Physical Biochemistry Fluorescence Microscopy
Quantum Efficiency (Q.E.) = emitted photons / absorbed photons
Molar Extinction Coefficient () is the absorbance of a 1 molar solution in a 1cm lightpath at a
specified wavelength
Fluorescence microscopy is based on illuminating a sample at a certain
wavelength then recording light at another wavelength corresponding to the fluorescence
spectrum.
Confocal Microscopy:
An imaging technique that is used to increase the optical resolution and contrast of a micrograph
by using point illumination and a spatial pinhole to eliminate out of focus light derived from parts
of the sample that are out of the focal plane.
1. Laser light of a set wavelength is deflected through the beam splitter to the objective
Laser light is parallel - the objective focuses parallel light down to a point in the focal plane
2. Sample is illuminated fluorescence is generated
3. Beam splitter allows fluoresced light to pass through (it is not reflected like the laser light)
Fluorescence light is at a different wavelength to laser light
The reflective surface of the beam splitter will reflect everything below a certain
wavelength and let everything above a certain wavelength pass through
4. Fluorescent light from the focal point will be parallel so the 2nd lens will focus it and it will
subsequently be recorded by the detector
If the light entering the 2nd lens is not parallel its will not be focused at the focal point of the
second lens (it will be focused before or after it)
By placing a physical aperture (spatial pinhole) at the focal point the aperture will only allow
light that is focused to pass freely
Out of focus light will be severely attenuated
This allows imaging of a specific point (small region around the focal point) in the sample
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Physical Biochemistry Fluorescence Microscopy
The sample can be scanned in 3D by moving it or the beam (using mirrors) allowing a 3D
reconstruction of the specimen to be obtained (virtual sectioning).
Example: The glomerulus (~200 μm)
Serial sectioning is used in conjunction with confocal microscopy to image the entire structure
At regions below 20 μm there are effects such as scattering that reduce resolution
20 μm regions were studied then a milling machine chopped that portion off.
The main problem with the technique is speed scanning a 3D specimen point by point takes time
It can take minutes to scan complex specimens making the technique useless for scanning fast
phenomena.
Hybrid techniques have been developed that compromise of confocality to gain speed
e.g. scan an entire line of the sample at a time. There is some loss of resolution in the z direction
however it is not pronounced.
e.g2. Scan multiple points (that are well separated in space) in parallel simultaneously
Confocal Resolution:
Lateral resolution is similar to that of Rayleigh resolution apart from a small difference in factor.
(Either is ok in the exam)
Lateral Resolution:
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Physical Biochemistry Fluorescence Microscopy
Axial Resolution:
z plane refers to depth
Axial resolution is related to depth perception
Numerical aperture is squared for axial resolution so the resolution in the axial axis will always be
less than that of the lateral axis
Cross-Talk:
Specimens are often studied using multiple fluorescent labels
e.g. one label for DNA another for protein
Multiple detectors are required to detect fluorescence from the different labels
Conventionally the different wavelengths are separated – usually by placing a filter
One detector will detect everything below a certain wavelength the other above it
Detector 1 should detect green
Detector 2 should detect red
The fluorescence spectrum is not usually well defined it has long tails
The detectors will not exclusively detect one fluorescent molecule (known as cross talk)
If the green fluorescent label is at a higher concentration than the red then it will be scaled up
against the red peak. Its tail could exceed the peak of the red fluorophore.
Cross talk is often initially measured then compensated for computationally (calibration)
When we draw fluorescence spectra we usually assume each peak height will be 1 – this is not
always the case.
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Physical Biochemistry Fluorescence Microscopy
Fading (Photobleaching):
Not well understood however free radicals are thought to be involved
Molecule is excited to an excite state it then undergoes a chemical change so that it does not
fluoresce anymore.
Anti-fading agents are now available – free radical scavengers reduce this effect
Can be very cyto-toxic which limits their use in live cell imaging
Two-Photon Microscopy:
It is possible to use two photons of half the excitation energy to excite an electron
High intensity is required as both photons need to interact with the molecule at roughly the same
time. Special lasers provide the high intensity light in short (femtosecond) pulses
The only change in the system is the absorption
Intensity is only high enough in the focal region – 3D imaging can be provided without the need
for a confocal system. Specimen is moved in relation to the beam in order to scan. Less time
consuming than a confocal system no pinhole used.
A longer wavelength will cause less scattering. IR is used
which allows penetration into deeper tissue
o Less scattering
Bleaching and phototoxic effects are limited to the focal
region – conventionally the whole specimen is illuminated
Slightly worse resolution than confocal microscopy
Requires lasers capable of generating intense short pulses
Multi-photon microscopy also exists where three photons are used at one third of the excitation
energy. Provides equal resolution to confocal but requires more expensive higher intensity lasers
Deconvolution Microscopy:
Several images of the specimen are taken while moving the specimen towards the objective (z-
axis) no scanning in the x-y plane or pinholes are required.
The smudging of information in the z-axis is mathematically compensated for computationally
Simple, cheap and quick – can use a standard microscope
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Physical Biochemistry Fluorescence Microscopy
For many complicated specimens, not as
good as confocal or two-photon microscopy
Mathematical (computational) techniques are
used to combine the images to generate a 3D
structure – deconvolution. This process is
not perfect especially in tissues but its good
for individual cells.
Most spectroscopic techniques that we have studied can be combine with microscopy to perform
3/4D studies
FRET – Fluorescence Resonance Energy Transfer
FRAP – Fluorescence Recovery After Photobleaching
Antibody Labelling:
Antibodies are conjugated with fluorophores
Direct: Antibody binds directly to antigen of interest
Indirect: Primary antibodies are raised against the antigen of interest
The 2ndary antibody is raised against the primary antibody – it will bind to it
The 2ndary antibody has a bound fluorophore
Has multiple 2ndary antibodies / primary antibody (amplification of fluorescent signal)
Labelling of 2nd antibody can result in unspecific binding
Direct labelling is more specific but less intense
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Physical Biochemistry Fluorescence Microscopy
FISH (Fluorescence In Situ Hybridisation):
Useful for chromosomal studies, especially when combined with multiple labelling
Sample DNA is denatured
A small fluorescent label will hybridise with the sequence of interest
This can be visualised using a fluorescence microscope
Used to show chromosomal disorders such as polyploidy
Both antibody labelling and FISH are used on fixed specimens
GFP (Green Fluorescent Protein):
In vivo reporter from a jellyfish
The FP gene is fused with the gene of interest
When the fusion protein is expressed it can be localised in living cells as it is tagged
GFP has a relatively high Q.E. and is quite robust in terms of photobleaching
Possibly due to barrel-like structures protecting and isolating the buried chromophore
A number of fluorescent proteins are now available (red, blue, yellow)
Important technique for live cell imaging
Concentration Measurements:
Calcium gradients in neurons and Mg, K elsewhere
High temporal and spatial resolution is incompatible with precise measurements
Specialised fluorophores are sensitive to ca2+
Confocal microscopy is too slow to study calcium gradients across an entire cell (ms timescale)
In order to carry out fast measurements the whole cell needs to be studied at once
A small area is studied at a time
Studying Living Cells:
Live cell imaging – specimen needs to be kept alive
Tissue is sensitive to buffer conditions and temperature
Microscope systems have been developed to study live systems
Often inverted – objective is under specimen – leaves more room to manipulate specimen
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Physical Biochemistry Fluorescence Microscopy
Constant buffer status and temperature
Non-cytotoxic wavelengths (no UV) and non-cytotoxic anti-fading agent
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