BOT 205: Microscopy and Histology

Lecture 3: Image & image characteristics

Khayriyyah Mohd Hanafiah, PhD
kye@usm.my
 • EMR is classified by wavelength
• Visible light is the only one human eyes can see
• EM waves are characterized by amplitude,
frequency, and wavelength
• Duality of light refers to its wave-like and particle-
like properties, of which wave-like properties are
most relevant in microscopy
• Light interacts with matter through: transmission,
Revision absorption, refraction, dispersion, reflection,
diffraction
Learning
outcomes
At the end of this lecture,
students will be able to
explain:

• Real vs Virtual Images

• Image formation for convex
lenses
• Magnification, contrast,
resolution
1.Real Images
• are images which form when outgoing light
rays actually come from a point i.e. light
actually converges
• can be caught/made visible on a screen
placed in the plane of the image
• e.g. image seen on cinema screen
(source – projector, with film), camera
• can be produced by concave mirrors &
converging/convex lenses only if object is
placed outside (further away) focal length
of mirror/lens
• Image may be magnified or reduced
depending on position of object
E.g. looking directly at Benedict Cumberbatch
enjoying a cuppa in front of you
• Ray tracing gives the location, size, orientation, and type of image that
is formed by the double convex lens
• Real light rays represented by full solid lines
• Real images are inverted!
• Position of image can also be determined using lens equation

u v

The image here is larger than the object because the

object is between f and 2f away from the lens; if the
lens were farther away than 2f, the image would be
closer to the lens than 2f, and would be smaller than
the object.
 Ray 1 Ray 1
Ray 2 Ray 2

Ray 3 Ray 3

Incidents rays from the object:

• Ray 1: travels parallel to the principal axis of a converging lens, refract
through the lens and pass focal point on the opposite side of the lens.
• Ray 2: passes through the center unrefracted.
• Ray 3: pass through the focal point on the way to the lens, refracts through
the lens and travel parallel to the principal axis.
2. Virtual Images
Virtual image: Image which forms when
light rays do not actually come from a point
on an object but they "seem" to come from
that point
• Rays only perceived to converge i.e.
outgoing rays from that point always
diverge
• E.g. When looking at something’s
reflection in a mirror, you cannot touch
the object in the mirror because it isn't
actually there
• image exists in a space that is not
real i.e. virtual
• appears to be as far behind mirror as
object is in front of mirror
• image not magnified (same size as
object)
• Virtual images are formed by diverging (concave) lenses or by placing an
object inside the focal length (<1F) of a converging (convex) lens.
• If the ray-tracing exercise is repeated for a virtual image:
• Rays represented by dashed lines
• Virtual images are upright

u
v

*focal length f is positive for a convex lens

(enlarges), negative for concave lens (shrinks)
 Ray 1

Ray 3

Ray 2

Incidents rays from the object:

• Ray 1: travels parallel to the principal axis, refracts through the lens and travel in
line with the focal point (i.e., in a direction such that its extension will pass
through the focal point).
• Ray 2: passes through the center of the lens unrefracted.
• Ray 3: traveling towards the focal point on the way to the lens will refract
through the lens and travel parallel to the principal axis.
Image Type Real Image Virtual Image
Ray source Comes from object Does not come from object
(only seem to)
Rays where image Converge Diverge (but appear to
forms converge)
Capture on screen Possible Not possible without lens
Image orientation Inverted Upright/same as object
Appearance in ray Solid lines Dashed lines
diagrams

Real vs Virtual Image

A VIRTUAL IMAGE
“When you look into a microscope, you are not
looking at the specimen, you are looking at
the image of the specimen. The image appears to
be "floating" in space about 10 millimeters below
the top of the observation tube (at the level of
the fixed diaphragm of the eyepiece) where the
eyepiece is inserted. The image you observe is
not tangible; it cannot be grasped.
It is a "map" or representation of the specimen
in various colors and/or shades of gray from black
to white.”
https://micro.magnet.fsu.edu/primer/anatomy/m
agnification.html
3. Sign Convention for Lenses

OR

u = distance between lens and object Ensure that sign is consistent when calculating!
v = distance between image and lens
D = least distance of distinct vision from lens or eye
f = focal length of lens
Question: If an object is 6cm away from a convex lens of focal length 3cm
where will the image be?
(Draw a ray diagram of how this image is formed to see if your answer is
plausible.)
Answer:
The question states that u = 6 cm and f = 3 cm.
This can be substituted into the lens equation as follows:
 • Definite relationship between image
characteristics & location of object when placed in
front of a double convex lens
• Object-image relationships best summarized into 5
cases:
• Case 1: object located beyond 2F point
• Case 2: object located at 2F point
• Case 3: object located between 2F point & F
4. Convex • Case 4: object located at F
• Case 5: object located in front of F
Lens Action
in Image
Formation
 • Case for camera for ordinary scenic photography
• Object located beyond 2F point
• Image always located somewhere in between 2F
point & focal point (F) on the other side of lens
opposite object
• Image smaller than object, real & inverted
• magnification number with absolute value < 1
• real image can be captured on film or other
4.1 Case 1: imaging device
• light rays converge at image location
Object located
beyond 2F point
4.2 Case 2: Object located at 2F

• Case for so-called 1:1 photography

• When object located at 2F point
• image also located at 2F point on other side of
lens
• image inverted & real image
• light rays actually converge at image location
• image size equals to object size
• absolute value of magnification exactly 1
• Case for ordinary portrait photography
• When object closer to front of lens but still > 2F
in front of lens
• image found further behind lens image larger
than in case 1 but still smaller than object
• image inverted & real
 • Describes function of finite tube lengths objectives
in microscopy
• When object located in front of 2F point
• image always located beyond 2F on other
side of lens
• image inverted & real - light rays converge at
image location
• image larger than object dimension
4.3 Case 3: • absolute value of magnification > 1
Object located • Finite tube length objectives project a real,
inverted, magnified image into body tube of
between 2F & F microscope
• image designed to be focused at plane of
fixed diaphragm in eyepiece
 • When object is located at focal point
• no image appears (formed at infinity?)
• refracted rays neither converge or
diverge
• refracting light rays travel parallel to each
other & cannot produce an image

4.4. Case 4:
Object
located at F
 4.5 Case 5:Object located in front of F
• Describes function of eyepiece of microscope
• When object located at a location in front of focal point
• image always located somewhere on same side of lens as object but image
located behind object
• image is upright/erect, enlarged & virtual
• magnification > 1
• since image is virtual it cannot be captured on screen because light does not
pass through image location

Object situated at front focal plane of eyepiece is the

magnified, inverted, real image projected by objective.
It is virtual and appears as if 25cm from eye. Eyepiece
has similar function to magnifying glass, magnification
depends on lens curvature.
 4.6 Summary of Image Formation
Nature of
Position of object Position of image Size of image Application
image
Objective lens of
Beyond 2F1 Between F2 & 2F2 Inverted real Diminished
a telescope
Photocopying
At 2F1 At 2F2 Inverted real Same size
camera
Photographic
camera,
Between 2F1 & F Beyond 2F2 Inverted real Magnified terrestrial
telescopes,
Photocopier
At F1 At infinity Inverted real Magnified Search lights
On same side of Magnifying lens,
Between O & F1 Erect virtual Magnified
lens eye piece
5. Tasks of a microscope

• Produce a magnified image of the specimen (magnification)

• Separate the details in the image (resolution)
• Render the details visible to the human eye or camera (contrast)
5.1
5.2
5.3
 5.3.1 Diffraction Barrier
• Resolution limitations often referred to as the diffraction barrier
• Optical instruments have circular apertures (pupils, diaphragm & lenses of
microscope)
• produce diffraction patterns similar to those described earlier, except the
pattern has a circular symmetry
• Size of central spot in Airy disk relates to the wavelength of light and the aperture
angle of the objective:

Abbe Resolutionx,y = λ/2NA

λ =average wavelength of illumination in transmitted light.

The objective numerical aperture (NA = n•sin(θ)) is defined by
the R.I (n) of the imaging medium (e.g. air or oil) multiplied by
the sine of the aperture angle (sin(θ))
 The effect of objective aperture angle
on the size of a diffraction spot:
• Difference in point source and its
conjugate (P) in the image plane
where wavefronts converge and
undergo constructive interference
for objectives with high & low NA
• The point P1 is moved laterally in
the focal plane until destructive
interference at a certain distance
(dictated by the objective
numerical aperture)
• defines the location of the
first diffraction minimum and
thus the radius of the
diffraction spot.
Figure 1 - Resolution Limit Imposed by Wave
Nature of Light
Recap & Take Home Messages
• There are definite relationships between image characteristics & location
of object when placed in front of a double convex lens
• Real images are inverted, light ray converges to a point
• Virtual images are upright, lights do not converge to a point (diverge)
• Image formation can be summarized in 5 cases, pertaining to
distance of object from focal point
• Tasks of microscope include magnification, contrast and resolution
• Diffraction barrier determine limit of resolution
References
• http://micro.magnet.fsu.edu/primer/anatomy/magnification.html
• http://www.tutorvista.com/content/physics/physics-ii/optical-
instruments/simple-microscope.php
• http://www.tutorvista.com/content/physics/physics-iv/optics/compound-
microscope.php
• http://www.physicsclassroom.com/class/refrn/Lesson-5/Converging-Lenses-
Ray-Diagrams
• Dr Suriyati Mohamad’s previous lecture notes

"Nature composes some of her

loveliest poems for the
microscope and the telescope."
-Theodore Roszak-