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Tutorial on Microscopy September 15, 2007. Why the need to study microscopy ? It is a tool complementary to molecular biology It has become an indispensable tool for many biologists and pathologists to check sterility of cultures to study the histology of biopsies

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slide1

Tutorial on Microscopy

September 15, 2007

slide2

Why the need to study microscopy?

  • It is a tool complementary to molecular biology
  • It has become an indispensable tool for many
  • biologists and pathologists
      • to check sterility of cultures
      • to study the histology of biopsies
      • to study the developmental program of organ
      • to follow the movement of a protein from
      • the cytoplasm to the chloroplast
slide3

Students need to understand the microscope

  • to get a sense of size,
  • to get the best image possible,
  • to learn how to enjoy using it,
  • to use it for cool and fun purposes,
  • to keep it in good shape,
  • to be able to share the instrument with others.
slide4

MOTIC

ZEISS

slide5

Eye-piece or ocular

Revolving nose

with objectives

slide6

Eye-piece or ocular

Revolving nose

with objectives

Stage and its controls

Focussing knob

slide7

Eye-piece or ocular

Revolving nose

with objectives

Stage

and its controls

Condenser

Light source

Focussing knob

slide8

Light and lenses are the two pieces of equipment

which are used to manipulate the light. Both are

inherent to the microscope you used; they cannot

be changed.

slide9

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular
slide10

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular
slide11

The condenser: a combination of lenses

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

slide12

The condenser: a combination of lenses

  • The simplest condenser
  • Role: to condense and
    • focus light onto the
    • specimen

Also called an iris

diaphragm

Allows more or less

light to enter the condenser

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

slide13

The condenser: a combination of lenses

  • Will possess specific
  • characteristics (correction,
  • numerical aperture and
  • others) depending on
  • manufacturer specifications

http://www.zeiss.com/C1256B5E0047FF3F?Open

slide14

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular
slide15

The objective: a combination of lenses

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

slide16

The objective: a combination of lenses

Motic Zeiss

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

slide17

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

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

slide18

Achromat:

Good color correction – exactly for two wavelengths.

Field flatness in the image center, refocusing also covers

the peripheral areas. Designed for fields of view up to

18 mm diameter.

Versions for phase contrast. Budget-priced objectives.

Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism.

http://www.zeiss.com/C1256B5E0047FF3F?Open

slide19

The effect of chromatic aberration

Rays of longer λ focus further away that those of shorter λ.

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

slide20

The effect of chromatic aberration

Rays of longer λ focus further away that those of shorter λ.

Rays of longer λ focus further away that those of shorter λ.

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

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

slide21

Motic

Plan and Epiplan:Improved Achromat objectives with good image flatness

for fields of view with dia. 20 or even 23 mm. Therefore

ideal for photomicrography.

Zeiss

Achromat:

Good color correction – exactly for two wavelengths.

Field flatness in the image center, refocusing also covers

the peripheral areas. Designed for fields of view up to

18 mm diameter.

Versions for phase contrast. Budget-priced objectives.

Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism.

http://www.zeiss.com/C1256B5E0047FF3F?Open

slide22

Field curvature: the sharpest focus of a lens is on a curved surface rather than on a flat plane.

Plant microtechnique and microscopy. E. Ruzin

slide23

http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffieldhttp://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

slide24

http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffieldhttp://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

slide25

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

4x / 0.10

10x / 0.25

40x / 0.65

Magnification / Numerical Aperture

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

slide26

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

Magnification / Numerical Aperture

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

slide27

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

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

slide28

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

∞ / 0.17 ∞ / -

∞ / 0.17

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

slide29

All the objectives mentioned here are members of the family

of ICS-Optics (ICS: Infinity Color-corrected System). These

objectives project their images to “infinity” first. Only the tube

lens produces an intermediate image – to be more precise, at

a distance of approx. 164.5 mm behind the tube lens. This

distance was chosen to comply with the classical tube length.

slide30

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

∞ / 0.17 ∞ / -

∞ / 0.17

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

slide31

A coverslip is

  • part of the image-forming system,
  • a lens element,
  • its power has been taken into account by the manufacturer,
  • its thickness and the making of the glass will affect the
    • deviation of the light.
slide32

Klosevych, 1989

The thickness of the coverslip and the refractive index of the glass will have an effect of the light path

1 thickness= 0.13 to 0.17 mm

Satisfactory for NA ≤ 0.4.

slide33

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

∞ / 0.17 ∞ / -

∞ / 0.17

Any questions?

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

slide34

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular
slide35

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

Eyepieces are not just simple lenses, but are corrected optical systems consisting of several lenses.

slide36

http://www.zeiss.com/C1256B5E0047FF3F?Open

1. Position of the intermediate

image (also for the reticle)

2. Limit of the field of view

(black edge of image)

3. Eye-piece optics

(Ramsden ocular)

4. Position of the eyepiece pupil

(pupil of the observer’s eye)

5. Focusing ring for the diopter

compensation

Eyepieces: magnifiers to view the intermediate

Image produced by the objective and the tube lens.

slide37

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular

There will be specifications on these lenses

WF for Wide Field of view

PL to match the objective correction

Magnification 10X

Field number which refers to

the diameter (in mm) of the fixed

diaphragm in the eyepiece.

Motic

WFPL 10x / 20

Glasses symbol

slide38

The lenses: three types in your microscope

  • The condenser
  • The objective
  • The eye-piece or ocular

There will be specifications on these lenses

Zeiss

PL 10x / 18

Glasses symbol

Designed for eyeglass wearers.

The exit pupil is at a considerable

distance from the eyepiece.

slide39

Final image

Intermediate

image

Specimen

slide40

What is the main purpose of the microscope?

  • The condenser
  • The objective
  • The eye-piece or ocular
slide41

What is the main purpose of the microscope?

  • The condenser
  • The objective
  • The eye-piece or ocular
  • To condense and focus light onto the specimen
slide42

What is the main purpose of the microscope?

  • The condenser
  • The objective
  • The eye-piece or ocular
  • To condense and focus light onto the specimen
  • To form a clear intermediate image
slide43

What is the main purpose of the microscope?

  • The condenser
  • The objective
  • The eye-piece or ocular
  • To condense and focus light onto the specimen
  • To form an intermediate image
  • To form the final image
slide44

Final image

Intermediate

image

Specimen

Any questions?

slide45

What is the main purpose of the microscope?

  • Magnification: apparent increase in size
  • Resolution: the minimum distance between
  • 2 dots that can be discerned
slide46

Airy disc: defined as

the region enclosed

by the first minimum of

the Airy pattern and

contains 84 % of the

luminous energy.

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

slide47

Resolution: the minimum distance between 2 dots that can be discerned

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

slide48

The smaller the diameter of the Airy disc produced by

a lens, the higher is the resolving power of that lens,

the better you can separate two distinct points.

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

slide49

Resolution = (0.61 λ) / Numerical Aperture

The larger the numerical aperture of a lens,

The smaller the Airy disc,

The smaller and better the resolution.

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

slide50

Airy disk sizes vary with changes in objective

numerical aperture and illumination wavelength.

Resolution = (0.61 λ) / Numerical Aperture

Resolution = (0.61 λ) / (n x sin θ)

λ: wavelength of light

n: refractive index of the medium in the object space

Θ: angular aperture

slide51

Resolution = (0.61 λ) / Numerical Aperture

Resolution = (0.61 λ) / (n x sin θ)

λ: wavelength of light

n: refractive index of the medium in the object space

Θ: angular aperture.

slide52

Objective

= θ

Specimen

  • Angular apertureis a measure of the number of the highly
  • diffracted image-forming light rays captured by the
  • objective
  • expressed as the angle between the microscope optical
  • axis and the direction of the most oblique light rays
  • captured by the objective.

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

slide53

Resolution = (0.61 λ) / (n x sin θ)

In theory, θ cannot be superior to 90o

sin(θ) cannot be superior to 1.

In the best microscopes, θ is about 70o

with a sine of0.94.

The other limitation for the objective is

the refractive index of the medium;

generally, it is air with n=1.

slide54

Resolution = (0.61 λ) / (n x sin θ)

If in air and maximum θ, thenr = (0.61 λ) / 0.94

where 0.61 represents the degree to which image points can overlap and still be recognized by an observer as separate points

  • the lower the wavelength,
  • the lower and better the resolution,
slide55

Resolution = (0.61 λ) / (n x sin θ)

If in air and maximum θ, thenr = (0.61 λ) / 0.94

Monochromatic light better

Possibility to use coloured filters

If blue light, λ = 450 nm, r = 274.5 / 0.94 = 292 nm

If green light, λ = 550 nm, r = 335.5 / 0.94 = 356 nm

If UV light, λ = 250 nm, r = 152.5 / 0.94 = 162 nm

Because the λ of an e- is much shorter than a that of a photon, resolution is much greater in an e- microscope (0.2 nm).

  • The shorter the λ,
  • The better the resolution
slide56

Resolution = (0.61 λ) / (n x sin θ)

To further improve resolution, to visualize

minute specimens, one could use a medium with

a n higher than that of air.

slide57

Use of immersion lenses

allows more light to be

collected

More points to

form an image,

Better resolution

There is no diffraction

of light because of the

homogeneity of the

refractive indices.

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

slide59

Resolution = (0.61 λ) / (n x sin θ)

If maximum θ and blue light, thenr = 274 / (n x 0.94)

If air (n = 1), r = 274 / 0.94 = 292 nm

If 50% glycerol, r = 274 / (1.4 x 0.94) = 208 nm

If generic imm. oil, r = 274 / ( 1.515 x 0.94) = 192 nm

If permount, r = 274 / (1.525 x 0.94) = 191 nm

If Canada balsam, r = 274 / (1.545 x 0.94) = 189 nm

  • The larger the refractive index of the medium,
  • The better the resolution.
slide60

angular aperture

NA = n • sin(θ)

The larger the angular aperture,

The higher the numerical aperture,

The more light the lens can capture,

The more information the lens can transmit.

The larger the numerical aperture of a lens,

The smaller the Airy disc,

The better the resolution.

Any questions?

slide61

The objective: a combination of lenses

Motic Zeiss

Plan CP-Achromat

5x / 0.12

10x / 0.25

40x / 0.65

100x / 1.25 oil

4x / 0.10

10x / 0.25

40x / 0.65

∞ / 0.17 ∞ / -

∞ / 0.17

r = (0.61 λ) / NA

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

slide62

It would be a pity if the intermediate

image produced with such sophisticated optics were to be impaired just before it reaches the eye because of poor handling of the microscope.

Koehler illumination

slide63

Koehler or Köhler illumination

  • Switch on the microscope
  • Check that the light is on

http://www.zeiss.com

slide64

Koehler or Köhler illumination

  • Open wide the field diaphragm:
  • the spot of light should be at
  • its maximum diameter

http://www.zeiss.com

slide65

Koehler or Köhler illumination

  • Open wide the iris
  • diaphragm of the
  • condenser. The small
  • light spot shows its
  • maximum brightness

http://www.zeiss.com

slide66

Koehler or Köhler illumination

  • Place slide on stage
  • Reduce brightness of light if need be
  • Position the oculars so that you are comfortable when looking through them
  • Adjust your view by focussing the diopter compensation ring

http://www.zeiss.com

slide67

Relaxed viewing is important

First, look into the distance with your eyes relaxed

and then into the eyepieces – without changing the

setting of your eyes. Only then should you set the

inter-pupillary distance of the eyepieces via the folding

bridge until you see only one circle instead of two.

Remember to use both your eyes for viewing.

slide68

Keep your distance

Microscopes for teaching labs are usually designed for eyeglass wearers. Therefore, the exit pupil of the eyepiece is at a considerable distance from the eyepiece. Users who do not wear eyeglasses should also keep this distance to permit the entire light from the microscope to find its way to the iris of the eye. If you slowly move your head to and fro in front of the eyepieces, you will soon find the optimum, relaxed posture allowing you to see the entire circle of the field of view.

slide69

Koehler or Köhler illumination

  • Focus grossly on the specimen

http://www.zeiss.com

slide70

Koehler or Köhler illumination

  • Close the field diaphragm
  • Focus the condenser by
  • using the focussing knob
  • on the left of the microscope

http://www.zeiss.com

slide71

Koehler or Köhler illumination

When the condenser is

focussed, you will see the

sharp edges of the field

diaphragm

http://www.zeiss.com

slide72

Koehler or Köhler illumination

  • Center the light with
  • the centering knobs
  • placed below the stage

You should now see a

centered image of the

specimen surrounded by

black

http://www.zeiss.com

slide73

Koehler or Köhler illumination

  • Open now the field diaphragm
  • Do open just enough to fill the field of view

Do not touch again the condenser knob,

because your condenser is now focussed

slide74

I was taught to set up Koehler illumination with

a focussed slide on the microscope stage but

with the specimen out of the field-of-view so that

its staining could not interfere with the light source.

I was also taught to use whenever possible a blue

filter so that a monochromatic light with a low λ

is obtained.

slide75

Koehler or Köhler illumination

  • Remove gently one of the eye-pieces (us. the left).
  • Look down the tube to see the back focal plane of
  • the objective

http://www.zeiss.com

slide77

Koehler or Köhler illumination

  • Three elements essential for optimal results:
    • circular outline of the objective aperture,
    • iris opening of the aperture diaphragm of the condenser,
    • image of the light source.

http://www.zeiss.com

slide78

Koehler or Köhler illumination

  • Three elements essential for optimal results:
    • circular outline of the objective aperture,
    • iris opening of the aperture diaphragm of the condenser,
    • image of the light source.
  • Adjust the size of the light disc by swinging
  • the iris diaphragm of the condenser

http://www.zeiss.com

slide79

Koehler or Köhler illumination

  • A compromise between resolution and contrast
  • 9/10: best resolution
  • 2/3: best contrast
slide80

For research purposes,

you should set-up Koehler each time you change objectives.

slide81

9/10 position of aperture diaphragm

Maximum resolution

2/3 position of aperture diaphragm

Maximum contrast

Maximum depth of detail

1/2 position of aperture diaphragm

Creation of artefacts

Klosevych, 1989

slide82

Microscopy and photomicrography. R.F. Smith, CRC Press

1

3

1: Aperture diaphragm = NAobj

lack of contrast and low visibility

2: Aperture diaphragm at 90%

better contrast &

excellent resolution

3: Aperture diaphragm at 50%

excessive diffraction &

creation of artefacts

2

slide83

Tips given by Zeiss:

  • Relaxed viewing is important
  • Keep your distance
  • Exclusively for eyeglass wearers: a little test
  • Avoid the use of force
  • Protect your investment: the dust cover
  • Please avoid “do-it-yourself” work on the microscope
slide84

Two invaluable web-sites to visit

when you want to learn more about microscopy:

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

http://www.zeiss.com

(Microscopy from the beginning)