Plant and Mammalian Tissue Culture
Fluorescence Microscope Basics and Beyond
- much taken from Nikon’s Microscopy U tutorial
www.microscopyu.com
-…and Olympus Microscopy Primer
www.olympusmicro.com
Reporter Genes
 Genes used to
measure
transfection
efficiency.
 One of the most
common is Green
Fluorescent Protein
Green Fluorescent
Protein
 GFP
 Easily viewed under a
fluorescence
microscope.
 Excitation Maximum at
395 nm
 Emission Maximum at
509 nm
 Use Green Fluorescent Protein to test the
ability to genetically alter animal cells in
culture. (Collaboration with Aldevron)
 Panel A - Gwiz High Expression Vector GFP
 Panel B - pGreen Lantern Expression Vector
Fluorescence
 Fluorescence is the absorbance of a high energy
photon, excitation of the molecule to a high energy
state with a release of energy at a lower energy in
photon of a lower (red shifted) wavelength.
 Length of time the molecule is in
the high energy state is the
fluorescence lifetime
 Loss of energy from absorbed
photon to released photon is
due to molecular vibrations and
heat
Fluorescence
 Fluorescence involves shining a light (broad spectrum or
focused filtered light) onto a sample and measuring the light
emitted.
 Emitted light is measured from a side or different angle than
the excited light
 Many fluorescent dyes contain
conjugated rings with electron
withdrawing nitrogen groups.
Fluorescence
 Excitation and Emission for each dye is
different and the spectras may overlap.
The specific spectra for each dye and
use of the filter sets on a microscope is
critical.
 Difference between max absorbance
(Excitation) and Emission is called the Stokes
Shift
 Small Stokes Shifts result in overlap and result in
difficult to measure dyes
 The efficiency of excitation is the extinction
coefficient – measured in quantum yield.
High quantum yields is related to the intensity
of given (emitted) light
Fluorescence
 Filters (more coming on filter
sets or cubes) are chemically
treated to allow or block all light
above a specific wavelength or
a narrow slice of light through.
 Knowing which filter set vs the excitation,
emission, stokes shift and overlap as well
as excitation coefficient is critical for
success in microscopy
Fluorescence
The excitation filter spectrum (red curve) exhibits a high
level of transmission (approximately 75 percent) between
450 and 490 nanometers with a center wavelength (CWL)
of 470 nanometers. The dichromatic mirror (yellow curve)
reflects wavelengths in the region of the excitation
spectrum, while passing higher and lower wavelengths
with relatively high efficiency. Note that zero percent
transmission on the dichromatic mirror curve corresponds
to 100 percent reflection. The pronounced dip in the
transmission profile between 450 and 500 nanometers,
which represents a peak in reflectance, serves to reflect
the band of wavelengths passing from the excitation filter
at a 90-degree angle and onto the specimen.
The final component in the optical train, an emission or barrier filter (white curve), transmits wavelengths in the
green visible light region, in the range between 520 and 560 nanometers. Boundaries between transmitted
and reflected wavelength bands of the various superimposed spectra are designed to be as steep as possible
to assure nearly complete separation of the reflected and transmitted wavelengths. A pattern of sinusoidally
rising and falling spikes appearing in the dichromatic mirror spectrum is a common effect of the thin-film
deposition process known as ringing. The performance of this filter combination is remarkable and is a clear
demonstration of the rapid advances being achieved in thin film interference filter technology.
Light Source
Condenser
Objective
Magnification
Fluorescence Microscopy
 The absorption and
subsequent re-radiation of light
by organic and inorganic
specimens is typically the
result of well-established
physical phenomena described
as being either fluorescence or
phosphorescence.
 The emission of light through
the fluorescence process is
nearly simultaneous
 the absorption of the excitation
light due to a relatively short time
delay between photon absorption
and emission
 ranging usually less than a
microsecond in duration
Fluorescence Microscopy
 The basic function of a
fluorescence microscope is to
irradiate the specimen with a
desired and specific band of
wavelengths, and then to
separate the much weaker
emitted fluorescence from the
excitation light.
 In a properly configured
microscope, only the emission
light should reach the eye or
detector so that the resulting
fluorescent structures are
superimposed with high
contrast against a very dark (or
black) background.
Fluorescence Microscopy
 The limits of detection
are generally governed
by two elements:
 The darkness of the
background
 The removal of excitation
light.
• Excitation light is typically
several hundred thousand
to a million times brighter
than the emitted
fluorescence.
Excitation and Emission
 Light of a specific wavelength (or
defined band of wavelengths), often in
the ultraviolet, blue or green regions of
the visible spectrum, is produced by
passing multispectral light from an arcdischarge lamp through a wavelength
selective excitation filter.
 Wavelengths passed by the excitation
filter reflect from the surface of a
dichromatic (also termed a dichroic)
mirror or beamsplitter through the
microscope objective to bathe the
specimen with intense light.
Excitation and Emission
 If the specimen fluoresces, the
emission light gathered by the
objective passes back through
the dichromatic mirror and is
subsequently filtered by a
barrier (or emission) filter,
which blocks the unwanted
excitation wavelengths of light.
 Epi-fluorescence illumination is
the overwhelming choice of
techniques in modern
microscopy
Excitation and Emission
 In most reflected light illuminators,
the excitation filter, dichromatic
mirror, and barrier filter are
incorporated into an optical block
(often referred to as a cube)
 Modern fluorescence microscopes
are capable of accommodating
between four and six fluorescence
cubes usually on a revolving turret
or through a slider mechanism
Quenching and
Photobleaching
 Quenching is the loss of florescence when a second molecule
interacts with the flourophore and induces non-radiative
relaxation from the excited state (no photon emission)
 This often requires close
proximity and can be
used to show interaction
 Photobleaching is a loss of
fluorescence (fading)
caused by vibrations in the
excited state which change
bonds – often involves
reaction with O2.
 Can be avoided with
addition of some reagents
Applications
The wide spectrum of fluorescent proteins
and derivatives uncovered thus far are quite
versatile and have been successfully
employed in almost every biological
discipline from microbiology to systems
physiology.
These unique probes have proven extremely
useful as reporters for gene expression
studies in both cultured cells and entire
animals.
Applications
 In living cells,
fluorescent proteins
are most commonly
utilized to track the
localization and
dynamics of proteins,
organelles, and other
cellular
compartments, as
well as a tracer of
intracellular protein
trafficking.
Bacterial
Contamination
 Live and dead bacteria
visualized on freshly
isolated human cheek
epithelial cells
 live bacteria with intact
cell membranes
fluoresce green and
dead bacteria with
compromised
membranes fluoresce
red.
Multicellular Eukaryotes
 Live and dead rat
kangaroo (PtK2) cells
stained with ethidium
homodimer-1 and the
esterase substrate
calcein-AM
 Live cells fluoresce a
bright green, whereas
dead cells with
compromised
membranes fluoresce
orange-red.
Lysosomes and
Mitochondria
 Bovine pulmonary
artery endothelial cells
 LysoTracker Red stains lysosomes red.
 MitoTracker Green stains mitochondria
green.
Mitochondria and Nucleus
 The mitochondria of
bovine pulmonary artery
endothelial
 stained with MitoTracker
Red
 The cells were
subsequently fixed,
permeabilized and
stained with SYTOX
Green nucleic acid stain.
Enodplasmic Reticulum
 Live bovine
pulmonary artery
endothelial cells
 stained with ERTracker Blue-White
DPX
Cytoskeleton
 Microtubules of fixed
bovine pulmonary
artery endothelial cells.
 Alpha-Tubulin: Green
 F-Actin: Red
 Endosomes: Blue