 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to near-field optical microscopy in which a
sample undergoing study is irradiated with light and the
reflection/transmission or absorption characteristics of the sample are
analyzed so that an image of the sample is generated. The invention also
relates to near-field optical (NFO) microscopes in which a near-field
optical probe is used to produce changes in the reflection, transmission
or absorption characteristics of a sample under study.
2. Discussion of the Related Art
Optical microscopy is one of the most widespread experimental techniques in
science because of its ease of use and its well established theoretical
background, which makes image interpretation relatively straight forward.
The major limitation of optical microscopy is on the resolution of the
produced images, which will generally be equal to the order of wavelength
of light which is used. This limitation remains despite the fact that
under certain circumstances distances or thicknesses can be measured with
much higher accuracy. In these cases, the sample geometry is restricted or
certain predetermined information about the sample is necessary in order
to interpret the images. Confocal microscopy and video enhanced microscopy
can increase lateral and vertical resolution by typically a factor of two
for some types of samples (see Pluta, M. Advanced Light Microscopy;
Elesevier: Amsterdam, 1989; volume 2).
An improvement in resolution is possible by using a near-field optical
microscope. The basic idea for this type of microscope originated in
Ireland in the beginning of the 20th Century and was demonstrated using
microwaves in the 1970's. The first application to optical light was
reported recently in Pohl, D. W.; Denk, W.; Lanz, M. Applied Physics
Letters, 1984, volume 44, pages 651-653. In near-field optical microscopy,
a sharp tip which is usually a sharpened glass fiber, is used as a light
source and raster scanned above the surface. Using the specially sharp
fibers, a resolution of about 12 nanometers can be achieved. The
sharpening of the fibers is usually done by pulling them in a very special
way which results in a very thin fiber. They are very fragile, yet need to
be scanned very close to the samples. Care needs to be taken to prevent
the fiber from touching the sample and breaking. One solution to this
problem involves looking at the lateral bending of the fiber and using
this signal as the input to a distance control circuit. Another problem
with using fibers is their low efficiency. Because of their small opening
angle, most of the light is absorbed by the metal coated fiber walls so
that only a small fraction of the light passes through the tip of the
fiber (signal levels of up to 50 nW have been reported). Despite these
problems, the benefit of the increased resolution has justified the
increased amount of work in this area and has yielded impressive images.
Another approach to the low resolution problem has been to make specially
designed microfabricated tips for scanning near-field optical microscopy
similar to those designed for scanning ion conductance microscopy. These
tips are not, however, available commercially and the microfabrication
process is too complex to make it practical for most researchers.
Near-field scanning microscopy has been performed in the prior art mainly
using optical fibers which have been tapered to a sharp tip so as to
project light onto the sample in order to measure light reflected by the
sample and/or transmitted through the sample and projected onto a detector
device for viewing and/or analysis by an operator. However, the use of
optical fibers for use in near-field optical microscopy has other
disadvantages in that these optical fibers are subject to easy breakage,
they can not be fabricated with current micro-machining technology and the
fibers can be destructive to the sample if they come in contact with the
sample, such as, by scraping the sample. Therefore, a complicated
mechanism for preventing the contact between the optical fibers and the
sample has been necessary. The use of optical fibers has another drawback
in that it is difficult to taper the optical fibers in order to create an
aperture small enough for performing high resolution imaging.
In order to overcome such disadvantages associated with optical fibers,
there has been proposed a method of performing near-field optical
microscopy using a cantilever having a sharp tip formed of silicon nitride
(see van Hulst, et al, "Operation Of a Scanning Near Field Optical
Microscope in Reflection in Combination With a Scanning Force Microscope",
SPIE Vol. 1639, pp. 36-43, 1992). In this conventional device as shown in
FIG. 1, a micro-fabricated SiN cantilever 131 having an integrated
pyramidal tip 123 is fixed at an angle to the upper surface of a sample
121. The tip is coated with an opaque material so that only light focused
on the aperture of the tip passes through to the sample surface. The
sample is placed on a mount 127 which is movable with respect to the fixed
cantilever via a piezoelectric tube scanner 119. As can be seen from FIG.
1, light from a source 143, such as a mercury discharge lamp, passes
through lenses 141 and 139, and is reflected by a dichroic mirror 137 and
focused by objective lens 111 onto the back surface of the sample to
illuminate the sample portion under study so that it can be viewed by an
observer. Light from a laser source 125 is projected through a dichroic
mirror 135 and is deflected by reflector 129 and focused by lens 133 into
the back portion of the silicon nitride tip of the cantilever 131. Light
which passes through the opening of the tip 123 is reflected off the
sample back through the silicon nitride tip, reflected by elements 129 and
135, passed through a filter 113 and diaphragm 115 and is then received by
a detector 117. These detected reflections are then used to generate an
image of the sample which has been scanned across its entire surface.
However, the foregoing apparatus has an important disadvantage in that the
light which is projected onto the back surface of the cantilever tip is
also reflected back to the detector device along with the light which
passes through the tip of the cantilever, strikes the surface of the
sample and is then reflected back through the tip to the detector device.
Because of this, it is difficult to distinguish the small changes due to
the light which is reflected off the sample since there is a significant
amount of reflected light which constitutes "noise" generated by the
reflections off the back surface of the cantilever tip.
Another known method of performing near-field optical microscopy relies
upon local modification of an evanescent field which is an extremely thin
(of micron proportions) region of electromagnetic energy which exists
adjacent to a back surface of a prism where a beam of light has been
projected through a front surface of the prism and this light beam is
totally internally reflected off the back surface of the prism so that no
visible light is emitted through the back surface of the prism into the
far field. The introduction of a sharp probe tip into this evanescent
field, however, will cause an induction of some of the electromagnetic
energy to be emitted from the back surface of the prism. This will result
in the emission of light into the far field. This light which is emitted
from the back surface of the prism will be modified by the different
properties of the sample which is disposed on the back surface of the
prism where the back surface serves as a mount for the sample and the
probe tip follows the sample topography. An image of the sample can be
obtained by detecting the changes in light emitted through the sample into
the far field as the probe tip follows the surface topography of the
sample. However, the main disadvantage of using evanescent field
modification is, as noted above, that these fields extend only a very
small distance above the back surface of the prism. Therefore, if it is
desired to view an object such as a cell, for example, which is of
relatively thick dimensions, the foregoing prior art devices will not
provide satisfactory results, since the evanescent field would not extend
completely through the relatively thick cell under study so as to be able
to be locally modified by the probe tip.
Another known method of performing near-field optical microscopy using a
probe is disclosed in U.S. Pat. No. 5,105,305 to Betzig et al. According
to this method and apparatus, a microscope is provided with a probe having
a narrow aperture which includes a plurality of minute particles which are
impregnated with fluorescent dye so that upon irradiation of these
particles with light, the particles emit fluorescent light to be projected
onto the sample. Then, by moving the probe aperture into close proximity
to the sample surface, the fluorescence emitted from the probe aperture
will be quenched due to dipole-dipole interactions between the particles
and the sample surface. The Betzig et al method and apparatus also
provides transmission and reflection modes of scanning using the
fluorescent light emitted from the probe tip in order to generate images
of the sample surface or composition of the sample. Such a microscope has
a main disadvantage, however, that the probe apparatus is not finely
controlled with respect to its positioning in close proximity to the
sample surface which could thereby lead to the possibility of a large
contact force with the sample surface which may damage the sample.
Another known probe for performing near-field optical microscopy is
disclosed by Pohl in U.S. Pat. No. 4,604,520. Such a probe is formed by
providing a transparent tip having a metal coating around the outside
walls thereof in order to provide a small opening for performing the
optical scanning in a transmission or reflection mode. However, the Pohl
apparatus has the same disadvantages described above with respect to
Betzig et al, i.e., there will be the possibility of causing damage to the
sample if the probe tip comes too close to the sample surface.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide an apparatus
for performing near-field optical microscopy which overcomes the foregoing
disadvantages of the prior art.
Another object of the present invention is to provide a near-field optical
microscope for scanning an object in order to produce a high resolution
map of a fluorescing characteristic of the sample under study.
Another object of the present invention is to provide a method and
apparatus for performing near-field optical microscopy which avoids the
disadvantages associated with using optical fibers and which utilizes
micro-machined cantilevers for determining the locations of proteins or
other cell structures within a sample based on the fluorescence
characteristics of these structures.
A still further object according to the present invention is to provide a
method and apparatus for performing near-field optical microscopy which
utilizes a micro-machined cantilever member having a sharp tip which
enables local modifications of the optical properties of a sample exposed
to light from a light source in order thereby to generate an image of the
sample under study.
These and other objects are achieved according to the present invention by
providing a novel apparatus for performing near-field optical microscopy
on a sample supported on a mount, including a light source for irradiating
the sample with light to cause a portion of the sample to fluoresce; a
probe having a tip provided with an optically active element in order to
locally discharge a portion of the energy imparted to the sample by the
light source; and a detecting means for detecting changes in the optical
properties of the sample due to movement of the optically active element
into close proximity to the sample.
In a preferred embodiment, the apparatus of the invention includes means
for producing relative movement between the optically active element of
the probe tip and the sample, and the detecting means detects changes in
the optical properties of the sample at a plurality of locations on the
sample during the relative movement between the sample and the optically
active element of the probe tip.
In another embodiment, the fluorescence imparted to one or more portions of
the irradiated region is enhanced by the interaction of an optically
active element disposed at the tip portion of the cantilever probe,
thereby to produce sharper images with greater signal-to-noise ratios. The
apparatus according to the present invention is also used to measure the
reflection, transmission or absorption characteristics from a sample
region within a distance of one wavelength of light away from the sample
surface. The apparatus of the present invention also includes means for
producing a relative scanning motion between the sample and the probe such
as by raster scanner or circular scanning, for example.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention and many of the attendant
advantages thereof will be readily obtained as the same becomes better
understood by reference to the following detailed description when
considered in connection with the accompanying drawings, wherein:
FIG. 1 illustrates a conventional near-field scanning microscope;
FIG. 2 illustrates a first embodiment according to the present invention;
FIG. 3 illustrates an alternative embodiment according to the present
invention whereby the fluorescence of different portions of the sample is
enhanced by the interaction of electromagnetic energy with a conductive
particle included at the tip portion of the cantilevered probe;
FIG. 4 illustrates a further embodiment according to the present invention
which illustrates a reverse-field scanning technique;
FIG. 5(a) illustrates an enlarged view of a cantilever with a tip portion
coated with an optically active element;
FIG. 5(b) illustrates an enlarged view of the transparent tip having a
conductive element disposed at an apex portion thereof;
FIG. 5(c) illustrates an enlarged view of the transparent tip having an
opaque material coated around the external walls of the cantilever tip in
order to produce a point source of light having a cross-sectional diameter
equal to the diameter of the apex portion of the tip not coated with the
opaque material;
FIG. 6 illustrates a feedback control system associated with AFM techniques
which is used for controlling the cantilever tip in near-field microscopy
according to the invention;
FIG. 7 illustrates a schematic diagram of the scanning near-field optical
microscope according to the present invention shown in combination with a
detector and piezoelectric translation tube for moving the sample relative
to the cantilever probe;
FIG. 8(a) illustrates a near-field image obtained using the apparatus of
the present invention; and
FIG. 8(b) illustrates a graph showing results of analysis of the near-field
image shown in FIG. 8(a).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention involves the use of near-field optical microscopes
which use tips that are normally employed in the field of atomic force
microscopy (AFM). These tips are commercially available (Digital
Instruments, Santa Barbara, Calif.) and are microfabricated hollow
pyramids made of silicon nitride with a typical height and width of four
microns attached to long, soft cantilevers. The opening angle is typically
20.degree. and the radius of curvature at the tip can be as low as 20
nanometers. These tips can be scanned while in contact with a sample
because they can easily bend and are thus resistant to breakage. In
addition, an optical lever detection system, similar to the ones used in
atomic force microscopy, can be used to minimize loading forces in the
same manner as an atomic force microscope.
Referring now to the drawings, wherein like numerals designate identical or
corresponding parts throughout, and more particularly to FIG. 2 thereof,
there is shown a side view of the apparatus according to one embodiment of
the present invention. As shown in FIG. 2, the apparatus includes a
cantilever 3 having a tip 10 which includes, at a distal end portion
thereof, an optically active element 13 which can be used, for example, to
quench fluorescent light emitted from a portion of the sample 15 upon
irradiation of the sample with light energy, as will be explained in
detail later. Attached to an opposite end of the cantilever 3 is a first
translator device 16 coupled to the cantilever, preferably through a chip
17 for ease of handling. The first translator device 16 is used in one
embodiment of the invention to perform oscillation of the tip 10 in the z
direction, as indicated by the arrow shown in FIG. 2, as will be described
in detail below.
The sample under study is supported by a transparent mount 23 which is
connected at one end thereof to a second translator device 24 through a
boom 25. The second translator 24 provides the scanning movement of the
sample with respect to the stationary probe tip. Alternatively, the probe
is scanned with respect to a stationary sample. Although two translators
16 and 24 are shown, a single piezo tube scanner or any other type of
scanning device may be used for producing x, y raster scanning, circular
scanning and z direction movement, i.e., the sample can be moved up and
down rather than the probe tip, if desired.
Below the mount 23 is an optical lens 20 for focusing a light beam 18 from
a light source 30 which is reflected by a partially silvered or dichroic
mirror 19 and is focused onto region 14 from the back surface of the
sample in order to impart energy thereto and thus cause the sample to
fluoresce. The region 14 is normally the entire field of view to be
imaged. It can, however, be expanded to be the entire sample, but this
will result in a detection light having a reduced intensity.
Alternatively, the region 14 can be narrowed to a fraction of the field of
view near the optically active element 13 when higher intensity is
required. An objective 26 enables the operator to view the sample while
the probe is being positioned into close proximity to the sample surface.
Light source 30 is a mercury arc discharge lamp or a laser, for example.
The light source 30 is generally positioned on the opposite side of the
sample from the cantilever 3 as shown in FIG. 2. It can, however, be
positioned on the same side if the tip 10 is transparent, i.e., the front
surface of the sample 15 is irradiated by projecting the light beam
through the transparent tip and onto the sample surface.
Irradiation of the sample imparts energy to the molecules of the sample.
This energy is discharged by means of the release of one or more photons
of fluorescent light 22 which is detected by an optical detector 7. The
light used for irradiating the sample has a wavelength which is typically
less than 500 nm, while the fluorescent light will generally have a
wavelength greater than 500 nm such as, for example, 600 nm or higher.
These different wavelengths facilitate the detection of changes in the
amount of fluorescence by enabling the filtering out of the light from the
irradiation source 30, thereby leaving only the fluorescent light for
detection. In a preferred embodiment, the optically active element 13 is
silicon, gallium arsenide, iridium, and iridium-platinum alloy, or any
other suitable conductive material.
In FIG. 2, the quenching of the fluorescent light emitted by portion 21
will occur when the optically active element 13 attached to the apex of
probe tip 10 comes into close proximity with the fluorescing portion 21.
When this occurs, the electromagnetic energy imparted to portion 21 will
be absorbed by the optically active element which is slightly heated in
the process, thereby quenching the energy of portion 21 and preventing
fluorescent light from being further emitted therefrom. This will cause a
dark portion to exist where portion 21 was previously fluorescing. This
change in the amount of fluorescence output by portion 21 is detected by a
detector 7, which can be a photomultiplier tube (PMT) and a lock-in
amplifier. For portions 21 of the irradiated region 14 which fluoresce
more brightly than other portions 21, there will be larger changes in the
overall light received from the irradiated region 14 by the detector 7.
The brighter the fluorescence, the greater the amount of fluorescent light
which will be quenched when the tip comes close to the sample surface.
Thus, by simply analyzing how much the fluorescent light output varies
upon introducing the probe tip into close proximity to the fluorescing
region 21 as a function of where the tip is over the surface of the
sample, the apparatus of the present invention provides a microscopic
mapping of the fluorescing characteristics of the sample.
In the above embodiment, in order to maintain the probe tip within
sufficient proximity to the sample surface in order to perform the
quenching of fluorescing regions, the probe tip is first moved into
position while an operator monitors the tip position by viewing the tip
through the microscope objective 26 so as to contact the sample surface
and the tip is then raised slightly a controlled distance above the sample
surface. The probe tip is thus maintained a slight distance (on the order
of nanometers) from the surface of the sample at certain portions of the
sample surface while at the same time coming into contact with other
portions of the sample surface depending upon the topography of the sample
surface, i.e., there will be hills and valleys due to the irregular
topography of the sample surface. In an alternative embodiment, the tip is
set so as to contact the sample surface and is permitted to passively
follow the topography of the sample due to the resiliency of the flexible
cantilever. In this case, since the cantilever is flexible, the probe tip
will move up and down along with the topography of the surface of the
sample while maintaining contact therewith without damage to the sample,
as will be described in more detail later.
In another embodiment, quenching is performed by oscillating the tip up and
down at, for example, several KHz at the surface of the sample and then
monitoring the corresponding variation in the light signal. In this case,
if the probe tip is over a portion of the sample which is brightly
fluorescing, a large signal at the cantilever oscillation frequency will
be detected since a large light signal will be quenched each time the
cantilever comes close enough to the sample to quench the light. If, on
the other hand, the probe tip is over a region that is not fluorescing,
there will be no output signal at all since there will be no light from
that region of the sample and therefore nothing to quench. In this manner,
by using an optical detector which is tuned to detect the frequency of the
output light so as to monitor how great the received AC signal is at the
frequency of cantilever oscillation as a function of the position, an
image of the fluorescence of the surface is generated.
As another embodiment according to the present invention, the fluorescence
quenching described above is replaced with a fluorescence enhancement
technique, as illustrated in FIG. 3. This is achieved by substituting the
quenching element with an optically active element which serves to enhance
the fluorescence of the different portions of the irradiated region 14.
Silver or gold particles are preferably used as the material for the
optically active element here, as such metals have been shown to enhance
electric fields in their vicinity and thus enhance fluorescence. See, for
example, Aravind et al, "The Interaction Between Electromagnetic
Resonances And Its Role In Spectroscopic Studies Of Molecules Adsorbed On
Colloidal Particles Or Metal Spheres" Surface Science, Vol. 110 (1981),
pp. 189-204. The wavelength of light is selected so as to cause the
greatest plasmon resonance of the particle, and therefore the greatest
fluorescence enhancement. While this wavelength will depend upon the size
of the particle, it is typically in the visible wavelength range for
particles in the size range of 10-100 nanometers.
As shown schematically in FIG. 3, fluorescing portion 21 is excited by
incoming electromagnetic radiation 4 and the fluorescence generated is
received by detector 7. The incident electromagnetic radiation 4 and the
outgoing light 5 is imparted or detected from any direction desired. The
optically active element 13, in this case a small metal particle, coating
or element, is provided at the tip portion of the cantilever 3 so as to be
within the electromagnetic field imparted to the sample, and the
interaction of the electromagnetic radiation with the optically active
element 13 causes a greatly increased electromagnetic field in the
vicinity of the conducting particle 13 due to the plasmon resonance
phenomenon. In particular, as described in Adams et al, "Possible
Observation of Local Plasmon Modes Excited by Electrons Tunneling through
Junctions", Physical Review Letters, April, 1979 and Hayashi et al,
"Surface Plasmon Resonances in Gas-Evaporated Ag Small Particles: Effects
of Aggregation" Solid State Communications, Vol. 76 No. 8, pp. 1067-1070,
1990, electromagnetic fields in the vicinity of a small metal particle can
be enhanced by surface plasmon modes in the particle. The enhanced
electromagnetic fields enhance Raman scattering, and also enhance light
emission from a tunnel junction. According to the present invention, the
enhanced fields are used as local probes for near field optical
microscopy. The enhanced fields are used to locally, i.e., in the
near-field region of the particle, excite fluorescence or increase the
excitement of the fluorescence. The optically active particle 13 is used
to create the locally enhanced field. Such use is enabled because the
enhanced fields decay away from the particle with a decay length shorter
than the wavelength of light. Thus the enhanced fields exist only in the
immediate vicinity of the particle and changes in the optical properties
of the sample which are stimulated by these fields are localized within a
region smaller than a wavelength of light, i.e., smaller than the decay
length. The enhanced fields are produced by first analyzing spectra of the
particles to be used and determining the optimum wavelength light for
exciting the particles by looking for the wavelength which results in the
greatest scattering of the incident light. Such scattering is due to the
excitation of the plasmons and their subsequent radiation decay in many
directions. This phenomenon has been described in Aravind et al, "The
Effects of the Interaction Between Resonances in the Electromagnetic
Response of a Sphere-Plane Structure; Applications To Surface Enhanced
Spectroscopy", Surface Science, Vol. 124 (1983), pp. 506-528. The optimum
wavelength is then used to effect the plasmon resonance of the particle
during imaging. The enhanced electromagnetic fields further excite the
fluorescing portion 21 which has been excited by the initial
electromagnetic radiation 4 so that it will fluoresce even more brightly
in the vicinity of the optically active element. In this manner, the
optically active element 13 functions to provide a greater signal-to-noise
ratio for the outgoing light 5 which is received by detector 7. FIG. 3
shows an enhanced electromagnetic field X in a region surrounding the
optically active element 13. The enhancement of the electromagnetic field
can be up to 100 times that which exists at the sample surface due to the
illumination light 4. The signal-to-noise ratio for the embodiment shown
in FIG. 3 will be B/A where A is equal to the total amount of fluorescent
light detected by detector 7 due to the emitted light from portion 21, and
B is the maximum amount of fluorescent light which can be quenched in
region X, i.e., B is the amount of fluorescence from region X being
collected. Also, if in the enhancement design, with average enhancement C,
the enhanced fluorescence detected from region X is BC and the S/N ratio
becomes BC/A which is C times better in terms of enhancement. The present
invention generates a map of the fluorescence excitability of the sample
having a resolution substantially equal to the size of region X shown in
FIG. 3, i.e., a near-field optical microscope. The optically active
element 13 acts like a small antenna scanning over the surface of the
sample and a microscopy is performed by looking at the light output
intensity as a function of the location of this small antenna.
Conventional near-field optical microscopes include a small aperture at
the tip of the probe which is smaller than an optical wavelength and the
fields at the tip can be calculated. It should also be noted that the
largest output signal is not recorded when the probe is directly above the
molecule. Rather, a complicated dipole-dipole interaction between the
probe tip and the fluorophor is created which makes each fluorophore
produce two irregular patches of high intensity. This will also occur for
an enhancement near-field optical microscope using such an optically
active element 13. The excitability of each fluorophore depends upon the
orientation and distance from the near-field optical probe. For isolated
particles, the fluorophore itself "images" the electric field distribution
of the probe because the fluorophores are significantly smaller than the
conventional near-field optical probes. It is also possible in theory to
reduce the probe down to another fluorophore and look at "resonance energy
transfer" (RET) between the fluorophores.
As a further embodiment according to the present invention, there is
illustrated in FIG. 4 a reverse-field near-field microscope which operates
in essentially the opposite manner from that of the embodiment of FIG. 2.
For example, the surface of the sample 15 is coated with a layer 8 of
fluorescence quenching material such as, for example, a liquid or gel
doped with conducting particles, such as a ferro-fluid. The layer 8 is
locally displaced by a nonconducting element located at the tip portion of
cantilever 3 to permit fluorescence in a small region in close proximity
to the tip of the probe. The embodiment of FIG. 4 results in a greatly
reduced background signal as fluorescence will only be detected when the
nonconducting tip displaces the quenching liquid or gel away from the
surface of sample 15. In this embodiment, when the ferro-fluid is
displaced by the cantilever tip, the ferro-fluid will not quench the
fluorescence as strongly and therefore brighter fluorescence will be seen.
When the ferro-fluid is displaced approximately 10 or 20 nanometers
radially from a sample region, quenching will be reduced by an amount
equal to approximately two orders of magnitude. Also, in the case where
the sample 15 is opaque, light 2 from the light source will be irradiated
from the front surface of the sample from a location above the transparent
cantilever tip.
The fluorescence quenching or enhancement techniques described above may be
used in biological applications, where it is desired to evaluate a cell
with specific proteins of interest located on the surface of the cell.
These specific proteins are tagged with materials which will fluoresce
more brightly than other portions of the irradiated sample in order to
make those porti | | |
