Microscopy basics - I

BT-2062 (Lecture 1)
1st August 2023
Santhosh Sethuramanujam
Today…
• Microscopy
• Magnification
• Limitations of the eye
Brain – reticular theory vs neuron doctrine
• In the 19th century, cell theory was well-
established.
• But, the brain was an enigma. (Can a structure
that defines us be made of cells?)
• Observation was cell bodies connected by long
processes
• There was “no beginning and no end”.
• Hence, it was thought that the whole brain is
made of a continuous reticular network.
• Electrical information from one part of the brain
flows to any other part through this network.
• Camillo Golgi – greatest proponent
Microscopy to prove the neuron
doctrine.

• Ramon y Cajal used staining developed by

Golgi.
• Only 1/5 neurons are stained – individual
“neurons” can be seen
• Ends of the processes clearly visualized
• Proposed the neuron doctrine. Cells that
talk to each other (later discovery of
synapse, again using microscope)
Cajal’s
drawings
• Pyramidal
neurons/
interneurons
in cortex
• Purkinje
neurons in
cerebellum.
Whole networks were identified on the cellular
level

• Retina and hippocampus

• Newer techniques – better
resolution. But, his drawings are
still used today!
Magnification
• Magnification atleast by
400X
• 10 micron structure will look
like a 4 mm structure.

• How does a microscope

work?
• Think how you make objects
big visually…
• Why cant we keep bringing
the object closer?
Limitations of our vision
• Cross section of eye.
• Light path  cornea 
pupil  lens  retina.

• To understand the
limitations of our vision,
we have to understand
the way images are
created on the retina.
• Bigger the image on the
retina, bigger will be our
perception.
Lens magnify the
object
• Bi convex lens can magnify
the object under certain
conditions.

• Biconcave lens shrinks the

object.
Refraction

• Light slows down when passing from a medium of lower to higher density.
• Light bends towards the normal when entering a denser medium, and
bends away from the normal when entering lighter medium
Snell’s law – angle of refraction

• q – angle to the normal

• v – velocity of light
• N – refractive index.

• n2/n1 is a constant
 Lens – use refraction to focus light
• Convex lens
• Converging at the focal
point
• Concave lens
• Diverging from a focal
point.

• Note that as light enters

the denser medium, it
bends towards the
normal, while it exits the
denser medium, it moves
How does the curvature of lens affect focal point?
away from the normal
https://researchpedia.info/difference-between-convex-and-concave-lens/
Factors that change the focal length of the
lens…
• Increasing angle of
incidence will increase the
angle of refraction.
• Consequently, the focus will
become shorter and shorter
• This is achieved by
increasing the curvature of
the lens.
• This is how the eye works.
• How does this help the eye?
What is the relation between radius of curvature and focal point?
How else can you modify the focal point?
Lens equation

• Focal length decides

the distance at which
an image is formed
given the object
distance.

Why is the image of point A formed only at E?

what happens if you move it front or back?
Location of image formation with change in
distance.

• Case 1: u = 2f
v = 2f
• Case 2: u = 1.1f
v = 11f
• Case 3: u = 1f
• Case 4: u = .9f v = inf

• Case 5: u = .5f v = -9f

v = -1f

Usually f is fixed, and u and v will be changed

 Virtual and real images

• Object distance greater than f  real image convergence of the light.

• Object distance at f  image at infinity.
• Real images – inverted. Virtual images – upright.
• Object distance less than f  virtual image rays diverge, but our brain cannot
understand that light is refracted (or reflected)  so, an image is created where the brain
thinks the object is. (point of convergence)
The lens in the eye
2.5 cm
https://webvision.med.utah.edu/
• Cross section of eye.
• Light path  cornea 
pupil  lens  retina.
• Chambers filled with fluid
(not our concern here).
• Here v is fixed! So focus is
changed to view objects at
different distances.
• f = 2.27 – 2.5 cm
• Lens can be relaxed or
constricted to change the
focal length.
Limits of the eye…
• Case 1: Max distance at which object can be
viewed.
1
• f = 2.5 cm, v = 2.5 cm=
1 1
+
• u = inf 𝑓 𝑢 𝑣
2.5 cm

• Case 2: Min distance at which objected can be

viewed.
• f = 2.27 cm, v = 2.5 cm
• u = 25 cm
What happens at shorter distances….
• Minimum focal length of the lens in the eye is 2.27 cm. Where will the
image be formed when the object is 10 cm from the eye?
•

10 cm 2.94 cm
Magnification of the lens.
(linear)

• Magnification is the fold

increase in image size.
• Equal to the ratio of the
distance between object
and image
 Notice that u and v are not independent of each other.
A given u can only have one v, so magnification is a property of f

Region of maximum magnification.

• Case 1: u = 2f, v = 2f M=1

• Case 2: u = 1.1f, v = 11f M = 10

• Case 3: u = 1f, v = inf M = inf

• Case 4: u = .9f, v = -9f M = 10

• Case 5: u = .5f, v = -1f M=2

Region of maximum magnification.

• Multiply by u

u really close to f  magnification is hugebut image is formed at a great distance

Magnification of the eye… Hence, we do not have the focal length to allow
us to bring the object closer to the eye. At this
point our resolution is about 100 microns

• Case 1: Focal length of lens, when focusing objects at infinity.

• u = inf; v = 2.5
• f = 2.5 cm
• M=0

2.5 cm
• Case 2: Focal length when focusing the nearest possible
object
• u = 25 cm; v = 2.5
• f = 2.27 cm
• M = 0.1 (Images are smaller than the object)
 How can you magnify objects?

• In the eye, v is fixed. So, bring

u closer.
• This is what magnifying
glasses do.

How much closer do you have to bring the object,

before the image become larger than the object?

Notice that although the object is closer to us than 25 cm, our brain cannot
comprehend this. So it assumes that the image is at 25 cm and huge