Monica Feole
PhD Student
Translational Neuroscience and Aging
Program
Seminar Masaryk University 14th October
2022
Picture credit: human neurons differentiated 40 days in vitro, imaged in a
confocal microscope
Advanced microscopy
techniques
Microscopes allowed us to see the invisible
• Thanks to Golgi staining technique he was able to illustrate multiple structures of
the nervous system, with particular attention to the multitude of neurons found
in our brain
Marcello Malpighi (1628-1694)
• The first to observe capillaries in animals;
• Described the complex pulmonary structure;
• Santiago Ramon y Cajal (1852-1934) Nobel prize 1906 “in recognition
of his work on the structure of the nervous system”
Evolution of microscopes helped us to improve our knowledge on both microorganisms
and our own anatomy
1625
First use of
term
“microscope”
1873
Abbe’s law and
Electron
Microscopy
1957
Confocal
microscopy
principles
1978
Confocal LSM
1986
Physics Nobel
Prize for EM
and STM
1990
Multiphoton
microscopy
1993
Super-
resolution
1994
SIM and STED
techniques
2006
PALM and
STORM
2014
Chemistry
Nobel Prize
for Super-
resolution
• Microscopy is one of the most powerful tool in many research fields;
• It gave us the possibility to unravel what is beyond the capacity to be seen by
our eyes;
• Since the beginning of microscopy history breakthrough discoveries have
been made (e.g., electron microscopy);
• Over the past two decades methods that can overcome the diffraction limit
and allow the imaging of complex structures have emerged;
Fluorescence microscopy
Electron microscopy
Super-resolution microscopy
1mm 100 µm 10 µm 1 µm 100 nm 10 nm 1 nm 1Å
Small organisms
Plant cell
animal cell
Mitochondria/
bacteria
Animal viruses
proteins
Small molecules
Techniques for different scales
Scanning probe
microscopy
Many microscopy techniques are available for different case-study
fluorescence
confocal
multiphoton
Super-resolution
Optical
Transmission (TEM)
Electron
Scanning (SEM)
Scanning probe
Atomic force (AFM)
Scanning tunneling (STM)
Microscopy
techniques
Super-resolution microscopy
• Multiple color labeling
• Sample preparation straightforward (as in a simple
immunocytochemistry approach)
• Temporal resolution may be a bit slower than a confocal (going from
speed acquisition of msec to sec)
• Important improvement in spatial resolution >> 20-150 nm
• Define small structures (e.g., cytoskeletal components and organization;
protein-protein interactions)
• Live imaging for better acquisition of single molecule movement;
• 3D imaging for better understanding spatial organization of proteins
(e.g., dsDNA interactions with surrounding proteins);
Nobel Prize in Chemistry, 2014
Eric Betzig Stefan Hell William Moerner
Abbe’s law
Structured Illuminated
Microscopy (SIM)
Stimulated Emission
Depletion (STED)
Single Molecule Localization
Microscopy (SMLM)
Structured Illuminated microscopy (SIM)
• A sample with an unknown frequency pattern is illuminated with
light of a KNOWN high-frequency pattern (moiré pattern);
• Angle changes to determine different patterns to apply for
acquisition (15-25);
Different frequencies
Different patterns
Processed image
(overlap of many
pattern/frequencies images)
Komis et al., 2015
GFP-labelled Arabidopsis
thaliana microtubules
What can we do with SIM?
Visualization of proteins at high resolution levels
Li et al., 2015
Actin
Clathrin-coated pits
Actin
Cortical actin and internal filaments in COS7 cells
3D-SIM of DNA-damage repairing proteins
Ochs et al., 2019
DNA-proteins visualized in Retinal epithelial human cells
SIM-Live imaging of dendritic spines
Turcotte et al., 2019
Cytosolic GFP expressed in mouse brain neurons
Can we go a little bit further ? Organization of cytoskeletal proteins in an in vitro model of
Trauma Brain Injury
Unravel spatial organization of
proteins and how it can change
in specific conditions
Pozo Devoto et al., 2022
Figuring out details about proteins
organization can help analyzing
parameters such as orientation,
periodicity of the protein in the cell,
changes in conformation due to a
pathological event
What are the main limitations of SIM?
RESOLUTION
It can only be doubled compared to the classical diffraction
limit of point scanning microscopy
SPEED
The acquisition rate is limited by the movements of the illumination
patterns (depends on the grating rotation options of the machine);
SAMPLE QUALITY
Poor quality sample could lead to a wrong or no modulation of the
grating pattern, requiring higher exposure, which can damage the
sample;
Stimulated Emission Depletion
Microscopy (STED)
• Confocal microscope;
• The system needs an additional laser beam that quenches
”unnecessary” light;
• Spatial resolution unlimited-bypass diffraction-limit;
• Doesn’t require computational processing;
Vicidomini et al., 2011
STED applications and achievements
• Structural analysis;
• Protein-protein interactions;
• in vivo-imaging;
Actin rings periodicity at the dendrites
D’Este et al., 2015
Berning et al., 2012
In vivo imaging in a mouse brain of its synapses
Wegner et al., 2020
Dual-color STED imaging for post-synaptic protein dynamic studies
STED is a powerful but challenging super-resolution technique
• Multicolor imaging can be done, but it can be
difficult since for each excitation wavelength
an associated depletion beam is needed;
Solovei and Cremer,2010
• Sample preparation needs to be very accurate,
both in the choice of fluorophores and the
“density” of their staining;
• High resolutions require massive laser intensities, which can
conduct to photobleaching and phototoxicity;
photobleached
• Expensive technique, since requires a
multilaser system;
Where is the center?
Are we confident about the exact center of
this molecule?
Some microscopy techniques can push beyond the
boundaries of resolution limit to visualize for single
molecule imaging
A single Alexa Fluor 647 dye
Gaussian fit to find
the centerx
Point Spread Function (PSF)
Light waves converge and
interfere at the focal point to
produce a diffraction pattern
of concentric rings
Single Molecule Localization Microscopy (SMLM)
• Employs conventional wide-field excitation and achieves super-resolution by localizing individual molecules;
• Can achieve spatial resolution of ca. 20-50 nm and sometimes even better (10x
more than conventional microscopy!);
• SMLM methods are relatively easy to implement, but require a careful choice of fluorophores;
Principle(1):
Spatial coordinates of fluorescently labelled molecules can be determined with
high precision if their PSFs do not overlap;
Principle(2):
Photoswitching of fluorescent molecules is applied via multiple acquisition over
time of different molecules;
Many ways to acquire single molecules in our samples
PALM
Photoactivatable
Localization
Microscopy
Sequential imaging of fluorophores subsets and
consequent reconstruction of their positions via multiple
frame acquisition (the “blinking” image);
STORM
Stochastic Optical
Reconstruction
Microscopy
PAINT
Points Accumulation for
Imaging in Nanoscale
Topography
The fluorophores used for all these techniques switch from a
“bright” state to a “dark” one
Stochastic Optical Reconstruction Microscopy (STORM)
Conventional microscopy
Lelek et al., 2021
STORM
Individual
molecule
activation
Individual
molecule
detection
(gating)
Localization of
single molecules
(coordinates)
Many fluorophores are available for different approaches
➢ Fluorophores used in SMLM techniques fall into one of
five different classes based on how they exchange their
ON<>OFF status
➢ Fluorescent dyes rather than fluorescent proteins have
higher photon count therefore allowing shorter imaging
times and higher localization precision
➢ Fluorescent proteins are more suited for live-imaging
applications (PALM), but premature bleaching or poor
levels of expression in the specimen may limit the
structural resolutionLelek et al., 2021
STORM applications
and milestones
Since its discovery in 2006 this microscopy
technique has evolved continuously guaranteeing
a revolutionary approach to study proteins and
nucleic acids at nanoscale resolution
Unraveling nano-structures in cell nucleus
Ricci et al., Cell 2015
STORM images of H2B in human
fibroblasts
Tens to hundreds of nucleosomes along
the chromatin fiber
Thevathasan et al., Nat. Methods 2019
STORM imaging of single molecules for different nuclear
pore proteins present at the nuclear level of Nup96 cells
RNApol II and Histone
protein 2B interaction
Cytoskeletal proteins ultrastructure and synaptic receptor organization
Siddig et al., Science 2020
Nanoscale organization of metabotropic
glutamate receptors at presynaptic active
zone
Xu et al., Science 2012
For the first time spatial organization of the
cytoskeletal proteins of the axon was
reconstructed thanks to STORM
A fancy technique with different limitations…
1. Susceptibility to artefacts upon image
reconstruction;
2. Difficulties in imaging thick samples: not suitable
for tissue samples;
1. Low-throughput due by the small field of view
required for single molecule localization and
imaging;
Minimize with
good software
algorithms
Electron microscopy
First application 1931
when Ernst Ruska built
the first electron
microscope, for which he
was awarded with the
Nobel Prize in Physics in
1986;
“for his fundamental work
in electron optics, and for the
design of the first electron
microscope”
Basic concepts of Electron microscopy
• The source allowing the images to be built is not a beam of light
but of electrons, captured under vacuum condition usually on a
phosphorescent screen;
• Resolution and magnification capacities go beyond the classic
diffraction limit, down to nm scale;
• The main concept is based on the theory of Louis De Broglie
(1924) that matter behaves like a wave exactly as light does;
• the wavelengths of a beam of electrons is small such to don’t
have a remarkable impact on daily activities;
• The smaller the wavelength the higher the resolution.
General “workflow” of an electron microscope
Electron gun generates the beam
2 sets of condenser lenses direct the electron
beam toward the specimen;
To move electrons down the column an
accelerating voltage is applied (100-1000 kV)
The electron beam passes through the sample,
which based on its density will differently scatter
the electrons
The beam then passes through the objective lens,
forming the intermediate magnified image
Finally, another set of lenses produces the further
magnified image
How are the images generated in electron microscopy?
EMs work using signals arising from the interaction of
an electron beam with the specimen
2 main EM techniques Transmission and
Scanning Electron Microscopy (TEM and SEM
respectively)
TEM (inner structures) SEM (outer structures)
Analogous to a compound microscope where a system of multiple lenses are
placed one after the other
TEM was the first non-optical technique to give a remarkable resolution improvement
Powerful for visualizing many kind of small molecules:
- Arrangement of proteins,
- Lipids
- Nucleic acids (DNA, RNA, ribosomes)
Contrast for visualization of different structures is achieved often by using
metal compounds
- Osmium tetroxide (fixative, good lipid-binding)
- Uranyl acetate (good for nucleic acid contrast)
- Lead
Samples are dehydrated upon fixation and then stained;
Ultra-thin sections of 20 – 100 nm are then cut before imaging;
After the cut the thin layers are placed/embedded on a copper grid that
goes than to the microscope;
In the final image, the denser regions will reflect less and so they will be
darker, while the less density will allow less absorption of electrons, thus
generating a “brighter” image
TEM was the first non-optical technique to give a remarkable resolution improvement
Contrast for visualization of different structures is achieved often by using
metal compounds
- Osmium tetroxide (fixative, good lipid-binding)
- Uranyl acetate (good for nucleic acid contrast)
- Lead
Samples are dehydrated upon fixation and then stained;
Ultra-thin sections of 20 – 100 nm are then cut before imaging;
After the cut the thin layers are placed/embedded on a copper grid that
goes than to the microscope;
In the final image, the denser regions will reflect less and so they will be
darker, while the less density will allow less absorption of electrons, thus
generating a “brighter” image
Nucleus
microtubules
mitochondria
centriole
Nucleolus
Golgi
Lysosome
• a focused beam of high-energy electrons
generates a variety of signals at the surface of
solid specimens;
• signals deriving from electron-sample
interactions reveal
- external morphology
- chemical composition
- crystalline structure and orientation of
materials;
• magnification range >> 20-60000X
• spatial resolution >> 50-100 nm
Scanning Electron Microscopy (SEM)
• accelerated electrons carry significant amount of kinetic
energy, then dissipated as a variety of signals:
> secondary electrons (produce SEM images)
> backscattered electrons
> diffracted backscattered electrons (used to determine
crystal structures and orientations of minerals)
samples
dehydrated
solid
Coated with Ag, Au,
Pt, others
An example of SEM application
Workflow to establish swelling formation in human neurons, a
mechanism occurring after a traumatic impact in the brain.
In this experiments the number of events (swellings) forming after
injury were quantified by immediately fixing the cells and then scan
images in an electron microscope
Pozo Devoto et al., 2022
Trauma brain injury model in vitro
SEM images of ctrl and Injured axons
SEM image of full MF system Quantification of swellings in SEM
images before and post-injury
TEM and SEM: advantages and limitations
High magnification and
resolution
Material rarely distorted during
specimen preparation, which is
not complicated
Allows the investigation of deep
structures but also surface
interactions
Live samples cannot be
imaged
TEM samples must be cut in
ultra-thin slices
Samples need to be properly dried
to favor the internal vacuum
conditions
Expensive to maintain (very
sensitive to vibration and external
magnetic fields)
PRO >>
<< CONS
…. And more.. Scanning Probe Microscopy (1980s)
• Nanoscale surfaces and structures, including
atoms;
• Resolution sometimes <1nm;
• A physical probe is used to scan back and
forth over the sample surface;
• Probe tip mounted at the end of a cantilever;
• The tip can be as sharp as an atom;
• The user doesn’t see the surface directly, is
the tool that “feels” it;
• A computer collects the data to generate an
image;
• SPMs can measure deflections caused by
many forces
- mechanical contact
- electrostatic
- magnetic
- Van der Waals
• Images are colorless since they are
measuring properties rather than
reflection of light
AFM
Electrostatic forces between
the cantilever tip and the
sample;
Mechanical properties of
cells/tissues
STM
The electrical
current flowing
between the
cantilever tip and
the sample
AFMSTM
Conclusions
• Microscopy helped us to unravel the invisible;
• Nowadays advanced microscopy techniques have a variety of applications in many fields, being extremely important for
biology and understanding of structure and interactions between proteins;
• Super-resolution microscopy allows multiple color imaging by breaking the boundaries of the diffraction limit;
• single molecule imaging allowed the visualization of cellular nanostructures never resolved before with light
microscopy;
• Electron microscopy was the earliest technique able to reach resolutions that light microscopy had never reached;
• TEM and SEM are still of large use for describing composition of structures and microorganisms;
• SPM techniques achieved the ability to image a structure just based on the atomic composition and the forces involved
in the interaction between a surface and a scanning probe;
• The use of fluorophores for staining cell cultures and tissues both for basic research purposes or diagnostic ones
(histopathology) is in continuous evolution and the offer on the market improves very quickly;
Thank you for the attention!
My contacts:
monica.feole@fnusa.cz
monicafeole@gmail.com