Integrated microscope providing near-field and light microscopy

Apparatus are disclosed that perform, on demand, both near-field microscopy (NFM) and light-microscopy (e.g., bright-field microscopy or phase-contrast microscopy) of a specimen. The apparatus comprises an NFM microscope including an NFM probe, a first condenser lens system that converges a center portion of an illumination-light flux at a first terminus of the NFM probe, and a second condenser lens system that converges an annular portion of the illumination-light flux at a specimen to illuminate the specimen by Kohler illumination. Evanescent light from a distal terminus of the NFM probe passes to a locus on the specimen. Downstream optics capture light transmitted and/or scattered from the specimen from the NFM probe and from the Kohler illumination to produce both a light-microscope image of the specimen and an NFM image of the specimen.

FIELD OF THE INVENTION 
The present invention pertains to near-field microscopes (NFMs) and light 
microscopes, especially to optical instruments that are operable to 
perform near-field microscopy in addition to observations of specimens by 
light microscopy, e.g., bright-field and phase-contrast microscopy on 
demand. 
BACKGROUND OF THE INVENTION 
In an near-field microscope (NFM), illumination light is made incident on a 
first end of a glass rod or optical fiber referred to as an NFM probe. The 
other end (i.e., the distal end) of the NFM probe is sharply pointed to 
form a point source of light having dimensions typically much smaller than 
the wavelength of the illumination light. When the distal end of the probe 
is maneuvered into close proximity to a specimen (i.e., a distance from 
the specimen smaller than the wavelength of the illumination light), light 
waves spanning the gap from the distal end to the adjacent locus on the 
specimen are evanescent. This evanescent light permits observation of the 
locus with a degree of resolution substantially greater than otherwise 
would be possible with most other light microscopy methods using the same 
wavelength of illumination light. The region of the specimen that can be 
observed using NFM, however, is very small. 
FIG. 3 schematically shows certain features of a conventional NFM system. 
An illumination-light flux F generated by a high-intensity light source 
(not shown) is restricted by passage through an aperture diaphragm 30 (the 
light source is situated above the aperture diaphragm 30 in the figure). 
The illumination-light flux F then passes through a condenser lens L3 
(comprising lens elements L3a, L3b) that converges the light flux to a 
point at an entrance end 20 of an NFM probe N. (The entrance end 20 is 
situated at a focal point of the condenser lens L3.) The distal end 10 of 
the probe N is positioned near the specimen O as mentioned above. A piezo 
element P or analogous scanner is attached to the NFM probe N. The piezo 
element P is operable to cause the NFM probe N to scan two-dimensionally 
(in the X-Y plane) with respect to the specimen O. 
The evanescent light propagating from the terminus 10 of the NFM probe N to 
the specimen O is scattered or transmitted by the specimen and is 
converged by an objective lens (not shown in the figure) to form an NFM 
image of the specimen. The NFM image can then be observed and/or otherwise 
analyzed. 
Because an NFM image must be constructed from a series of scans in the X-Y 
plane, significant time is required to obtain each NFM image. In addition, 
each NFM image is of a very small region of the specimen. It is frequently 
necessary to obtain a number of NFM images of the specimen, thereby 
consuming much time, before finding a desired locus on the specimen for 
further NFM examination. Thus, there is a need for a way to simultaneously 
observe a substantially larger region of the specimen than is observable 
by NFM so as to facilitate more rapid finding of the desired locus on the 
specimen for further NFM examination. 
The NFM probe N normally has a length of only a few centimeters due in part 
to the need to attach it to the scanner P. Unfortunately, the probe N, the 
scanner P, and the condenser lens L13 in a conventional NFM, while useful 
for performing NFM microscopy, are obstructions that would block 
sufficient illumination for larger-scope observations of the specimen by 
light-microscopy methods such as bright-field microscopy or phase-contrast 
microscopy. 
Furthermore, because time is required to obtain each NFM image, a 
conventional NFM cannot be used for instantaneous on-demand observation of 
a specimen. Thus, there is a need for an integrated apparatus operable to 
perform both NFM and at least one light-microscopy method capable of 
producing on demand a larger-scope image of the specimen than NFM. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide an NFM integrated 
microscope that is operable to solve the above-referenced shortcomings of 
the prior art. A more specific object is to provide an NFM integrated 
microscope that is operable to perform NFM observations of a specimen as 
well as larger-scope ("total") observations such as bright-field 
observations and phase-contrast observations of the specimen on demand. 
According to one aspect of the present invention, an integrated microscope 
is provided. The integrated microscope comprises, on an optical axis, an 
irradiation optical system and an observation optical system. The 
irradiation optical system, which is operable to produce an 
irradiation-light flux for irradiating a specimen, comprises an NFM probe, 
a first condenser lens system, and a second condenser lens system. The NFM 
probe extends along the optical axis and has an entrance terminus and a 
distal terminus. The first condenser lens system is operable to cause a 
central portion of the irradiation-light flux to converge on the entrance 
end of the NFM probe so as to form an evanescent wave of light from the 
distal end of the probe to a locus on the specimen. The second condenser 
lens system is operable to cause a peripheral portion of the irradiation 
light flux to converge at a region on the specimen. The observation 
optical system comprises a first observation lens system operable to 
collect light from the evanescent wave that is scattered or transmitted by 
the locus on the specimen, and a second observation lens system operable 
to collect light scattered or transmitted from the region on the specimen. 
The integrated microscope can include a light source operable to produce 
the irradiation light flux and to propagate the irradiation light flux 
along the optical axis. 
The integrated microscope can also include a stop that defines a central 
aperture and a coaxial annular aperture. The central and annular apertures 
are preferably situated so as to produce, from the irradiation light flux 
propagating from the light source, the central and peripheral portions of 
the irradiation light flux, respectively. 
Preferably, the first condenser lens system comprises first condenser lens 
and a second condenser lens, and the second condenser lens system 
comprises the second condenser lens. For example, the second condenser 
lens system preferably comprises two positive lenses, and the first 
condenser lens system preferably comprises the second condenser lens 
system and a third positive lens (the third positive lens preferably spans 
the central aperture). Thus, the first condenser lens system preferably 
has a shorter focal length than the second condenser lens system. 
In the integrated microscope summarized above, the observation optical 
system preferably includes, on the optical axis, an objective lens and a 
beam-splitting prism. The beam-splitting prism is operable to reflect 
light along a first branch of the optical axis to the first observation 
lens system and to transmit light along a second branch of the optical 
axis to the second observation lens system. In such a configuration, the 
objective lens is operable to form a first primary image of the specimen 
on the first branch of the optical axis and a second primary image of the 
specimen on the second branch of the optical axis. The first observation 
lens system preferably includes a photomultiplier situated on the first 
branch of the optical axis, and the second observation lens system 
preferably includes a relay lens and an eyepiece lens to permit 
observation by user of the second primary image. 
According to another aspect of this invention, an apparatus is provided for 
performing near-field microscopy and light microscopy of a specimen. The 
apparatus comprises, on an optical axis, a stop, a near-field microscope 
subsystem, and a light-microscope subsystem. The stop defines a central 
aperture and a coaxial annular aperture. The stop is operable to produce, 
from an illumination light flux propagating along the optical axis, a 
central light flux and an annular light flux coaxial with the central 
light flux. The near-field microscope subsystem comprises, on the optical 
axis, a first condenser lens system, an NFM probe, an objective lens, and 
a photosensor. The first condenser lens system is operable to converge the 
central light flux onto the entrance of the NFM probe. The NFM probe is 
operable to produce, from the central light flux, evanescent light that 
passes from a distal terminus of the NFM probe to a locus on the specimen. 
The objective lens is operable to produce, from the evanescent light 
interacting with the locus, an image of the locus analyzable by the 
photosensor. Finally, the light-microscope subsystem comprises, on the 
optical axis, a second condenser lens system that is operable to converge 
the annular light flux onto the specimen. The objective lens also produces 
a viewable image of a region of the specimen on which the annular light 
flux is incident. 
In the foregoing apparatus, the first condenser lens system preferably has 
a fixed first focal length and the second condenser lens system has a 
second focal length that is preferably longer than the first focal length. 
It is also preferable for the first condenser lens system to include a 
first condenser lens having a positive refractive power and for the second 
condenser lens system to include a second condenser lens having a positive 
refractive power. 
The foregoing and additional features and advantages of the present 
invention will be more readily apparent from the following detailed 
description, which proceeds with reference to the accompanying drawings.

DETAILED DESCRIPTION 
As used herein, "light microscopy" shall refer to microscope methods, other 
than NFM, involving illumination of a specimen using light. 
General features of a preferred embodiment of an NFM integrated microscope 
according to the present invention are illustrated in FIG. 1, which 
represents the current best mode. Components shown in FIG. 1 that are the 
same as shown in FIG. 3 have the same reference designators. 
Arranged on an optical axis AX are the following components: a light source 
S, a collector lens L1, a first mirror M1, a first condenser lens L2 
having positive refractive power, a stop or diaphragm RS defining a 
preferably circular central aperture A.sub.c and a separate coaxial 
annular (ring-shaped) aperture A.sub.r, a second condenser lens L3 having 
positive refractive power and preferably comprising a first positive lens 
L3a and a second positive lens L3b, an NFM probe N, a specimen O, an 
objective lens L4, a beam-splitting prism M2, a second mirror M3, a first 
relay lens L5a, a third mirror M4, a second relay lens L5b, a reflective 
prism M5, and an eyepiece lens L6. A primary image of the specimen O is 
formed at each of the locations designated I.sub.11 and I.sub.12, and an 
observation image of the specimen is formed at the location designated 
I.sub.2. Also situated on a branch portion of the optical axis AX split by 
the beam-splitting prism M2 is a photomultiplier 1 and a monitor 2. An 
observer's eye would be situated at the location designated E. 
A first condenser system C1 comprises a combination of the first condenser 
lens L2 and the second condenser lens L3. A second condenser system C2 
comprises the second condenser lens L3 but not the first condenser lens 
L2, as will become clearer below. The first condenser lens L2 and the stop 
RS are preferably integrally constructed, with the first condenser lens L2 
being situated so as to span the central aperture A.sub.c and the annular 
aperture A.sub.r in surrounding relationship to the central aperture 
A.sub.c. In addition, the stop RS and the first condenser lens L2 are 
preferably freely insertable and removable with respect to the optical 
axis AX. 
The light source S, collector lens L1, and first mirror M1 collectively 
function to supply an illumination light flux in a conventional manner. 
Specifically, the light source S is operable to provide an 
illumination-light flux for both NFM microscopy and for any of various 
"larger-scope" light-microscopy techniques that can be performed using the 
apparatus of FIG. 1, such as light-field microscopy, dark-field 
microscopy, and phase-contrast microscopy. After being converged by the 
collector lens L1, the illumination-light flux is reflected by the first 
mirror M1 and propagates, preferably as parallel rays, toward the first 
condenser lens L2. 
The first condenser system C1 is operable to converge light propagating in 
the central region of the illumination light flux to a point at the 
entrance end of the NFM probe N. The second condenser system C2 is 
operable to converge light propagating at the periphery of the 
illumination light flux on the specimen O. In other words, the 
illumination light flux is simultaneously converged at two points, one at 
the entrance end of the NFM probe N and the other at the specimen O. As a 
result, an NFM observation image of a locus on the specimen can be formed 
from illumination light irradiated at the specimen from the sharp end of 
the NFM probe N, and a light-microscope image of a larger region of the 
specimen can be simultaneously formed from illumination light that 
bypasses the NFM probe N before irradiating the specimen. 
The foregoing is more clearly illustrated in FIG. 2, wherein the 
illumination light flux F propagates as substantially parallel rays 
(relative to the optical axis AX) toward the stop RS. As can be seen, the 
stop RS defines a central aperture A.sub.c and an annular aperture 
A.sub.r. These apertures are separated from each other by an annular ring 
R. The first condenser lens L2 can be supported by the annular ring R so 
as to span the entire central aperture A.sub.c. 
The illumination light flux F, upon encountering the stop RS, is divided 
into two coaxial light fluxes; an annular flux A resulting from passage of 
light through the annular aperture A.sub.r, and a central flux B resulting 
from passage of light through the central aperture A.sub.c. The central 
flux B is also refracted by the first condenser lens L2 while the annular 
flux A is not. Rather, the annular flux A propagates as substantially 
parallel rays until encountering the second condenser lens L3 (preferably 
including separate first and second positive lenses L3a, L3b, 
respectively). The second condenser lens L3 converges the annular flux A; 
thus, the annular flux A illuminates, by Kohler illumination, the specimen 
O positioned at the rear focal point of the second condenser lens L3. This 
Kohler illumination is used to illuminate the specimen for light 
microscopy of relatively large regions of the specimen. 
Meanwhile, the central flux B, after being refracted by the first condenser 
lens L2 and passing through the central aperture A.sub.c, is further 
converged by the second condenser lens L3 to a point located at the 
entrance terminus 20 of the NFM probe N. 
The distal terminus 10 of the NFM probe N is sharply pointed. The position 
of the distal terminus 10 is adjacent (and very near as customary in NFM 
microscopy) the specimen O. 
Thus, the first condenser lens L2 and the second condenser lens L3 together 
comprise a first condenser system C1 operable to cause the central flux B 
of illumination light from the light source S to converge at a point on 
the entrance terminus 20 of the NFM probe N. After passing though the NFM 
probe, this light flux B passes as an evanescent light wave from the 
distal terminus 10 of the NFM probe N to the specimen O, thereby becoming 
illumination light for NFM observation of the particular locus on the 
specimen O immediately adjacent the distal terminus 10. 
A piezo element P or analogous scanning means is attached to the NFM probe 
N. The piezo element P is operable to displace the NFM probe N in two 
dimensions (i.e., in the X and Y plane) so as to scan the specimen O over 
a defined range. 
Referring further to FIG. 1, light from the annular light flux A 
illuminating the specimen O via Kohler illumination and transmitted 
through the specimen, as well as light (from the central light flux B) 
passing as evanescent light from the distal terminus 10 of the NFM probe N 
and transmitted through and/or scattered by the specimen, both propagate 
to the beam-splitting prism M2 via the objective lens L4. The light 
originating as evanescent light from the distal terminus 10 of the NFM 
probe N, after passing through the specimen O, has an intensity profile 
dependent on the shape and material properties of the specimen. 
Light transmitted through the beam-splitting prism M2 is reflected by the 
second mirror M3 and forms the primary image I.sub.11 of the specimen O 
usable for light-microscopy observation of the specimen. To such end, the 
light from the primary image I.sub.11 passes through the first relay lens 
L5a, is reflected by the third mirror M4, passes through the second relay 
lens L5b, is reflected by the reflective prism M5, and forms the 
observation image I.sub.2. The observation image I.sub.2 is viewed by an 
observer using the eyepiece lens L6. Thus, the observer can perform any of 
various light-microscopy methods, e.g., bright-field microscopy or 
phase-contrast microscopy, of the specimen. 
Light reflected by the beam-splitting prism M2 forms the second primary 
image I.sub.12 of the specimen O that can be used for light-microscopy 
observation of the specimen as well as NFM observation of the specimen. 
The second primary image I.sub.12 can be analyzed by any of various 
devices such as the photomultiplier 1 or analogous image-analyzing means. 
For NFM observation of a locus on the specimen, the annular aperture 
A.sub.r is preferably blocked to prevent formation of the annular flux A. 
Then, while the locus on the specimen is being scanned by the evanescent 
light from the distal terminus 10 of the NFM probe N, the resulting 
primary image I.sub.12 is analyzed by the photomultiplier 1. Thus, an 
NFM-produced distribution of light intensity is produced at the location 
of the primary image I.sub.12. The distribution of light is based on 
physical attributes of the locus of the specimen in regions adjacent the 
distal terminus of the probe N. The information generated by the 
photomultiplier 1 can be visualized using the monitor 2. 
Thus, it is now possible, using the same instrument, to simultaneously 
perform NFM-microscope observations of small loci on a specimen and 
ordinary light-microscope observations (e.g., bright-field microscopy or 
phase-contrast microscopy) of relatively larger regions on the specimen on 
demand without having to move the specimens or any of the condenser lenses 
or other components of the apparatus. Also, it is now possible, using the 
same instrument, to employ light microscopy to find a desired locus on a 
specimen for examination by NFM, and then to perform NFM at that locus 
without having to move the specimen or any of the components of the 
apparatus. In addition, the obtaining of light-microscope images of the 
specimen as well as NFM images of selected loci on the specimen can now be 
performed quickly and simply. 
Furthermore, because the first condenser lens L2 and the stop RS are 
preferably freely insertable and removable with respect to the trajectory 
of the illumination light flux, phase-contrast observations of the 
specimen are possible at different magnifications by exchanging the 
current stop RS with another having a differently sized annular aperture 
A.sub.r. 
In the FIG. 1 embodiment, light from the specimen O is divided by the 
beam-splitting prism M2. Alternatively, for example, the primary image 
I.sub.11 or the primary image I.sub.12 can be formed by replacing the 
beam-splitting prism M2 with an analogous light-switching means such as a 
freely insertable and removable mirror. 
Further with respect to the FIG. 1 embodiment, the primary image I.sub.12 
is preferably detected and analyzed by the photomultiplier 1. 
Alternatively, for example, to perform NFM observations, the intensity of 
light transmitted through the specimen O from the evanescent light from 
the distal terminus 10 of the NFM probe N can be detected. This allows the 
intensity of the transmitted light from the specimen O to be detected at 
an optimum position within the light path without restricting the position 
where the primary image of the specimen O is formed. 
Furthermore, with respect to the FIG. 1 embodiment, each of the first and 
second condenser systems C1, C2 comprises one or more refractive lenses 
having positive refractive power. Alternatively, it is possible to produce 
images at two points by means of such systems that comprise one or more 
reflective elements. 
Therefore, an NFM integrated microscope is provided that permits, on 
demand, both NFM observations and light-microscopy (e.g., bright-field 
microscopy and/or phase-contrast microscopy) observations of a specimen to 
be performed. These observations can be performed without moving lenses or 
other components of the apparatus. In addition, it is now possible to 
quickly obtain a light-microscopy image of the specimen as well as an NFM 
image of one or more desired loci on the specimen. 
Whereas the invention has been described in connection with a preferred 
embodiment, it will be understood that the invention is not limited to 
that embodiment. On the contrary, the invention is intended to encompass 
all alternatives, modifications, and equivalents as may be included within 
the spirit and scope of the invention as defined by the appended claims.