Principles of Microscopy

Lecture outcome
• Know the basic buildup of light and electron microscope
• Understand how light properties (diffraction and interference) affect resolution
• Understand the difference between resolution and magnification
• Know formula for resolution, be able to evaluate resolution under different
conditions (different NA, different wavelengths of light)
 Microscopes

• Instrument producing magnified

images of objects

https://micro.magnet.fsu.edu/primer/anatomy/introduction.html
 Optical
principles
of
microscopy
• source of
illumination
• specimen
• system of lenses
(condenser – to
illuminate specimen;
objective – to detect
light)
 Magnification
Magnification is the extent to which the image of the specimen,
viewed under the microscope, is enlarged

64X enlarged (magnified); no

more details are observed
 Magnification
• If object is placed at a distance greater than the focal length away from a convex
lens, an inverted real image of the object on the opposite side of the lens will be
formed.
Focal point of the lens
 Optical principles of microscopy

Placed in the path of light or electron beam, specimen changes the physical
characteristics of a beam

- phase shift due to difference in refractive index

- amplitude change due to light absorbance (for unstained biological specimen – minor)
- interference
Phase shift and amplitude shift

amplitude shift – change in brightness

phase shift –
undetected by eye;
but can be
converted to
amplitude shift in
phase-contrast
microscope
 Light properties
• Electromagnetic radiation
• Wave-particle dualism (Christian Huygens (XVII)- wave nature of light, Newton
– corpuscular theory)

https://www.olympus-lifescience.com/en/microscope-resource/primer/lightandcolor/particleorwave/
 Wave – like nature of light
• spherically expending wave - traverses through space in a manner similar to the
ripples spreading across the surface of a still pond after being disturbed by a
dropped rock (travels in all directions)
 Characteristics of light wave

• λ (wavelength) = c/ν; c ~ 300000000 m/s

• frequency (ν) - number of sinusoidal cycles (oscillations or complete
wavelengths) that pass a given point per second
Visible spectrum
Refractive index
• Refractive index (n) -
measure of change in the
velocity of light as it passes
from one medium to
another
• ratio of the speed of light in
vacuum (c) to the speed of
light in that medium (v)
• n=c/v
Refractive index
• water – 1.33 (1.33 times slower in water than in a vacuum)
• glycerin - 1.47
• immersion oil - 1.51 (similar to glass refractive index)
 Light waves diffract and interfere
• Diffraction - bending of a wave around the edges of an
opening or an obstacle
• occurs at edges and when light goes through round opening
comparable in size to the light wavelength (! think about
size of intracellular organelles)
• fringes (set of parallel lines) are seen when light passes the
edge of a solid object
• when light passes through a small round hole, pattern of
concentric rings (Airy discs) is observed
• image of a point source of light appears as a set of
concentric rings due to light diffraction
 Airy disk
• Airy Disc appears as a bright point of
light around which are concentric
rings or ripples are seen (Airy
diffraction Pattern)
• The diffraction pattern is determined
by the wavelength of light and the
size of the aperture (circular
opening) through which the light
passes
• George Biddell Airy (1801-1892) was
an English mathematician and
astronomer
Airy disk and Point
spread function
• The three-
dimensional
representation of
the Airy Pattern is
also known as the
Point-Spread
Function (PSF)

Figure 1 from Super-resolution optical microscopy for studying

membrane structure and dynamics
Erdinc Sezgin 2017 J. Phys.: Condens. Matter 29 273001
doi:10.1088/1361-648X/aa7185
Size of the Airy disk depends
• Wavelength of light
• Numerical aperture of objective lens
 Interference
• light waves of the same wavelength and frequency arriving simultaneously at the same point in space interfere
with one another
• light waves in phase combine – constructive interference – amplitude increases – perceived brightness increases
• light waves out of phase cancel each other partly – destructive interference – amplitude decreases – brightness
decreases
 Young’s double-slit experiment
destructive interference – dark stripes
constructive interference – light stripes

https://www.britannica.com/video/179685/experiment-Thomas-Young
“the microscope image is the interference effect of a
diffraction phenomenon”

Ernst Abbe, 1873

 Resolution vs magnification
resolution – minimum distance at which two separate points are seen as
separate

Factors affecting resolution:

- light properties (wavelength of illumination source, diffraction) – can’t be

changed
- lens properties (angular aperture, aberrations)
- refractive index of the medium surrounding the specimen
Factors affecting resolution
• Given type of radiation can’t be used to probe structures much smaller
than its own wavelength

• Can you see a ribosome (~30 nm in diameter) using light of 450 nm

• Can you see a bacterium (~1 um in diameter) using light of 550 nm

In general, spatial resolution of an optical system is approximately

equal to half the wavelength of light at which illumination is
performed
Point spread function is crucial for resolution

- only if the distance between two objects is larger than resolution limit, they are seen as two
separate objects
- several criteria for resolution limit exist (Rayleigh, Abbe, Sparrow)
 Abbe criterion Rayleigh criterion
d=0.61λ/Naobj
d=0.5 λ/Naobj d=1.22λ/(Naobj +
Nacon)

In general, spatial resolution of an optical system is approximately equal

to half the wavelength of light at which illumination is performed
 Rayleigh criterion
- uses the size of the Airy disc to
describe the theoretical 0.61λ/NA
resolving power of an optical
system
- Rayleigh criterion is satisfied
when the maximum of one
PSF coincides with the first
minimum of the second PSF
- Used as a standard definition
for
theoretical resolution limit
- Radius of an Airy disk
Rayleigh criterion When objective lens also
serves as a condenser
d=0.61λ/NAobj (fluorescence microscopy)

d=1.22λ/(Naobj + Nacon)
 Resolution
- Given type of radiation can’t be used to probe structures much smaller than
its own wavelength
- In general, spatial resolution of an optical system is approximately equal to half the wavelength of
light at which illumination is performed
- with blue light, the resolution limit is approximately d = 0.2 μm
- with red light, around d = 0.35 μm
 Lens properties affecting resolution
• angular aperture –half-
angle of the cone of
light entering the
objective lens of the
microscope from the
specimen
 Numerical aperture
- NA = n sin α
- where n is the refractive index of the medium filling the space between
the object and the lens, and α is an angular aperture
- Angular aperture - half-angle of the cone of light entering the objective
lens of the microscope from the specimen

d=0.61λ/Naobj
 Numerical aperture

d=0.61λ/NAobj

NA determines the size of cone of light that enters the

objective
 Refractive index and
NA

- Objectives with high NA have short working distance

- immersion oil creates continuity between coverslip glass and objective and prevents light refraction (refractive index of
immersion oil is same/similar to glass)
- BE CAREFULL WORKING WITH HIGH NA OBJECTIVES
Where to find NA of objective?
 Question
• Using Rayleigh criterion find theoretical resolution to
distinguish two GFP-tagged proteins. Assume you are
observing them using 20X objective with NA = 0.4. GFP emits
green light of 500 nm. Would you see 2 molecules separately
if they are within 300 nm of each other?

• What if using 100X immersion oil objective with a NA of 1.4

 Light
microscopy
• Brightfield – illumination with transmitted white light
• Fluorescent
• Superresolution
 Brightfield
microscopy
• transparency of most of
biological specimens
• need to use dyes
• need to fix (chemically
preserve) and stain =>
samples are dead
 Specialized Light
Microscopes
• A variety of special optical techniques have been developed for
observing living cells directly
• These include:
– Phase-contrast microscopy
– Differential interference contrast microscopy
– Fluorescence microscopy

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 Phase-contrast microscopy
• converts phase difference into brightness difference
• Phase contrast and differential
interference contrast microscopy
make it possible to see living cells
clearly
• The phase of transmitted light
changes as it passes through a
structure with different refractive
indexes
• These types of microscopy
enhance and amplify these
slight changes
 DI
C
- Differential interference contrast
- polarized light is used
Fluorescence microscopy
 Fluorescence
•
Microscopy
Fluorescence microscopy allows detection of molecules using
fluorescent dye (molecule that absorbs light of one wavelength and
then re-emits it at a longer wavelength)

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Absorption and emission spectra
 Fluorescent Proteins
Green fluorescent protein (GFP) is derived from jellyfish A.victoria

© 2016 Pearson Education, Inc.

 Method of Fluorescent Microscopy
Fluorescence: Fluorescent microscope:

Specific fluorescent tagging:

 Immunostaining
(direct and indirect)

• An antibody is a protein that binds a particular target molecule, called an antigen

• The antibody can be coupled to a fluorescent molecule, which emits light wherever
the target molecule is bound by the antibody
 GFP-tagged proteins
EBFP2 fused to actin-binding peptide domain known
as Lifeact reveals filamentous actin network

•Green fluorescent protein

(GFP) and it derivatives
can be used to study the
mEmerald fused to alpha-tubulin
temporal and spatial
distribution of proteins in
a living cell

EBFP2 fused to C-Src

 Confocal fluorescence microscopy
• Produces optical sections by
excluding out-of-focus light
• Instead of illuminating the whole
specimen at once, light is focused
onto a single point at a specific
depth in the specimen (due to
pinpoint illumination by a light that
passes through a pinhole)
• Before the detector – second
pinhole – at a position where rays
from illuminated point come to
focus
 Confocal vs widefield microscopy

Wide field (A) and confocal (B) image of a triple-labeled cell aggregate (mouse intestine section).

Image is from “Advanced fluorescence microscopy techniques—FRAP, flip, flap, FRET and flim”
Molecules 17(4):4047-132
© 2016 Pearson Education,
 Superresolution fluorescent microscopy

• Stimulated Emission
Depletion (STED) microscopy
• Single Molecule Localization
(SML) microscopy
- PALM (Photo-Activated
Localization Microscopy)
- STORM (Stochastic Optical
Reconstruction Microscopy)
 STED

https://www.sciencedirect.com/science/article/abs/pii/S0959438804001291?via%3Dihub
PALM/STORM

https://www.uni-frankfurt.de/49962827/Methods
 • YFP fused to sequence
targeting it to ER

• immunostained
microtubules
Questions
• A fluorescent molecule, having absorbed a single photon of light at
one wavelength, emits it at a longer wavelength
 Electron Microscopy
• The electron microscope -
uses a beam of electrons
rather than light
• The wavelength of electrons
at 100 keV is 3.70 pm
• Practical resolution limit is
~0.1 nm

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Inc.
 Electron Microscopy

In transmission electron microscopy (TEM), electrons are

transmitted through the specimen

© 2016 Pearson Education,

Inc.
 Electron Microscopy
In scanning electron microscopy (SEM), the surface of a specimen is
scanned by detecting electrons deflected from the outer surface
Method of Electron Microscopy
Electron Microscope

Light Microscope
 Bacteria E. coli

HIV virus

Yeast cell, S. cerevisiae