Brightfield and phase contrast microscopy
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Brightfield and Phase Contrast Microscopy Microscope: Micro = Gk. “small” + skopien = Gk. “to look at” "Microscope" was first coined by members of the first "Academia dei Lincei" a scientific society which included Galileo Microscopes Upright Inverted Köhler Illumination

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Slide2 l.jpg

Microscope: Micro = Gk. “small” + skopien = Gk. “to look at”


Microscopes l.jpg

"Microscope" was first coined by members of the first "Academia dei Lincei" a scientific society which included Galileo

Microscopes

  • Upright

  • Inverted

  • Köhler Illumination

  • Dissecting (Stereoscopic)

J. Paul Robinson – Purdue University


Euglena viridis green in the middle and before and behind white antony van leeuwenhoek 1674 l.jpg
Euglena viridis "Academia dei Lincei" a scientific society which included Galileo - “green in the middle, and before and behind white”Antony van Leeuwenhoek - 1674


Earliest microscopes l.jpg
Earliest Microscopes "Academia dei Lincei" a scientific society which included Galileo

  • 1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor city official.

  • Leeuwenhoek is incorrectly called "the inventor of the microscope"

  • Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683

  • Could reach magnifications of over 200x with simple ground lenses - however compound microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke claimed they were too difficult to use - his eyesight was poor.

  • Discovered bacteria, free-living and parasitic microscopic

  • protists, sperm cells, blood cells, microscopic nematodes

  • In 1673, Leeuwenhoek began writing letters to the Royal

  • Society of London - published in Philosophical Transactions

  • of the Royal Society

  • In 1680 he was elected a full member of the Royal Society,

  • joining Robert Hooke, Henry Oldenburg, Robert Boyle,

  • Christopher Wren

J. Paul Robinson – Purdue University


So why are imaging systems needed l.jpg
So why are imaging systems needed? "Academia dei Lincei" a scientific society which included Galileo

  • Every point in the object scatters incident light into a spherical wave

  • The spherical waves from all the points on the object’s surface get mixed together as they propagate toward you

  • An imaging system reassigns (focuses) all the rays from a single point on the object onto another point in space (the “focal point”), so you can distinguish details of the object


Pinhole camera is simplest imaging instrument l.jpg
Pinhole camera is simplest imaging instrument "Academia dei Lincei" a scientific society which included Galileo

  • Opaque screen with pinhole blocks all but one ray per object point from reaching the image space

  • An upside-down image is formed

  • BUT most of the light is wasted (it is stopped by the opaque sheet)

  • Also, diffraction of light as it passes through the small pinhole produces artifacts in the image


Imaging with lenses doesn t throw away as much light as pinhole camera l.jpg
Imaging with lenses: doesn’t throw away as much light as pinhole camera

Collects all rays that pass through solid-angle of lens


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Refractive index is dependent on a ray of illumination pinhole camera

entering a medium of differing density causing the

beam to bend

Classical optics: The refractive index changes abruptly at a surface and is constant between the surfaces. The refraction of light at surfaces separating media of different refractive indices makes it possible to construct imaging lenses. Glass surfaces can be shaped so that the angle at which the ray strikes it can differ.


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In light optics this is accomplished when a pinhole camera

wavelength of light moves from air (optical density of 1.0) into glass (O.D. 1.4 – 1.6).


Thin lenses part 1 l.jpg
Thin lenses, part 1 pinhole camera


Thin lenses part 2 l.jpg
Thin lenses, part 2 pinhole camera


Ray tracing with a thin lens l.jpg
Ray-tracing with a thin lens pinhole camera

  • Image point (focus) is located at intersection of ALL rays passing through the lens from the corresponding object point

  • Easiest way to see this: trace rays passing through the two foci, and through the center of the lens (the “chief ray”)


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Resolution pinhole camera ½ 


Slide16 l.jpg

0.61 pinhole camera

R.P. = ----------

N.A.

Ernst Abbe

1840 - 1905

 = wavelength of illumination

N.A. = n (sine α)

n = index of refraction

α = half angle of illumination


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In light microscopy the N.A. of a lens and therefore resolution can be increased by:

a) increasing the half angle of illumination,

b) increasing the refractive index of the lens by using Crown glass and

c) decreasingthe wavelength

() of illumination.

0.61 

R.P. = ----------

N.A.


Object resolution l.jpg
Object Resolution resolution can be increased by:

  • Example:

    40 x 1.3 N.A. objective at 530 nm light

.00053

= 0.20 m

=

2 x 1.3

2 x NA

40 x 0.65 N.A. objective at 530 nm light

.00053

= 0.405 m

=

2 x .65

2 x NA

J. Paul Robinson – Purdue University


Slide19 l.jpg

Images reproduced from: resolution can be increased by:

http://micro.magnet.fsu.edu/

Please go to this site and do the tutorials

J. Paul Robinson – Purdue University


Microscope objectives l.jpg
Microscope Objectives resolution can be increased by:

Standard Coverglass Thickness

#00 = 0.060 - 0.08

#0 = 0.080 - 0.120

#1 = 0.130 - 0.170

#1.5 = 0.160 - 0.190

#2 = 0.170 - 0.250

#3 = 0.280 - 0.320

#4 = 0.380 - 0.420

#5 = 0.500 - 0.60 mm

Microscope

Objective

60x 1.4 NA

PlanApo

Oil

Stage

Coverslip

Specimen

J. Paul Robinson – Purdue University


Slide21 l.jpg

Refractive Index resolution can be increased by:

Objective

n = 1.52

n = 1.5

n = 1.0

Oil

n = 1.52

Air

n = 1.52

n=1.52

Coverslip

n=1.33

Specimen

Water

n=1.52

J. Paul Robinson – Purdue University


The issues between simple and compound microscope l.jpg
The issues between simple and compound microscope resolution can be increased by:

  • Simple microscopes could attain around 2 micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration

  • In the 1730s a barrister named Chester More Hall observed that flint glass (newly made glass) dispersed colors much more than “crown glass” (older glass). He designed a system that used a concave lens next to a convex lens which could realign all the colors. This was the first achromatic lens.

J. Paul Robinson – Purdue University


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Converging (positive) lens resolution can be increased by: : bends rays toward the axis. It has a positive focal length. Forms a real

inverted image of an object placed to the left of the first focal point and an erect virtual image of an

object placed between the first focal point and the

lens.


Real and virtual image formation by biconvex lenses l.jpg
Real and virtual image formation by biconvex lenses resolution can be increased by:

  • Lens focal point

  • For an object further away than the lens focal point, an inverted, real image will be formed on the opposite side of the lens

  • For an object closer than the focal point, a virtual image will be formed on the same side of the lens

  • http://micro.magnet.fsu.edu/primer/java/lens/bi-convex.html


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Diverging (negative) lens resolution can be increased by: : bends the light rays

away from the axis. It has a negative focal length.

An object placed anywhere to the left of a diverging

lens results in an erect virtual image.


Compound microscope l.jpg
Compound Microscope resolution can be increased by:

  • The compound microscope uses at least two lens systems

    • The objective forms an intermediate real image of the object at the objective tube length

    • The ocular forms a virtual image of that intermediate image to the retina of the eye

    • If we are dealing with a photodetector, we must use a projection lens to form a real image from the intermediate image


Slide27 l.jpg

The compound microscope differs from the simple, single lens microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).

Today the Objective lens is a multi-element lens, thus the number of lenses in a modern microscope can easily exceed 20.


Ray tracings in the microscope l.jpg
Ray Tracings in the microscope microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).


K hler l.jpg
Köhler microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).

  • Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light

  • Two conjugate image planes are formed

    • one contains an image of the specimen and the other the filament from the light

J. Paul Robinson – Purdue University


K hler illumination l.jpg
Köhler Illumination microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).

eyepiece

condenser

Specimen

Field stop

Field iris

retina

Conjugate planes for image-forming rays

Specimen

Field stop

Field iris

Conjugate planes for illuminating rays

J. Paul Robinson – Purdue University


Current microscope objective tend to be infinity corrected l.jpg
Current microscope objective tend to be infinity corrected microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).

  • Infinite tube length

  • Require an additional lens in objective to converge beam

  • Advantages

    • Objectives are simpler

    • Optical path is parallel through the microscope body:


Infinity correction l.jpg
Infinity correction microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).


Other lenses l.jpg
Other lenses microscopes in that it consists of a minimum three lenses (condensor, objective, and projector).

  • Collector

  • Condenser

    • Allow us to use point light sources instead of parallel illumination

    • Also (later) increase the resolution of the microscope

  • Ironically, van Leeuwenhoek, who used simple non-compound, single-lens microscopes, was using the lens of his eye as a projection lens!


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Light that passes both around and through the specimen undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.

micro.magnet.fsu.edu/primer/anatomy/image.html


Airy disc l.jpg
Airy disc undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.


Lens resolution l.jpg
Lens Resolution undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.

  • Geometric optics predicts lenses of infinite resolution

  • However, because of the phenomenon of diffraction, every point in the object is converted into an Airy disc

  • Diameter of Airy disc:

    D = 1.22 X λ / n sin α, or

    D = 1.22 X λ / NA


We cannot resolve objects whose airy discs overlap by 20 l.jpg
We cannot resolve objects whose Airy discs overlap by ~20% undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.

As a consequence, Abbe’s rule is that d=λ/NA

http://micro.magnet.fsu.edu/primer/java/microscopy/airydiscs/index.html


Objective markings l.jpg
Objective markings undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.


Reading an objective l.jpg
Reading an objective undisturbed in its path is called direct light or undeviated light. The background light passing around the specimen is also undeviated light. Some of the light passing through the specimen is deviated when it encounters parts of the specimen. Such deviated light is rendered one-half wavelength or 180 degrees out of phase with the direct light that has passed through undeviated. The one-half wavelength out of phase caused by the specimen itself enables this light to cause destructive interference with the direct light when both arrive at the intermediate image plane at the diaphragm of the eyepiece.

http://micro.magnet.fsu.edu/primer/anatomy/specifications.html


For a typical 1 3 na lens at 525 nm the limit of resolution is 400 nm l.jpg
For a typical 1.3 NA lens at 525 nm, the limit of resolution is ~ 400 nm

  • How to improve?

    • Larger NA (lenses, immersion fluid)

    • Shorter λ

  • Add a condensor:

    D = λ / (NAobj. + NAcond.)

  • So, for a 1.3 NA lens and condensor, D drops to ~200 nm


Abberations l.jpg
Abberations is ~ 400 nm

  • Spherical aberration

    • Most severe

    • Immersion fluid

  • Field curvature

  • Chromatic aberration

  • Astigmatism, coma

  • http://micro.magnet.fsu.edu/primer/lightandcolor/opticalaberrations.html


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Lens Defects is ~ 400 nm

The fact that wavelengths enter and leave the lens field at different angles results in a defect known as spherical aberration. The result is that wavelengths are brought to different focal points .


Slide43 l.jpg

Spherical aberrations are worst at the periphery of a lens so a small opening aperture that cuts off the most offensive part of the lens is the easiest way to reduce the effects of spherical aberration but throws away a lot of the available illumination (i.e. pinhole camera)


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Lens Defects so a small opening aperture that cuts off the most offensive part of the lens is the easiest way to reduce the effects of spherical aberration but throws away a lot of the available illumination (i.e. pinhole camera)

Since the focal length f of a lens is dependent

on the strength of the lens, if follows that different wavelengths will be focused to different positions. Chromatic aberration of a lens is seen as fringes around the image due to a “zone” of focus.


Slide45 l.jpg

Lens Defects so a small opening aperture that cuts off the most offensive part of the lens is the easiest way to reduce the effects of spherical aberration but throws away a lot of the available illumination (i.e. pinhole camera)

In light optics wavelengths of higher energy (blue) are bent more strongly and have a shorter focal length

In the electron microscope the exact opposite is true in that higher energy wavelengths are less effected and have a longer focal length


Slide46 l.jpg

The simplest way to correct for chromatic aberration is to use illumination of a single wavelength!

Such illumination is called monochromatic .


Slide47 l.jpg

Lens Defects use illumination of a single wavelength!

In light optics chromatic aberration can be corrected by combining a converging lens of one O.D. with a diverging lens of a different O.D. This is known as a “doublet” lens


Brightfield microscopy l.jpg
Brightfield microscopy use illumination of a single wavelength!

  • Generally only useful for stained biological specimens

  • Unstained cells are virtually invisible

Brightfield

Phase contrast


Oblique illumination l.jpg
Oblique illumination use illumination of a single wavelength!


Oblique l.jpg
Oblique use illumination of a single wavelength!


Darkfield l.jpg
Darkfield use illumination of a single wavelength!


Radiolarian in darkfield l.jpg
Radiolarian in Darkfield use illumination of a single wavelength!

http://micro.magnet.fsu.edu/primer/techniques/darkfield.html


Phase contrast l.jpg
Phase contrast use illumination of a single wavelength!

http://microscopy.fsu.edu/primer/techniques/phasegallery/chocells.html


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