Microscopy

Tutorial 1
BIO102
 Types of Microscopes

• Simple Microscope
• Compound Microscope
• Stereo Microscope
• Fluorescence Microscopy
• Confocal Microscopy
• Scanning Electron Microscopy
• Transmition Electron Microscopy
• Super-Resolution Microscopy
Fluorescence microscopy
 Fluorescence microscopy - Principle

• The conventional microscope uses visible light (400-700 nanometers) to

illuminate and produce a magnified image of a sample.

• A fluorescence microscope uses a much higher intensity light source

which excites a fluorescent species in a sample of interest. This
fluorescent species in turn emits a lower energy light of a longer
wavelength that produces the magnified image instead of the original light
source.
 Fluorophores

• When fluorescent compounds in ‘ground state’ absorb light energy (photons),

alterations in the electronic, vibrational and rotational states of the molecule can
occur. This transition to an ‘excited state’ occurs very rapidly (in femtoseconds).
• Some of this energy is lost as vibration but most is re-emitted at a longer
wavelength
 Fluorescence microscopy - Principle

• A portion of the energy absorbed by fluorophores is re-emitted as light.

• The emitted light is always of longer wavelength (lower energy).

• We can quantify fluorescence by the quantum yield, Q

Q = Number of photons emitted/Number of photons absorbed

Under a given set of conditions, Q will have a fixed value for a particular
fluorophore, with a maximum possible value of 1.

This gives fluorophores a characteristic fluorescence spectrum as well as a

characteristic absorption spectrum.
 Types of fluorophores

Intrinsic fluorophores are those that occur naturally. These include the
aromatic amino acids, NADH, flavins, derivatives of pyridoxyl, and
chlorophyll.

Extrinsic fluorophores are added to the sample to provide fluorescence when

none exists, or to change the spectral properties of the sample
(Or)
Organic fluorophores
1. Intrinsic fluorophores (source of autofluorescence)
2. Dyes
3. Fluorescent proteins

Inorganic fluorophores
1. Lanthanides
2. Quantum dots
 Intrinsic fluorophores

• Intrinsic fluorophores in proteins include Trp, Tyr & Phe. Trp & Tyr give
stronger spectra than Phe.

• The bases of DNA nucleotides & of some co-factors (e.g. NAD) are also
intrinsic fluorophores, although they produce weak spectra.

NADH
Absorbs ~340 nm
Emits at ~460 nm
 Extrinsic fluorophores

• Extrinsic fluorophores are fluorescent molecules that have been added to

the sample by the experimenter.
• These would include synthetic dyes such as Dyes, fluorescent proteins,
and quantum dots.
 Fluoroscent Dyes
• Fluorescent dyes are non-protein molecules that absorb light and re-emit it
at a longer wavelength.

• They are often used in the fluorescent labelling of biomolecules and can be
smaller or more photostable than fluorescent proteins but cannot be
genetically encoded.

Hemicaynine – 1
(HC-1) is a dye that
localizes to
mitochondria.

*Raja et al., Chemistry Select, 2017

 Fluoroscent Dyes

Confocal images of HC‐1 stained E14TG2a mouse ESCs either with Leukemia Inhibiting Factor
(LIF) (A and B (zoomed)) or after LIF withdrawal (C). LIF‐withdrawal causes spontaneous
differentiation (C) whereas cells with LIF retain stemness (A and B)

*Raja et al., Chemistry Select, 2017

 Green Fluorescent Protein (GFP) –fusion proteins

 GFP is a fluorescent protein - The

protein of interest can be linked to
GFP (either to its N- or C-terminus)
to make a fusion protein
 Also other spectral variants
(different colours) - allowing
simultaneous visualization of several
proteins in the same cell
 + No need for an antibody, imaging
in both fixed and live cells possible a
dynamic studies
 - Overexpression of the protein of
interest, sometimes GFP-fusion
impairs protein function
 Quantum Dots

• Quantum dots (QDs) are semiconductor nano-particles, which show

interesting phenomena, such as size dependent emission wavelength,
narrow emission peak and broad excitation range.
 Intrinsic Vs. Extrinsic fluorophores

• Intrinsic fluorophores have the advantage of being naturally present. The

disadvantages are that they are relatively dim and high energy.

• Synthetic dyes come in a wide variety of colors and brightnesses. The

disadvantage is that they must be covalently attached to the biomolecules
to be detected, and this presents additional challenges.

• Fluorescent proteins come in a smaller selection of colors than synthetic

dyes, but can be attached to proteins using molecular biology.

• Quantum dots come in a wide variety of colors and are very bright. The
primary disadvantage is that they are large and do not allow for
monovalent attachment to the biomolecule of interest.
 Confocal Microscopy

•It is a advanced variation of fluorescence microscopy for

increasing optical resolution and contrast

•Uses point-by-point illumination of the sample

•Eliminates out of focus light

•Only a small volume can be visualized by confocal

microscopes at once. Bigger volumes need time consuming
sampling and image reassembling.
Confocal Microscopy

• In confocal microscopy, a
pinhole between specimen
and detector is used to select
information from a single
focal plane, producing a
sharply focused optical slice
through the specimen.

• Taking a series of optical

slices from different focus
levels in the specimen
generates a 3D data set.
 Confocal Microscopy

Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer.
 Limitations of Confocal Microscopy

• Resolution: It has inherent resolution limit due to diffraction (Diffraction

Barrier). Maximum best resolution limit in confocal is around 200nm. It
has a relatively low axial resolution (z res) compared to its lateral
resolution (xy res). typically, the axial resolution is 2~3 worse than the
lateral resolution.

• Speed: A typical confocal uses raster scanning, which means it scans the
specimen point-by-point. comparing to a wide-field detection (taking a
snapshot of the whole field of view), it is quite slow.

• Photo damage: Instead of illuminating a specific plane (optical section),

confocal illuminates the entire column throughout your z-axis. samples
could be photo damaged. this applies to any long scan.
 The Diffraction Barrier

Unresolved yleigh Criterion Resolved

The minimum angular separation of two sources that can be distinguished by a

telescope depends on the wavelength of the light being observed and the diameter
of the telescope.
This angle is called the DIFFRACTION LIMIT.
 Breaking the Diffraction Barrier
Super resolution Microscopy
● Stimulated Emission Depletion – STED

● Stochastic Optical Reconstruction Microscopy - STORM

 Stimulated Emission Depletion Microscopy
A ground state (S0 ) fluorophore can absorb a photon and jump to the excited state (S1
). Spontaneous fluorescence emission brings the fluorophore back to the ground state.
Stimulated emission happens when the excited-state fluorophore encounters another
photon with a wavelength comparable to the energy difference between the ground and
excited state. This process effectively depletes excited-state fluorophores capable of
fluorescence emission
Confocal Vs. STED
 Confocal Vs. STED

Actin Fibers

*Leica Microscopy Images

Stochastic Optical Reconstruction Microscopy - STORM

The fundamental principle behind stochastic optical reconstruction microscopy

(STORM) and related methodology is that the activated state of a photoswitchable
molecule must lead to the consecutive emission of sufficient photons to enable precise
localization before it enters a dark state or becomes deactivated by photobleaching.
Stochastic Optical Reconstruction Microscopy - STORM

An antibody molecule is labeled with the green fluorophore Cy3 and the red
fluorophore Cy5. When Cy5 is exposed to red light, it emits fluorescence and then
switches to a dark nonfluorescent state. Illumination with green light, however,
causes Cy5 to revert to its fluorescent state by means of a short-range interaction
with the neighboring Cy3. Under illumination conditions allowing single-molecule
detection, this single-molecule fluorescent switch can be cycled on and off for
hundreds of cycles before permanent photobleaching occurs.
Stochastic Optical Reconstruction Microscopy - STORM

Microtubules and Mitochondria in BS-C-1 cells, Image Credits: Huang lab