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TECHNICAL FIELD
The invention relates to high resolution absorption microscopy and
spectroscopy
BACKGROUND ART
Various techniques for high resolution microscopy followed from the
scanning tunneling microscope and its progeny. Generically, scanning tip
microscopies are based on the ability to piezoelectrically control the
position of a tip to Angstrom accuracies using feedback techniques. In
this regard, see U.S. Pat. Nos. 3,973,122; 4,343,993; 4,522,510;
4,747,698; PCT/CH87/00166; Kinoshita, "Sons of STM", Scientific American,
July 1988; Hansma et al, "Scanning Tunneling Microscopy and Atomic Force
Microscopy: Application to Biology and Technology", Science, Oct. 14,
1988, page 209; Matey, "Scanning Capacitance Microscopy", SPIE., Vol. 897,
page 110, 1988 and Martin et al, "Tip Techniques for Microcharacterization
of Materials", Scanning Microscopy, Vol 2, No. 1, 1988.
Scanning absorption microscopy is a prior art technique. Typically, the
technique employs a pump radiation beam which is incident on the object to
be investigated. Some of this incident energy is absorbed by the object,
resulting in a temperature rise. The spatial variation of temperature
across the object represents variations in the absorption of the object
caused by the incident radiation. These temperature variations can be
mapped using a temperature-sensitive probe. One example of a
temperature-sensitive probe is a thermocouple; a more recent example of
bolometric measurement is found in the scanning thermal profiler of
Wickramasinghe and Williams Patent 4,747,698.
Notwithstanding the availability of these prior art techniques, there is a
desire for enhancing the resolution of the instrument. Thus it is an
object of the present invention to enhance or improve the resolution for
scanning absorption microscopy and spectroscopy by at least an order of
magnitude.
In accordance with one preferred embodiment of the invention, a tunneling
tip is employed, in part, for temperature sensing A sample (an object
under measurement) may be supported on a substrate and, if the sample is
not conductive, it carries on a surface, opposite the surface in contact
with the substrate, a thin conductive coating. An incident energy wave
(which might be acoustic, optical or other) impinges on the sample through
the substrate which is transparent to the incident radiation. The incident
energy is absorbed by the sample and causes local temperature variations
across its surface. If a surface coating is necessary (in the case where
the sample itself is not conductive), a suitable conductive coating might
be a thin (for example 10 Angstroms) film such as carbon or platinum. With
this or any other coating which is thin enough, the temperature variations
across the sample will be transferred through the conducting film to the
exposed surface of the film. A tunneling tip is brought close to the
available surface (either the surface of a conducting film, if present, or
a surface of the sample) and positioned about 5 Angstroms over the
available surface. Tunneling current is used as a control mechanism via a
feedback loop to maintain the separation between the tip and the available
surface. By reason of the small separation between the available surface
and the tunneling tip, the tip will be in thermal equilibrium with the
available surface at the point being investigated. The apparatus at this
point is suddenly switched out of the tunneling mode (the tunneling
feedback loop is opened) and into a mode where the junction potential
between the tip and the available surface of the sample is measured. The
measurement provides an accurate measurement of the temperature at the
point being investigated. The temperature of other points across the
sample can be mapped as the tip tracks over the sample surface after again
closing and then opening the tunneling feedback loop.
While it is possible to measure the DC junction potential (as a measure of
steady state temperature), it may be advantageous to dynamically measure
temperature by modulating the pump power at some frequency, for example by
using short pulses, or a burst of short pulses at some given repetition
rate (typically around a kilohertz) within the response of the tunneling
tip sensor and detecting the AC component of the junction potential at the
same repetition rate. The repetition rate is selected at a low enough
frequency so that enough time is left over for the one micron spot on the
sample to cool down through thermal diffusion into the high diffusivity
substrate (which might for example be sapphire, a diamond or diamond
coating) before the next heating period. This allows an increase in the
contrast of the thermal signals from the background and also can eliminate
effects due to temperature fluctuations caused by the environment.
Although in the embodiment just described, tunneling was employed as the
control mechanism to maintain the close separation between the measurement
tip and the available surface, use of the tunneling mechanism is not
essential to maintain this separation. Rather, other techniques such as
atomic force microscopy or capacitance microscopy can also be used. With
these techniques the separation between the tip and the available surface
can be continuously controlled without having to switch the feedback loop
off during measurement of the thermoelectric signal.
The thermal equilibrium between the measuring tip and the available surface
at the junction region comes about because the measurement tip is so close
to the available surface that the electronic states in the tip and the
surface are strongly coupled so the Fermi levels have to equalize at the
junction. If the metal of the measurement tip and the available surface
have different work functions, there will be a corresponding step in the
vacuum level at the junction. This step is precisely the contact potential
which is the difference in the work functions of the two metals. The
temperature sensitivity results from the fact that the work functions of
the two metals increase differently with temperature. The response time of
the junction potential to temperature is limited by electron diffusion and
in this case will be in the picosecond range although in practice the
electronic detection circuitry will limit the response time to much lower
values. For example, for a tungsten tip and a platinum film surface, the
sensitivity is around 10 microvolts per degree Centigrade and the typical
junction contact resistance is a few 10's of ohms for a contact diameter
of 10 Angstroms. The temperature spatial resolution which could be
achieved in this case can be as small as a tip diameter atomic dimension
such as two Angstroms) and will in practice be limited by the signal to
noise ratio. The absorption imaging resolution achievable as described, on
the other hand, will be limited by the thickness of the conducting film
deposited on the sample surface (if present) which transfers the thermal
image and can be made as small as 10 Angstroms.
Rather than using a cross.TM.section in which the measurement tip is
adjacent the available surface (either the surface of the sample if it is
conductive or a conductive film deposited on an upper surface of the
sample) and the sample is supported by the substrate, with the energy
directed at the sample through the substrate, a different arrangement may
be used. For cases such as samples comprising a thin biological film or a
thin dielectric film (typically less than 2 nm) or both, the cross-section
can be altered as follows. The thermal measurement tip is adjacent a
surface of the thin biological film which is supported on a conductive
film. The tunneling, in this case, takes place between the tip and the
conducting film through the biological sample. The energy for heating the
sample is directed toward the same surface of the biological sample
adjacent the measuring tip.
Embodiments such as those described above should achieve resolutions of a
nanometer (10.sup.-9 meters) or better for absorption microscopy.
Furthermore, by tuning the frequency of the incident radiation, absorption
spectroscopy with similar, nanometer spatial resolution is possible.
Accordingly, the invention provides apparatus for absorption measurements
comprising:
a sample with a conductive surface,
means for supporting said sample for measurement,
means for directing energy onto said sample to cause local heating of the
sample,
a thermal measurement tip,
support means for maintaining said thermal measurement tip within about 10
Angstroms of said conductive surface, and
measurement means for measuring a junction potential between said
measurement tip and said conductive surface.
The invention also comprises:
a method or effecting absorption measurements comprising:
(a) supporting a sample with a conductive surface for measurement,
(b) directing energy onto said sample to cause local heating of the sample,
(c) supporting a thermal measurement tip sufficiently close to said
conductive surface to equalize Fermi levels between said tip and surface,
and
(d) measuring a junction potential between said thermal measurement tip and
said conductive surface.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be further described so as to enable those
skilled in the art to make and use the same in the following portions of
this specification when taken in conjunction with the attached drawings.
In the attached drawings:
FIG. 1 is a schematic of one embodiment of the invention for performing
absorption microscopy using the tunneling mechanism;
FIG. 2 is an alternative suited for thin biological samples; and
FIG. 3 is a schematic similar to FIG. 1 but, by the addition of tuner 315,
allows for spectroscopy applications.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
FIG. 1 is a schematic of one preferred embodiment of the invention for
effecting absorption microscopy. In FIG. 1 a sample 10 to be investigated
is supported on a substrate 12. In the example shown in FIG. 1, the sample
10 is non-conductive and a thin conducting film 11 is coated on an
available surface of the sample 10. A thermal measurement tip 21 (which
has characteristics similar to the tunneling tip in a STM) is supported
for three-dimensional movement by a XYZ piezoelectric drive 20. A
conductor 22 is connected to both the measurement tip 21 and to a terminal
33 of a switching arrangement 30. A conductor 41 is connected to the
conductive film 11 and to a terminal 37 of the switching arrangement 30.
The switching arrangement 30 has two movable contacts 32 and 36 which move
together. With the contacts in the full line position, contact 32 connects
terminals 33 and 34 while contact 36 connects terminals 37 and 38. In the
dashed line position, contact 32 connects terminals 33 and 35 and contact
36 connects terminals 37 and 39. Terminal 38 is connected to ground while
terminal 39 is connected to a suitable potential. Terminal 35 is connected
via a current-voltage amplifier 54 and a sample and hold circuit 55 to the
input of circuitry associated with an amplifier 23 in a STM tunneling
feedback loop. The STM tunneling feedback loop includes the measurement
tip 21, the conductor 22, the terminal 33, the contact 32, the terminal
35, the current-voltage amplifier 54, the sample and hold circuit 55, the
resistor 25, the amplifier 23, the conductor 26, to the Z input terminal
of the XYZ piezoelectric drive 20. The function of the STM tunneling
feedback loop is to maintain the separation d between an extreme point of
the measurement tip 21 and the available surface of the sample. In the
example shown in FIG. 1, since the sample 10 has a film 11 supported
thereon, the separation d is measured between the extreme end of the
measurement tip 21 and the available surface of the film 11. In other
embodiments where the sample 10 is conductive, the film 11 would be absent
and thus the separation d will be measured between an extreme end of the
measurement tip 21 and the available surface of the sample 10. The STM
tunneling feedback loop maintains the separation d by comparing the
voltage developed at the resistor 25 (by the tunneling current driven by
the potential source v across the separation d and through the
current-voltage converter) with the voltage v.sub.ref coupled by the
resistor 24 to an input of the amplifier 23.
Based on the foregoing, and on the description of the operation of a
scanning tunneling microscope provided in U.S. Pat. No. 4,343,993 (the
subject matter of which is incorporated herein by this reference), it
should be apparent that so long as the contacts 32 and 36 are in the
dashed line position, the tunneling feedback loop will maintain the
separation d between an extreme end of the measurement tip 21 and the
available surface and/or film. Suitable separations d for purposes of this
invention are on the order of 10A, a distance small enough so that there
is thermal equilibrium between tip and surface.
The source 15 is provided to generate and focus radiation, represented at
16, onto the sample 10. For the geometry shown in FIG. 1, that energy is
incident on the sample 10 through the substrate 12; therefore the
substrate is arranged to be relatively transparent to the energy generated
by the source 15. The source 15 and its associated focusing structures can
take on a variety of forms. In one embodiment, the source 15 is a laser
with associated optical focusing arrangement. Alternatively, the source 15
could be a microwave generator (10 gHz for example) with conventional
focusing apparatus. As further alternatives, the source 15 could be an
x-ray source or an ultrasonic source with, again, associated conventional
focusing structures. The purpose for the incident radiation is to cause
local heating at the sample 10 to allow temperature variations in the
sample to be measured as a consequence of that local heating.
Timer 50 provides, along conductor 51, a modulating signal for modulating
the source (15) power at a repetition frequency selected at a low enough
frequency so that enough time is left over for the irradiated spot on the
sample to cool down through thermal diffusion into the high diffusivity
substrate 12, typically at a frequency of approximately 1 KHz. The timer
50 provides the modulating signal at the repetition frequency.
Alternatively, the timer may provide a burst of short pulses, where the
bursts repeat at the repetition frequency.
Timer 50 provides a control signal, over conductor 52, to control the
position of the switching arrangement 30. Thus at times the switching
arrangement 30 is controlled to alter the position of contacts 32 and 36
from the dashed line position to the full line position. Simultaneously
with the control signal over conductor 52, the timer 50 provides a control
signal along conductor 56 to the sample and hold circuit 55 to switch from
the continuous sampling mode (for v.sub.t) to the hold mode as the
switching arrangement 30 changes to the full line position. This allows
the integrating amplifier 23 to hold its voltage output to the Z-piezo and
consequently the Z position of the tip when the tunneling feedback loop is
opened and the junction potential is being measured. In the full line
position, contact 36 effectively grounds the film 11. At the same time,
the potential from the tip 21 is applied via the conductor 22 through the
contact 32, in its full line position, via the terminal 34 to the
amplifier 40. The amplifier 40 may be enabled by a control signal on the
conductor 53 simultaneous with the control of the switching arrangement to
its full line position. With the film 11 grounded, the potential measured
at the tip 21 (which is the potential applied to the input of the
amplifier 40) is the junction potential between the tip 21 and the
conductive surface 11. The output of amplifier 40 is provided to detector
45 such as a lock-in detector in order to detect the value of this
potential and the output of the detector 45 is applied as an input to the
display 60. Accordingly, with the switching arrangement 30 in the full
line condition, the display 60 will indicate the junction potential
(between the tip 21 and the film 11) at the x-y position of the tip, as
indicated by the signals on the x and y inputs of the display 60.
By moving the tip 21 across the surface of the sample, using signals input
to the x and y inputs of the xyz piezoelectric drive 20, with the
switching arrangement 30 in its dashed line position, the tunneling tip
can be caused to map the surface of the sample with the tunneling tip
maintained at the separation d. For each point at which a temperature
measurement is desired, the condition of the switching arrangement 30 is
cycled to its full line position and then back to the dashed line
position. In the full line position of switch 30, the junction potential
at that point can be measured so as to map the junction potential of the
available surface.
The control signals from timer 50 cycles the switching arrangement 30 at a
frequency at least ten times higher than the repetition frequency of the
modulating signal provided along conductor 51, typically at a frequency of
at least 10 KHz.
In the case of a conductive sample 10, there is no need for film 11 and so
the measurement depends on the tip to sample potential.
If .delta.T is the temperature excursion at a point on the sample caused by
the incident energy, .sigma. is the sensitivity of the junction potential
to temperature variation and R.sub.j is the tip to sample junction
resistance, we can write the S/N ratio (in the case where the contact
potential is measured by a high impedance amplifier) as:
(S/N).sub.v =.delta.T.sigma./.sqroot.4.pi.KTR.sub.j .DELTA.f
where K=Boltzmans constant, T=Absolute temperature and .DELTA.f=Detection
bandwidth. With .sigma.= 10 uv/deg C, R=100 Ohm, T=300K and S/N=1, we can
find the minimum detectable temperature, .DELTA.t.sub.min =0.0001 deg C.
The highest spatial resolution that can be achieved will depend on S/N
which in turn depends on the highest thermal contrast that can be
achieved. In order to get the highest temperature excursion from an
absorbing region of spatial extent .delta.a, the pulse width must be short
enough so that all the energy dumped into the absorbing region gets
absorbed without diffusion during the pulse interval This gives an
expression which relates the pulse width .delta.t to the resolution
.delta.a through the effective thermal diffusion length in the sample.
.delta.a=[2K.delta.t/.pi..rho.C].sup.1/2
where K is the thermal conductivite, .delta.t is the pulse width and
K/(.rho.C) is the thermal diffusivity. This suggests that with pulses in
the ps range, it should be possible to obtain absorption across nm regions
without thermal diffusion limiting the temperature rise. If .delta.a is
the differential absorption coefficient over the average value in the
sample at the point being investigated, I is the flux intensity incident
on the object over a time .delta.t, .rho. is the density and C is the
specific heat of the object, we can write an expression for the
temperature increment .DELTA.T:
.DELTA.T=.delta..alpha.I.delta.t/.rho.C
With ps pulses of 100 W peak power focused to 1 micron and consider a
biological sample with a differential optical absorption coefficient from
its surroundings of 100,000/meter, an estimated peak temperature rise of 2
degrees centigrade is obtained. This is four orders of magnitude higher
than the detection limit for temperature measurement. It should however be
pointed out that the system will also operate with much broader pulses in
the .mu.s or even ms range with reduced temperature rise.
Another way to detect temperature dependent work function changes would be
to vibrate the tip in the Z axis direction at a frequency outside the band
of the tunneling control loop while the loop is closed, and measure the
component of tunneling current at the vibration frequency. This signal
will be proportional to .sqroot.((.phi..sub.w +.phi..sub.pt)/2). However
this signal varies more slowly with temperature and in addition cannot
respond to temperature variations faster than the tip vibration frequency.
In FIG. 2 the relationship between the tunneling tip, the switching
arrangement 30 and the circuitry connected thereto, including the XYZ
piezoelectric drive 20 is identical to that of FIG. 1. FIG. 2 differs from
FIG. 1 in that the sample 10' being investigated can be a thin biological
film or a thin dielectric film (typically less than 2 nanometers) which is
in turn supported on a conductive film 11'. In the embodiment shown in
FIG. 2, the tunneling takes place between the tunneling tip 21 and the
conductive film 11'. The focused energy 16 emanating from the source 15 is
focused on the point of the sample 10' being investigated, i.e. that point
opposite the tunneling tip 21. In other respects, the apparatus of FIG. 2
operates in a fashion identical to that explained with respect to FIG. 1.
As mentioned, while the embodiments of FIGS. 1 and 2 employ STM techniques
for maintaining the separation of the tip 21, the use of STM techniques
for this purpose is not essential. Rather, other techniques such as atomic
force microscopy or capacitance microscopy can be substituted in lieu of
STM techniques. An advantage of using techniques other than STM is that
when using techniques other than STM, since the techniques do not rely on
tunneling current, the switching arrangement 30 can be eliminated.
Although the foregoing description has concentrated on microscopy
applications, those skilled in the art will understand that spectroscopy
applications can also be implemented using the same architecture by merely
providing for tuning of the source 15 FIG. 3 for example, which is similar
to FIG. 1, differs therefrom by the use of a frequency controller or a
tuner 315 for varying the frequency or wavelength of the energy generated
by the source 15. By tuning the frequency or wavelength of the energy
generated by the source 15, measurements can be made at different
frequencies or wavelengths.
Applications of the optical absorption microscope include optical
spectroscopy of single molecules and the location of dye molecules
selectively attached onto biological surfaces using immuno-fluorescence
methods. It should enable protein sequencing and DNA sequencing through
selectively staining the nucleotides with different dye molecules and
spatially identifying them with the absorption microscope.
While several specific embodiments of the invention have been described,
the invention should not be limited by that description and instead should
be construed by the claims appended hereto.
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