/s’=h”/h’

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Magnifying glass

Magnifier increases the angular size of the object

M=α2/α1

Magnification is defined by focal distance of lens

M=250/f

Maximum magnification of magnifying glass is 10x-20x

α1

α2

250 mmfobject

virtual image

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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

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3. How microscope works

Compound microscopeConvergent and infinite beam pathsComponents of microscope

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Compound microscope - convergent beam path

Sample is placed in front of objective focal plane. Intermediate imageis formed by objective and is observed through eyepiece.

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Disadvantage of a convergent beam path

Convergent beam

Beam is focused differentlyMore aberrations

Parallel beam

Beam is only shiftedLess aberration

Presence of parallel light beam is microscope light path is important for modern light microscope (for filters, and other optical elements)

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Compound microscope - infinity-corrected beam path

The sample is placed in the focal plane of the objective. Parallel light beams arefocused by the tube lens. The intermediate image is observed through the eyepiece.

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Objective

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

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The eyepiece acts as a magnifier of the intermediate image

Eyepiece

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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).

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Microscope Designs

Upright

Used in biology mostly for fixed specimens

Inverted

Widely used in biology for living cell imaging

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Main microscope components

camera

objective

eyepiece

filter cube turret

Hal lamp

stage

condenserHg lamp

field dia-phragm (f)

DIC slideraperture diaphragm (f)

focus

field dia-phragm (t)

aperture diaphragm (t)

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Two independentillumination paths:• Transmission• Fluorescence

Components forcontrasting methods:• DIC• Dark field• Phase contrast

Anatomy of microscope

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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

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4. Illumination and resolution

Koehler illumination Diffraction of lightNumerical apertureResolution

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Light sourcesHalogen lamp

• Continuous spectrum: depends on temperature• For 3400K maximum at 900 nm• Lower intensity at shorter wavelengths• Very strong in IR

Mercury Lamp (HBO)• Most of intensity in near UV• Spectrum has a line structure • Lines at 313, 334, 365, 406, 435, 546, and 578 nm

Xenon lamp (XBO)• Even intensity across the visible spectrum• Has relatively low intensity in UV• Strong in IR

Metal halide lamp (Hg, I, Br)• Stronger intensity between lines• Stable output over short period of time• Lifetime up to 5 times longer

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Requirements for illumination

Uniform over whole field of view Has all angles accepted by objectiveAllows optimize image brightness/contrast Allows continuous change of intensityAllows continuous change of field of viewChange in illumination and imaging parts do

not effect each other

Realized in Kohler illumination

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Conjugated planes in optical microscopyImage forming light path (Observed with eyepiece)1. Variable field diaphragm2. Specimen plane3. Intermediate image plane4. Image plane (camera, retina)

Illumination light path (Observed with Bertrand lens)1. Lamp (filament, arc)2. Condenser aperture diaphragm3. Objective rear (back) focal plane4. Eyepoint (exit pupil of microscope)

Conjugated = imaged onto each otherHas one diaphragm in every pathIf light at given plane is focused in onepath, it is parallel in other path

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Collector and condenser

Collector

gathers light fromlight source

Condenser

directs light onto the specimen

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How to set up Koehler illumination

Transmission• Focus on the specimen• Close field diaphragm• Focus condenser until field diaphragm is seen sharp• Center field diaphragm• Close field diaphragm up to 80 – 90 %• Remove eyepiece, look down to the aperture diaphragm• Center (if possible) aperture diaphragm• Open/Close aperture diaphragm up to 80 – 90 %

Start with low magnification objective. Repeat for every objective used

Fluorescence• Focus on the specimen• Swing in focusing aid (if available) • Focus image of arc sharply• Swing out focusing aid• Close field diaphragm• Center field diaphragm

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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

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0-1 +1

1st order (d = 5 λ)

Diffraction orders

d = 2 λ

0

+1

1st order (d = 1.5 λ)

d = 1 λfor small enoughstructures afirst diffractionmaxima isperpendicular tothe direct light

λα md =sinDirection of diffraction maxima depends on wavelength and period

Bigger period results in smaller diffraction angle

Bigger wavelength results in bigger diffraction angle

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0α0sin αnNA =

518.1=n

1=n

! !

The NA defines how much light (brightness) and how many diffraction orders (resolution) are captured by the objective.

Numerical aperture of objective

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Role of immersion

NA=nsinα

Refractive indices:Air - 1.003Water - 1.33Glycerol - 1.47 Oil - 1.52

Immersion media increase the NA of an objective or a condenserby bringing the beams with higher incidence angle into the light path

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Role of condenser in image formation

NAtot=NAobj+NAcond

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Airy disc

Image of a dot is not a dot (PSF)Airy disc is x-y section of PSF

NA=0.7

NA=1.3

r = 1.22λ/(NAobj + NAcond)

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Resolution of light microscope

Lateral resolution

Axial resolution

2NA2 nR

zλδ =

NA61.0 λδ =R

λ=540 nm, NA=1.4, n=1.52: 235 nm - lateral, 838 nm - axial

Shortest distance between two points on a specimen that can still be distinguished by the observer or camera as separate entities.

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Illumination and resolution: summaryChoice 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

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5. Microscope optics

Aberrations in opticsEyepiece engravingsObjective engravings Choice of magnification

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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

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Chromatic aberration

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

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Spherical aberration

Use cover slip 0.17 mm thick orUse objective with correction ringAvoid refraction index mismatch of

immersion and mounting media-42-



Eyepiece engravings

Field number (FN) – diameter of view field inmm measured in intermediate image plane.Magnification -10x, 16x, etc.Eyepiece typePl – gives plane imageW - wide field of viewAlso indicated: Diopter correction, use with glasses

Field Size = FN/(MobjxMint)

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Working distance and parfocal lengthParfocal distance

Distance from objective shouldertill 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 hundredsmicrometers. Special long working distance objective are available.

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Epi = observation from above (0 = no cover glass)

LD = long (working) distanceplan = 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)

Engravings on objectives

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NEOFLUAR optics is less color corrected than APOCHROMAT

W W Glyc Oil

Different immersion media under various cover glass conditions

Range of cover glass thickness

Ph = phase contrast (3 specifies matching condenser)

Objectives with correction collars

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Total microscope magnification

Defined by magnification of objective, eyepiece and intermediate magnification

Mtot=Mobj x Mint x Meyepiece

Objective magnification defined by focal lengths of tube lens and objectives

Mobj=ftl/fobj

Tube lens has a standardized value for specific manufacture Zeiss, Leica, Olympus 165 mm, Nikon 200 mm

Typical magnification rangies:• Mobj: 2x÷100x• Mint: 1.5x÷2.5x• Mobj: 10x÷25x

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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 twocell/pixel sizes).

Useful magnification = 500-1000 x NA of objective

Mobj Meyepiece NAobj Mtot Museful Magnification

10x 10x 0.35 100 175-350 low

40x 10x 0.70 400 350-700 ok

100x 10x 1.40 1000 700-1400 ok

100x 15x 1.40 1500 700-1400 empty

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Light budget in microscopeMicroscope 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, dichroicsUse clean optics

Image brightness (transmission) ~ (NA/M)2

Image brightness (fluorescence) ~ NA4/M2

Use high NA objectivesDo not use unnecessary high magnification

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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

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4. Contrasting methods

Dark fieldPhase contrastDICPlasDIC

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Amplitude and phase specimens

Amplitude specimen changes the intensity of incident lightPhase specimen changes the phase of incident light

Most unstained biological specimens are phase ones

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Examples of contrasting methods

Dark fieldBone thin section

DICNeurons

Phase contrastHEK cells

PlasDICHEK cells

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Darkfield contrast

5 – iris diaphragm4 - objective3 - sample2 - condenser1 - phase stopA - low NA objectiveB - high NA objective with iris

Required: special condenser, sometimes immersion oilPrinciple: direct light is rejected or blocked, only scattered light is observedDisadvantage: low resolution

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Interference

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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.



Phase Contrast Microscopy



Phase contrast microscopy9 - intermediate image8 - tube lense7 - indirect light6 - direct light5 - phase ring4 - objective3 - sample2 - condenser1 - 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

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Differential interference contrast9 - intermediate image8 - tube lens7 – analyzer7a - λ-plate6 - Wollaston prism5 – objective 4 – sample3 – condenser 2 – Wollaston prism1 - polariser

Required: special accessories in light path (prisms, polarizers). Principle: specimen is sensed with two linear polarized slightly shifted (<λ) light beams. Difference in optical path of the beams gives a contrast in image.Disadvantages: accessories are relatively expensive.

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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.

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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.

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Adjustment for DIC

(for inverted microscope)

• Set up Koehler illumination in transmission• Move specimen out of the light path• Insert polarizer, analyzer, lower Wollaston prism and remove eyepiece• Shift Wollaston prism to place dark line in the center • Turn polarizer until the line is seen mostly dark• Insert eyepiece back into eyetube• Insert upper Wollaston prism (in condenser)• Move the specimen back into the light path• Move lower Wollaston prism to get required contrast• Rotate the stage to highlight desired area in the sample• Insert the lambda plate if color staining is required

• Repeat procedure for each objective being used

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PlasDIC

Required: slit diaphragm, prism with polarizer, analyzer.

Principle: A slit diaphragm creates a pair of non-polarized light beams that are λ/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.

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Contrasting techniques: summaryDark fieldFine structural features at, and even below, the resolution limit of a light microscope. Highly suitable for metallographic and crystallographic examinations with reflected light.

Phase contrastUsed for visualizing very fine structural features in tissues and single cells contained in very thin (< 5 µm), non-stained specimens.

DICMethod shows optical path differences in the specimen in a relief-like fashion. The method is excellently suited for thick, non-stained specimens (> 5 µm). Can be used for optical sectioning.

PlasDICThe same specimen as conventional DIC but in plastic dishes.

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More about light microscopy1. Lecture

Biomicroscopy I + II, Prof. Theo Lasser, EPFL

2. Booksa) Digital microscopy, Sluder, G; Wolf, D.E., eds, Elsevier, 2003b) Optics, 4th ed., Eugene Hecht, Addison-Wesley, 2002

3. Interneta) http://micro.magnet.fsu.edub) b) Web sites of microscope manufactures

LeicaNikonOlympusZeiss

4. BIOpEPFL, SV-AI 0241, Sv-AI 0140http://biop.epfl.ch/

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http://micro.magnet.fsu.edu/�

http://biop.epfl.ch/�



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.

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