Cryo-electron microscopy is an emerging technique that uses electron microscopes to image biomolecules such as proteins that have been rapidly frozen to preserve their structure. It overcomes limitations of other techniques by not requiring crystallization and allowing study of molecules in their natural state. In cryo-EM, a sample is frozen at liquid nitrogen temperatures to vitrify it before being imaged with an electron microscope. This technique has helped determine structures of important proteins and revealed new details about their function. Recent advances in direct electron detectors, image processing, and specimen preparation have led to remarkable improvements in resolution with cryo-EM.

1. RECENT ADVANCES IN MICROSCOPIC
TECHNIQUES: CRYO-EM
PRESENTED BY- DANISH NIGAR

2. CONTENTS
• WHAT IS MICROSCOPY
• HISTORICAL BACKGROUND
• VARIABLES USED IN MICROSCOPY
• CLASSIFICATION OF MICROSCOPY
• RECENT DEVELOPMENTS IN MICROSCOPY
• CRYO ELECTRON MICROSCOPY
• THE RISE OF CRYO ELECTRON MICROSCOPY
• HOW THE CRYO ELECTRON MICROSCOPE WORKS
• SPECIMEN PREPARATION
• APPLICATION OF CRYO-EM
• PROS AND CONS OF CRYO-EM
• OTHER DEVELOPMENTS IN MICROSCOPY
• SUPER RESOLUTION MICROSCOPY
• INTERATIVE EXPANSION MICROSCOPY
• CONCLUSION
• REFERENCES

3. WHAT IS MICROSCOPY?
• Microscopy is a
technical field of
using microscopes by
which we can view
object that are not
within the resolution
range of normal eye.

5. A microscope is an instrument
that magnifies objects otherwise
too small to be seen, producing
an image in which the object
appears larger. Most photographs
of cells are taken using a
microscope, and these pictures
can also be called micrographs.

6. Giovanni Faber coined the
name microscope for the
compound microscope Galileo
submitted to the Accademia dei
Lincei in 1625 (Galileo had called
it the "occhiolino" or "little eye").
Giovanni Faber
(1574-1629)
HISTORICAL BACKGROUND

7. 1590 : Dutch lens grinders
Hans and Zacharias
Janssen make the first
microscope by placing two
lenses in a tube.

8. • 1675: Anton van Leeuwenhoek uses a
simple microscope with only one lens to
look at blood, insects and many other
objects. He was first to describe cells and
bacteria, seen through his very small
microscopes with, for his time, extremely
good lenses

9. • 1850s - John Leonard Riddell, Professor of
Chemistry at Tulane University, invents the first
practical binocular microscope.
• 1863 - Henry Clifton Sorby develops a
metallurgical microscope to observe structure of
meteorites.
• 1860s - Ernst Abbe discovers the Abbe sine
condition, a breakthrough in microscope design,
which until then was largely based on trial and
error. The company of Carl Zeiss exploited this
discovery and becomes the dominant microscope
manufacturer of its era.
• 1931 - Ernst Ruska starts to build the first electron
microscope. It is a Transmission electron
microscope (TEM).
• 1936 - Erwin Wilhelm Müller invents the field
emission microscope.
• 1938 - James Hillier builds another TEM.
• 1951 - Erwin Wilhelm Müller invents the field ion
microscope and is the first to see atoms.
• 1953 - Frits Zernike, professor of theoretical
physics, receives the Nobel Prize in Physics for his
invention of the phase contrast microscope.

10. • 1955 - George Nomarski, professor of
microscopy, published the theoretical
basis of Differential interference
contrast microscopy.
• 1967 - Erwin Wilhelm Müller adds
time-of-flight spectroscopy to the field
ion microscope, making the first atom
probe and allowing the chemical
identification of each individual atom.
• 1981 - Gerd Binnig and Heinrich
Rohrer develop the scanning
tunnelling microscope (STM).
• 1986 - Gerd Binnig, Quate, and Gerber
invent the Atomic force microscope
(AFM)
• 1988 - Kingo Itaya invents the
Electrochemical scanning tunnelling
microscope

11. On October 8, 2014, the Nobel
Prize in Chemistry was awarded to
Eric Betzig, W.E. Moerner and
Stefan Hell for "the development
of super-resolved fluorescence
microscopy," which brings "optical
microscopy into the
nanodimension”.

12. • In 2017 Nobel Prize in Chemistry has been awarded for work
that helps researchers see what biomolecules look like.
• Jacques Dubochet, Joachim Frank and Richard Henderson
were awarded the prize on 4 October for their work in
developing cryo-electron microscopy (cryo-EM), a technique
that fires beams of electrons at proteins that have been
frozen in solution, to deduce the biomolecules’ structure.

13. VARIABLES USED IN
MICROSCOPY
MAGNIFICATION
LIMIT OF RESOLUTION
RESOLVING POWER

14. MAGNIFICATION
• Magnification simply means that how much
the image become enlarge with respect to
the original size of the specimen.
• The total magnification obtained in a
compound microscope is the product of
objective magnification and ocular
magnification.
Mt = Mob X Moc
Where,
Mt = Total magnification,
Mob = Objective magnification and
Moc = Ocular magnification
Useful magnification:
It is the magnification that makes visible the
smallest resolvable particle. The useful
magnification in a light microscope is between
X1000 and X2000. Any magnification beyond
X2000 makes the image blurred.

15. FIGURE SHOWING MAGNIFYING IMAGES

16. LIMIT OF RESOLUTION
• It is the smallest distance
by which two objects can
be separate and still be
distinguishable as two
separate objects.
Limit of resolution (d) is given
by Ernst Abbe equation-
Where,
λ = Wave length of light and
NA= Numerical aperture of
the objective.
Limit of resolution = d = λ/2 NA

18. RESOLVING POWER
• It is the ability to distinguish two adjacent points as distinct as separate.
• It is the function of wavelength of light used and numerical aperture of the
lens.
• Numerical aperture(NA) of a microscope objective is a measure of its
ability to gather light and resolve fine specimen details at a fixed object
distance.
Where,
n = Refractive index of the medium between the object and the objective and
θ = Half aperture angle
NA = nsinƟ

19. CLASSIFICATION OF MICROSCOPY
OPTICAL
MICROSCOPY
ELECTRON
MICROSCOPY
SCANNING
PROBE
MICROSCOPY

20. OPTICAL MICROSCOPY TECHNIQUES
• BRIGHT FIELD MICROSCOPY
• DARK FIELD MICROSCOPY
• FLUOROSCENCE MICROSCOPY
• PHASE CONTRAST
MICROSCOPY
• DIFFERENTIAL INTERFERENCE
CONTRAST MICROSCOPY
• CONFOCAL SCANNING LASER
MICROSCOPY
• SUPER RESOLUTION
MICROSCOPY etc.

21. ELECTRON MICROSCOPY TECHNIQUES
• TRANSMISSION ELECTRON
MICROSCOPY(TEM)
• SCANNING ELECTRON
MICROSCOPY(SEM)
• REFLECTION ELECTRON
MICROSCOPY(REM)
• SCANNING TRANSMISSION
ELECTRON
MICROSCOPY(STEM)
• CRYO ELECTRON
MICROSCOPY(CRYO-EM)
etc.

22. SCANNING PROBE MICROSCOPY
• ATOMIC FORCE
MICROSCOPY(AF
M)
• SCANNING NEAR
FIELD OPTICAL
MICROSCOPY etc.

25. WHAT IS CRYO-ELECTRON MICROSCOPY?
• It is a technique that is used
for studying the architecture
of cells, viruses and protein
assemblies at molecular
resolution.
• It is based on the principle of
imaging radiation sensitive
specimens in a TEM under
cryogenic condition.
• In this technique the beams
of electron fires at proteins
that have been frozen in
solutions to deduce the
biomolecule structure.

26. THE RISE OF CRYO-ELECTRON
MICROSCOPY
HENDERSON(1970)-Bacteriorhodospin, proved unsuitable for crystallography.
Researchers turned to electron microscopy.
Produced first 3D model for protein
FRANK a Biophysist developed image processing software and converts that fuzzy
images into 3D molecular structure.
DUBOCHET(1980) worked to prevent water soluble biomolecules from drying out in the vacuum
of an electron microscopy allowing the molecules to retain their natural shape.

29. Cryo-electron microscopy of proteins such as this β-galactosidase enzyme has
progressed from the low-resolution density map on the left to the atomic coordinates
on the right

30. WHY IT IS USED?
• When we uses TEM, particularly
for biomolecules then these
molecules are not compatible with
the high-vacuum conditions and
intense electron beams.
• The water that surrounds the
molecules evaporates and the high
energy electron burn and destroys
the molecules.
• Besides that X-Ray crystallography
also used to resolve these
biomolecules but they requires
crystallise molecules and many
proteins won’t crystallise and in
some cases the process of
crystallisation alters the structure
of proteins.
A clearer picture of chromatin structure.

31. • NMR can also give very
detailed structures and
investigate conformational
changes or binding of small
molecules. But it is limited
to relatively small proteins
and usually soluble
intracellular proteins rather
than those embedded in cell
membranes .
• Cryo-EM doesn’t require
crystals and it also enables
scientists to see how
biomolecules move and
interact as they perform
their functions which is
much more difficult using
crystallography.
• Hence, Cryo-EM uses to
overcome these problems. The flexibility of supercoiled DNA revealed.

32. HOW THE CRYO-EM WORKS
A Cryo-EM is a TEM
with an additional
specimen holder
which:
• Enable the viewing
of the frozen-
hydrated specimen
• Maintains Liquid
Nitrogen or Liquid
Helium
temperatures.

33. SOURCE OF ELECTRON
THERMIONIC ELECTRON GUNS FIELD EMISSION GUNS

34. SAMPLE USED
• In Cryo-Electron Microscopy the sample under
observation is usually frozen (frozen-hydrated) for
preservation purposes. Here, a very thin slide of the
specimen may be rapidly plunged into a liquid ethane
bath and viewed in their natural state. Solvents like
water or a salt solution is used to ensure that the
sample remains stable.
• While liquid nitrogen can also be used for the freezing
process, ethane is used instead given that it has higher
heat capacity and is also liquid at temperatures slightly
above that of liquid nitrogen. As such, it is sufficiently
cold to freeze water rapidly and appropriately without
boiling off.

35. SPECIMEN PREPARATION
Two methods of specimen preparation are:
• Thin Film: Specimen is placed on EM grid and is rapidly frozen without
crystallizing it
• Vitreous Sections: Larger samples are vitrified by high pressure
freezing, cut thinly and placed on the EM grid

37. 1.VITRIFICATION
• Rapid Cooling is required to
avoid the formation of ice.
• Rapid cooling traps the water in
a vitrified state in which it does
not crystallize.
• Vitrified state is maintained by
keeping it at liquid nitrogen
temperature.
• Vitrified state can be
maintained for long periods.
• Sample is placed on carbon grid
and dipped into a bath of
ethane held in a container of
liquid nitrogen.

38. 2.CRYO-SECTIONING
• Whole cells and tissues are
too thick to be spread into
a thin layer
• First vitrify sample and
then cut into thin sections
using diamond knives
• Sectioning is a difficult
task, distortions are made
in sample
• These distortions cause a
loss in order of the
structure and makes it
difficult for images to
increase the signal-to-
noise ratio
Cryo Diamond knives

39. 3.OBSERVATION OF THE SPECIMEN
There are three methods of observing
and recording images
• Fluorescent Screen
• Photographic Film
• CCD Cameras

40. APPLICATIONS
• Nanoparticle Research
• Pharmaceutical Drug Research
• 3D Structure Visualization of:
Single Particles such as
Ribosome
tRNA
Viruses
Proteins
Macromolecules(Lipid Vesicles)

41. PROS AND CONS
• Advantage: Structure remains native and no
dehydration is required.
• Limitation: It is not possible to look at the
sample for a long time because of beam
damage and it causes poor resolution.

42. OTHER RECENT ADVANCES IN
MICROSCOPY
• SUPER RESOLUTION MICROSCOPY
• ITERATIVE EXPANSION MICROSCOPY(iExM)

43. SUPER RESOLUTION MICROSCOPY
• It is a form of light microscopy.
• The term super resolution refers
to a method that surpass the so
called “diffraction limit” that is
the limit of resolution which is
given by Ernst Abbes equation.
• Super-resolution techniques
allow images to be taken with a
higher resolution than the
diffraction limit.
• The recent unprecedented
technical innovation of super-
resolution microscopy has
changed the limits of optical
resolution from ~250 nm to ~10
nm.

44. TECHNIQUES USED IN SUPER
RESOLUTION MICROSCOPY
• Structured Illumination Microscopy
• Photoactivated Localization Microscopy
• Stochastic Optical Reconstruction Microscopy
and
• Stimulated Emission Depletion microscopy

45. APPLICATION OF SUPER
RESOLUTION MICROSCOPY
• Can analyse plant cell
structure such as pollen,
endosomes, chromatin
etc.
• In neuroscience such as
for the study of chemical
synapses in brain etc.
Chromatin organization in a
differentiated 8C A. thaliana leaf
nucleus

46. ITERATIVE EXPANSION MICROSCOPY(iExM)
• In this preserved biological sample are physically magnified
by embedding them in a gel.
• Then sample is expanded nearly 20 folds and enables about
25 nm resolution.
• A wide range of samples have been successfully expanded
and imaged using ExM, including synaptic proteins,
Escherichia coli bacteria , cultured mammalian cells etc.

47. CONCLUSION
• Cryo-EM is a form of Transmission
Electron Microscopy (TEM) where
the sample is studied in its native
state at cryogenic temperatures.
• It is used for 3D visualization of
biological molecules.
• Resolution of Cryo-EM is not high
enough but it is improving using
different computer techniques
• With the advancement of
technology, this technique will
certainly improve.

48. REFERENCES
• The University of Edinburgh (March 6, 2018). "What is
Microscopy?". The University of Edinburgh. Retrieved April 9, 2018
• https://www.microscope.com/education-center/microscopes-
101/history-of-microscopes/
• https://en.wikipedia.org/wiki/Timeline_of_microscope_technology
• https://www.slideshare.net/Madiheh/cryo-electron-microscopy
• https://www.nobelprize.org/educational/physics/microscopes/timeline/
• https://www.microscopyu.com/microscopy-basics/resolution
• https://www.chemistryworld.com/news/...is-cryo-electron-
microscopy/3008091.article
• https://www.nature.com/news/cryo-electron-microscopy-wins-
chemistry-nobel-1.22738
• https://www.microscopemaster.com/cryo-electron-microscopy.html
• Nat Methods. 2017 Jun;14(6):593-599. doi: 10.1038/nmeth.4261. Epub
2017 Apr 17.
(Iterative expansion microscopy. hang JB1,2, Chen F3, Yoon YG1,4, Jung EE1,
Babcock H5, Kang JS6, Asano S1, Suk HJ7, Pak N8, Tillberg PW4, Wassie AT3,
Cai D9, Boyden ES1)