Transcript Document

The studies of single human retinal lipofuscin granules using
atomic force and near-field optical scanning microscopy.
Astafiev A.A.1 *, Petrukhin A.N.1 **, Sarkisov O.M.1 Dontsov A.E. 2, Feldman T.B.2 , Ostrovsky M.A.2#
1. Institute of Chemical Physics, Moscow, Kosygin st., 4, 117977
2. Institute of Biochemical Physics, Moscow, Kosygin st., 4, 117977
E-mail:*[email protected]
**[email protected]
#[email protected]
Introduction: Lipofuscin (LF) is a conglomerate of lipids, proteins, organic molecules and metals which accumulates in tissues with high oxidative stress. Particularly it is present in Retinal Pigment Epithelium (RPE) cells in form of yellow-brownish granules of micron
size [1]. LF accumulation has a 99% correlation with a age-related macular degeneration – eye disease which causes vision distortion or even blindness among elderly people. This disease is thought to resulted from reactive oxygen intermediates (including hydrogen
peroxide, singlet oxygen and superoxide anion radical) generation in LF upon the blue-light excitation [2]. LF contain a variety of blue-absorbing orange-emitting fluorofores among them two: piridinium bis-retinoid A2E and its isomer iso-A2E have been identified as
being the major contributors to the orange fluorescence. It was shown that A2E has an ability to generate reactive oxygen species when exposed to blue light [4], yet it isn’t clear whether its contribution in phototoxicity of lipofuscin is dominant.The investigation of single
granule’s shape morphology and fluorophores distribution was aimed to clear up the role of the fluorescent compounds in LF phototoxic properties. Due to the small size of the granules this measurements were only attainable using atomic force and near-field scanning
probe microscopes
Retinal Pigment
Rod Outer
Epithelium (RPE) Segments
1
Electron micrograph of human RPE.
Lipofuscin granules are marked with
sick arrows.
2
1. Semicontact mode
(topography imaging)
2-5. Phase contrast
imaging mode
4
Structures of A2E and iso-A2E
The atomic force microscope (AFM) probes the surface of a
sample with a sharp tip, a 10-20 mm long with typical tip
curvature radius of 10 nm. The tip is located at the free end of a
cantilever that is 100 mm long. The system vibrates a stiff
cantilever near its resonant frequency (typically 250 kHz) with
amplitude of a few tens to hundreds of angstroms. Then, it
detects changes in the resonant frequency or vibration
amplitude as the tip comes near the sample surface.
The spacing between the tip and the sample is kept about
0.1-10 nm. By keeping the resonant frequency or amplitude
constant, the system also keeps the average tip-to-sample
distance constant. The motion of the scanner is used to
generate the data set (topography image).
In every scan of the sample with AFM phase image also being
taken. Phase imaging refers to the monitoring of the phase
shift between the signal that drives the cantilever to oscillate
and the cantilever oscillation output signal. Changes in the
phase shift reflect changes in the mechanical properties of the
sample surface properties such as elasticity, adhesion, and
friction that give additional information about sample surface
structure.
Atomic force
microscope
3
LF fluorescence spectrum at different
excitation wavelength.
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The
near-field
scanning
optical
microscope(NSOM) was used to detect
fluorescence signal from single LF
granules. NSOM probes the optical
properties of the sample with etched
optical silica fiber covered with
aluminium coating so that a small
aperture (~100nm) is formed at the very
tip, that is kept in close proximity to the
sample surface. The light has to
“squeeze” through the aperture when
illuminating the sample forming the
light spot of subwavelength size. The
resolution of aperture NSOM is
determined of the aperture diameter
and is typically in the range 50-200 nm,
that is smaller than the diffraction or
Rayleigh resolution limit of lens optical
microscopes in the visible range.
The distance between the probe tip and the sample surface is kept constant using the shearforce feedback: the fiber is glued to the silica tuning fork oscillating at the resonant
frequency, amplitude of the oscillation is damped when tip approaches the surface.
Amplitude is used as an input signal for the feedback loop that governs the voltage applied
to the piezo-scanner on which tuning fork with the probe is mounted, so that the amplitude
of the oscillations remains constant throughout the scanning.
The fluorescence of the sample was excited with second harmonic of femtosecond Tsunami
laser (SpectraPhysics) and was collected by the microscope objective lens and detected with
intensified CCD-detector (Pi-Max II, Roper Scientific).
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nM
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nM
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The procedure of single granule fluorescence
spectrum calculation by background subtraction
The spectrum was collected when illuminating the sample through the probe
aperture. The drawback of this method was the strong background signal
around 700 nanometers due to the fiber material fluorescence. Nevertheless
the inverse method (illumination of the sample through the objective lens
and collection of the emitting light by the probe) wasn’t applicable in this
case due to the spectrum distortion inflicted by the aperture and rather
weak magnitude of the signal. Fortunately the maximum of the LF
fluorescence lie in the other spectral area then background signal. That’s
why we were able to get the “pure” fluorescence signal by subtracting the
constant background measured when probe standing on the glass surface.
The resulting spectrum has maximum at most cases at 580 nm and width
about 150 nm as shown at the picture.
μm
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μm
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nA
nA
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nA
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Comparison of fluorescence spectrum obtained in different points of LF granule.
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nM
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Lipofuscin granules were isolated from human RPE cells and suspended in aqueous solution and then dispersed
on a glass cover slide by air-drying the diluted suspension. The resulting sample represented the micron-sized
single lipofuscin aggregates lying on a glass surface.
2
0
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2
μm
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0
The images of single lipofuscin granule obtained with atomic force microscope.
Three pairs of successive images of the same lipofuscin aggregate were received with time period of about a day. On the left there
are the topography images (color scale represents the sample surface height), the phase images are on the right (color scale
represents the cantilever oscillations’ phase). The large dark areas present conceivably the water solution as they are decreasing
in time when water evaporates. The lipofuscin granule (bright area) consist of the several subunits with no appreciable internal
structure
The topography image of single granule lying on the glass substrate is on the left, the fluorescence spectrum obtained in the points of
granule marked with numbers 1-6 is on the right.
The fluorescence signal spectrum was calculated using the procedure described above. Note that shape and magnitude and shape of
signal wasn’t constant in different points of granule. Namely while spectra 1-3 resembles each other the fluorescence in points 4-6 is
much weaker and spectrum 4 has the different shape with maximum at 680 nm. This results lead to consumption that the
distribution of fluorophores in granule is considerably nonuniform. That means that the most part of fluorescent material is
concentrated only in small part of granule. Also there are presumably a small fraction of fluorophores with different spectrum in the
other part of granule. The collection of the map of fluorescence which contain the map of emission in every point of granule will be
the next step which could help to clear up the distribution of light emitting compounds.
Conclusions
1. The shape and morphology of single lipofuscin granule were explored.
It was shown that granule consist of several homogeneous subunits.
2. The fluorescent spectrum was measured in different points of single
granule. It’s likely that fluorophores are contained in a rather small
part of granule’s volume.
3. The photobleaching of LF fluorescence under the light exposure was
detected, which is presumably due to the epoxide formation from A2E.
Fluorescence photobleaching under light exposure.
We observed the decreasing of fluorescence intensity due to the intense light exposure. Here on the left is the spectra kinetics under green light
(wavelength 532 nm) with the intensity before fiber coupling of about 10 mW. Intensity reduction with a characteristic time of several tens of
minutes is clearly visible. We believe this effect to be the result of epoxide formation from A2E under the action of light (see the right picture)
that was observed previously and is known to be accompanied by the absorption spectrum shift in to the ultraviolet area and hence the
reduction of emission upon green light excitation.
References:
1. N.M. Harlampus-Grynavski, L.E. Lamb, C.M.R.
Clancy, C. Skumatz, J.T. Sarna, J.D. Simon
“Spectroscopic and morphological studies of
human retinal lipofuscin granules” PNAS 2003
Vol.100 No.6 p.3179
2. J. Wassel, S. Davies, W. Bardsley and M. Boulton
“The photoreactivity of the retinal age pigment
lipofuscin” Journal of Biological Chemistry Vol.
274 No 34 p.23826
3. Laura E. Lamb, Tong Ye, Nicole M. HaralampusGrynaviski, T. Richard Williams, Anna Pawlak,
Tadeusz Sarna and John D. Simon “Primary
photophysical properties of A2E in solutions” J.
Phys. Chem. B 2001, 105, 11507-11512
4. L.E. Lamb and J.D. Simon “A2E: A component of
ocular lipofuscin” Photochemistry and
Photobiology 2004 79(2) p. 127