Resolution in microscopy and microlithography

In scanned optical systems such as confocal laser microscopes wherein a beam of light is focused to a spot in a specimen to excite a fluorescent species or other excitable species in the spot, the effective size of the excitation is made smaller than the size of the spot by providing a beam of light of wavelength adapted to quench the excitation of the excitable species, shaping this second beam into a pattern with a central intensity minimum, and overlapping this central minimum with the central intensity maximum of the focused spot, so that within the spot the intensity of quenching light increases with distance from the center of the spot, thereby preferentially quenching excitation in the peripheral parts of the spot, and thereby reducing the effective size of the excitation and thus improving the resolution of the system. In the preferred embodiment of the present invention, the central minimum of quenching light is narrowed further by creating the pattern of quenching radiation in the specimen by imaging onto the focal plane a plurality of pairs of sources of quenching light, arrayed at the vertices of a regular, even-sided polygon, the center of which is imaged in the specimen on the central maximum of exciting radiation, and such that the two members of each pair are on opposite vertices of the polygon and emit light mutually coherent and out-of-phase, and the light emitted by different pairs is incoherent with respect to each other.

FIELD OF THE INVENTION 
The present invention relates to scanned optical systems in which a beam of 
light is focused to the smallest possible spot in a specimen in order to 
selectively excite, within the illuminated spot, an excitable species such 
as a fluorescent dye, and more specifically to a method of improving the 
resolution of such systems. 
BACKGROUND OF THE INVENTION 
In many fields of optics, a light beam is focused to the smallest possible 
spot in a specimen in order to selectively photoexcite a molecular species 
in the illuminated spot. Such fields include scanned beam fluorescence 
microscopy, scanned beam microlithography, nanofabrication, and optical 
digital information storage and retrieval. The lenses in such resolution 
demanding applications often approach diffraction limited performance, and 
in view of the dependence of resolution on wavelength and numerical 
aperture of the objective focusing the light, these lenses are designed 
with the largest practical numerical apertures and used with light of the 
shortest practical wavelengths. 
Additionally, a variety of techniques have been devised to push resolution 
beyond the Abbe limit set by diffraction theory (S. Inoue, p. 85 in D. L. 
Taylor and Yu-li Wang, Fluorescence Microscopy of Living Cells in Culture, 
Part B, Academic Press, 1989). These techniques include placing annular 
and multiannular apertures in the aperture plane of the objective (Toraldo 
di Francia, Nuovo Cimento, Suppl. 9:426 (1952)) and using scanned confocal 
optics (M. Minsky, U.S. Pat. No. 3,013,467 (1961)). While in theory, such 
aperture plane apertures can allow arbitrarily narrow central maxima of 
the point spread function, any substantial narrowing of the central 
maximum is accompanied by dramatically less efficient light utilization 
and degraded image contrast. Although, as originally pointed out by the 
inventor of the present invention (Baer, U.S. Pat No. 3,705,755 (1972)), 
this problem of degraded contrast can be reduced by the use of such 
aperture plane apertures in a confocal scanning system, such a solution 
does nothing to improve the inefficient use of light actually reaching the 
specimen, so that in practice, light induced damage of the specimen or 
photobleaching of the fluorescent dye could limit the usefulness of such 
an approach. The technique of scanned probe, near field microscopy (Lewis 
et al U.S. Pat. No. 4,917,462) has had more success in achieving high 
resolution, but this technique is limited to the exposed surface of flat 
specimens. A related technique applicable only in the special case of 
optical disc recording and playback, involves the deposition, adjacent to 
the information containing layer, of an opaque layer which is be made to 
undergo a change an optical property such as transparency by a focused 
beam (Fukumoto and Kubota, Jpn. J. Appl. Phys. 31:529 (1992) Yanagisawa 
and Ohsawa, Jpn. J. Appl. Phys. 32:1971(1993), Spruit et al, U.S. Pat. No. 
5,153,873 (1992)). 
Though the variety of proposed superresolution techniques attests to the 
long recognized need to improve the resolution of the light microscope for 
applications such as the far-field imaging of typical specimens such as 
sections of biological tissue, it appears that the practical gains for 
such applications have been effectively limited to less than a doubling of 
resolving power relative to the Abbe limit. Thus any system which could 
increase resolution beyond the state of the art, and especially one which 
could work in conjunction with present superresolution techniques to 
further extend resolution performance, could be of great value in the 
field of light microscopy and other fields of scanned optics. 
OBJECTS AND ADVANTAGES 
It is the primary object of the present invention to improve resolution in 
optical systems such as scanned fluorescent microscopes, in which, at each 
moment, a beam of light is focused to the smallest possible spot in a 
specimen to excite an excitable species in the spot. 
Another object of the present invention, in such systems, is to minimize 
the light induced damage to a specimen resulting from photodynamic action. 
Another object of the present invention, in such systems, is to minimize 
light induced bleaching and photolysis of the molecules responsible for 
absorption and emission. 
Another object is to produce a method of fluorescent microscope resolution 
enhancement which is easily adapted to current laser confocal microscopes, 
two-photon excitation laser scanning microscopes, and fluorescent decay 
time contrast microscopes. 
Another object is provide a method of resolution enhancement which will 
work synergistically with known superresolution methods thereby increasing 
the resolution over these known techniques. 
Another object is to allow high resolution epiillumination imaging of 
living biological specimens at greater tissue depths from the surface than 
is possible with current techniques. 
Another object is to provide a resolution enhancement technology which can 
be adapted to the fields of high resolution photolithography, 
nanofabrication and digital computer memory storage and retrieval. 
Another object of the invention is to avoid image degradation due to 
coherence effects of laser illumination, while using such coherence 
instead to increase resolution. 
Still other advantages of the present invention will become evident in this 
disclosure. 
SUMMARY OF THE INVENTION 
The foregoing objects are achieved and the foregoing problems are solved in 
one illustrative embodiment of the invention, applied specifically to the 
field of fluorescence microscopy, although the principles embodied therein 
also apply to the other applications of the present invention discussed in 
this specification. This embodiment is an improvement of the laser 
scanning fluorescence microscope, wherein a scanned excitation beam is 
focused to a diffraction limited spot size and illuminates successive 
spots in a fluorescent specimen, exciting fluorescent dye molecules within 
these spots to fluorescence. Fluorescent light emanating from each of 
these illuminated spots is then electronically measured, and a spot of 
light the intensity of which varies in accordance with the measured 
fluorescence from these illuminated spots is scanned over a video monitor 
screen in correspondence with the scanning of the excitation beam over the 
specimen, to create a final image of the specimen. In the present 
invention, light of a wavelength adapted to quench fluorescent excitation 
of the excited dye molecules is focused in the specimen to a pattern 
containing a central minimum which is made concentric with the central 
maximum of the exciting radiation, the central points of the central 
maximum of the exciting beam and of the central minimum of the quenching 
beam substantially coinciding, so that within the central minimum region, 
the intensity of the quenching beam, and consequently the degree of 
quenching of the fluorescence, increases with distance from the central 
point, thereby decreasing the effective width of the distribution of 
probability of fluorescent excitation as a function of distance from the 
center of the illuminated spot, and consequently increasing the effective 
resolving power of the microscope. In the preferred embodiment of the 
present invention, the central minimum is narrowed by creating the pattern 
of quenching radiation in the specimen by imaging onto the focal plane a 
plurality of pairs of sources of quenching light, arrayed in a regular, 
even-sided polygon, such that the two members of each pair are on opposite 
vertices of the polygon and emit light mutually coherent and out-of-phase, 
and the light emitted by different pairs is incoherent with respect to 
each other.

DESCRIPTION OF THE INVENTION 
FIG. 1 shows an embodiment of the present invention employing continuous 
wave laser illumination. Light from excitation laser 10 is focused by lens 
12 onto pinhole aperture 13, and after passing through aperture 13, 
reflection from dichroic mirror beam splitters 14 and 15 and scanning by 
beam scanning means 16 (which may be a pair of orthogonal galvanometer 
powered scanning mirrors) the laser light is imaged by eyepiece 17 and 
objective 18 on or within the specimen 19 stained with fluorescent 
molecules excitable by light emitted by laser 10, to form a region of 
excited fluorescent molecules at the image of pinhole aperture 13. 
Quenching laser 11, which emits light of a wavelength adapted to quench, 
by means of stimulated emission, the fluorescent excitation caused by 
laser 10, is focused by toroidal lens 20 onto annular aperture 21. Light 
passing through aperture 21 passes through dichroic beam splitter 14, then 
is reflected by dichroic beam splitter 15 to pass through the beam 
scanning means 16, and is focused by eyepiece 17 and objective 18 onto 
specimen 19. In FIG. 1, for purposes of illustration only, rays are show 
emanating from the central point of annular aperture 21 to simplify the 
illustration and to show that this central point is conjugate, with 
respect to beam splitter 14, to the central point of pinhole aperture 13, 
and it should be understood that since this central region of annular 
aperture 21 is in fact opaque, rays do not actually emanate from this 
central point. In FIG. 2, a magnified detail showing laser 11, toroidal 
lens 22 and annular aperture 21, the rays are correctly shown focused by 
lens 22 onto and then emanating from the transparent ring of aperture 21. 
The mirror image relationship between the center of annular aperture 21 
and pinhole aperture 13 insures that the projected image in the specimen 
19 of annular aperture 21 is concentric with the image in the specimen of 
pinhole aperture 13. The diameter of annular aperture 21 is chosen so that 
the diameter in the specimen of the central ring of maximum intensity is 
the same as the diameter of the first minimum of an Airy disc point 
diffraction image which would be formed at the wavelength of the quenching 
laser 11. This means that the diameter in the specimen of the ring of 
maximum intensity for the quenching radiation is larger than the diameter 
of the first minimum of the point diffraction image of pinhole 13 by the 
ratio of the wavelength of the quenching laser to the wavelength of the 
excitation laser. 
Fluorescent emission from those excited fluorescent molecules in the 
specimen in the focus of the excitation beam, which have not been quenched 
by the quenching beam, is collected by objective 18, directed successively 
through eyepiece 17, beam scanning means 16, beam splitter 15, and 
blocking filter 27 adapted to block reflected excitation and quenching 
light, to viewing pinhole aperture 22, which is conjugate to pinhole 
aperture 13. Emitted light passing through aperture 22 is detected by 
photodetector 23 (which may be a photomultiplier tube) the output of which 
is directed to video frame store 24, which is synchronized by the scan 
drive circuit 25 which powers the beam scanning means 16. The information 
contents of the video frame store 24, as manipulated by appropriate image 
processing means, is displayed on video monitor 26, producing an image of 
the scanned plane of the specimen 19. 
FIG. 3 shows a perspective detail of a portion of the apparatus of FIG. 1. 
It should be noted that for purposes of illustration, the openings of 
pinhole aperture 13 and annular aperture 21 are shown larger than the 
scale of the rest of the elements in this figure. Although in principle, 
completely different optical systems could be used to project a central 
minimum of quenching radiation so its center coincides in the specimen 
with the central point of the central maximum of the exciting radiation, 
for use in a scanning microscope application, the sharing of focusing 
optics, made possible by the use of beam splitter 14, insures that 
provided the focusing and scanning systems are achromatic, the required 
coincidence between the central points of these images will be guaranteed 
even at the precision required in high resolution microscopy, once the 
apertures 13 and 21 are aligned at one scan position, because 
perturbations, for example due to inhomogenities in the specimen above the 
plane of focus, distort the excitation and quenching beams equally. In 
case focusing optics which have been achromatized for the choice of 
excitation and quenching wavelengths are unavailable, it is possible to 
longitudinally shift aperture 21 relative to the position conjugate to 
aperture 13 with respect to beam splitter 14, so that the images of the 
two apertures are coplanar and concentric in the specimen. Since such an 
arrangement will only provide correction for the axial focal point, 
scanning can be provided in such an arrangement, for example by lateral 
movement of the specimen. 
Why the present invention will cause an improvement in resolving power is 
shown in FIG. 4, which shows how quenching, applied according the present 
invention, can reduce the nominal width of the distribution indicating the 
probability that a given probe fluorescent molecule in the specimen will 
be in its excited state, as a function of the distance of this molecule 
from the center of the central maximum of the focused excitation beam. To 
simplify this illustration, it is assumed that this probability of 
excitation of the fluorescent molecule is proportional to the intensity of 
exciting illumination incident on the molecule. Secondly, the illustration 
is applied specifically to the imaging of the central spot of the imaging 
field, where the center of the spot intersects the optical axis of the 
objective 18. 
FIG. 4A shows the expected light intensity distribution of the "Airy disc" 
or diffraction image of excitation pinhole aperture 13 projected in the 
specimen 19 by objective 18. The use of the axial point as the example in 
this illustration means that the spatial coordinate in the distribution is 
simply the distance, in the plane of focus, from the axis of objective 17, 
this distance being shown by distance from the vertical line 31 common to 
FIGS. 4A, B, C and D. The vertical axis in these figures represents either 
light intensity or excitation probability, where the upward direction 
corresponds to increases. The description of imaging of non-axial points 
is somewhat more complicated than this present axial case, but resolution 
enhancement works in the same way. FIG. 4B shows the probability of 
excitation 30 of a fluorescent molecule as a function of the distance in 
the focal plane between the axis and that molecule. The assumption of a 
proportional excitation response by the fluorescent molecule means that 
this excitation probability distribution 30 shown in FIG. 4B is 
proportional to the diffraction image distribution shown in FIG. 4A. In a 
case where the excitation probability was not proportional to excitation 
intensity, for example because excitation saturated the population of 
fluorescent molecules causing the top of the central maximum of curve 30 
in FIG. 4B to be flattened with respect to the curve of FIG. 4A, the 
following arguments for the shrinkage of the width of the excitation 
probability curve are still valid. 
The nominal width of the central maximum of the excitation probability 
distribution (without any resolution improvement due to quenching) is 
shown by the double arrow 32, which measures the distance between the two 
points in the distribution where the probability of excitation is half 
maximal. (When this distribution is imagined in the two dimensions of the 
plane of focus, double arrow 32 indicates the diameter of the circle where 
the probability of excitation is half the maximum probability). The object 
of the present invention is to apply the quenching radiation in a pattern 
which preferentially decreases the probability of resulting excitation in 
peripheral portions of the central maximum of the excitation probability 
curve, while sparing, as much as possible, the probability of excitation 
in the central portion, thereby narrowing the nominal width of the 
probability of excitation curve. 
FIG. 4C shows the expected intensity distribution 33 of the image, 
projected in the specimen, of annular aperture 21, which is illuminated by 
the quenching beam. The mean radius of annular aperture 21 is such that, 
by diffraction theory, the diffraction image in the specimen resulting 
from the contribution from of each small section on the ring of annular 
aperture 21, taken in isolation, has an intensity of zero on the optical 
axis (i.e., the first minimum passes through the optical axis), so that as 
the light emanating from each of the small sections on the ring of 
aperture 21 summates to create an image of annular aperture 21 in the 
specimen, the sum, at this central axial point, of the zero intensities 
from each of the sections of the aperture 21 still adds to zero. However, 
scattering in the optics of the instrument and in the specimen, and 
reflections from lower lying layers of the specimen, causes the central 
minimum on the optical axis, in fact, to have a small but finite intensity 
I.sub.b shown by the horizontal line 35, which is above the zero intensity 
baseline 36. The term "central minimum" is used herein analogously to the 
more common expression "central maximum" and refers to the fact the image 
has a minimum at its center, even if the image as a whole is not centered 
with respect to the optical axis, as when non-axial points are imaged. 
In the following discussion, it is assumed that once a given fluorescent 
molecule is excited, it has a probability p of eventually emitting a 
fluorescent photon, and that for a wide range of initial conditions, such 
as different mixtures of exciting vs. quenching light, and different 
concentrations of fluorescent molecules, that adding to the existing light 
mixture the same intensity of quenching light, called I.sub.50, which is 
different for different species of fluorescent molecule, will reduce p to 
half its value before such addition. It should be emphasized that the 
assumption may not be completely valid in view of factors such as 
saturation, but it will nevertheless help illustrate several aspects of 
how quenching can improve resolution. 
For a given total power of the quenching beam and a given species of 
fluorescent molecule, it is possible to see approximately how much 
sharpening of resolution will result with the present system by 
determining the intensity of quenching radiation which must be mixed with 
an excitation beam to reduce its effective rate of excitation of the 
fluorescent molecules by 50%. This intensity is shown by the double arrow 
labeled I.sub.50 in FIG. 4C', which shows a detail from FIG. 4C in 
magnification. 
It is assumed that due to factors such as scattering in the optics and the 
specimen, there is a small but finite intensity of the quenching beam at 
the central point of the central minimum of the quenching beam, shown by 
the double arrow labeled I.sub.b. The addition of more quenching light of 
intensity I.sub.50 therefore will bring the total intensity of quenching 
light to the level shown by the line 37 which has a intensity of I.sub.b 
+I.sub.50. By definition of I.sub.50, then, at the distance from the 
central maximum where the intensity of the quenching beam is I.sub.b 
+I.sub.50, shown by the vertical lines 39 and 40, the efficiency of 
excitation in producing a latent image is half the efficiency in the 
center of the central maximum. Because the excitation beam is also most 
intense at the center, the full width at half maximum of the 
probability-of-fluorescent-emission curve, post quenching, is actually 
narrower than the distance between lines 39 and 40, shown by the double 
arrow 41. It can be seen that by simply increasing the total power of the 
quenching beam that the double arrow 41 can be arbitrarily reduced. FIG. 
4D shows the distribution 38 of the probability of fluorescent molecule 
excitation, subject to quenching by the quenching beam (the effective 
excitation, correlated with the probability of ultimate fluorescence 
emission), and it can be understood that this distribution can be 
arbitrarily narrowed, by reducing double arrow 41 by means of increasing 
the quenching beam power. 
Of course, there is a limit to how much quenching radiation can be directed 
onto the specimen before that layer is damaged by heating, therefore the 
ability of the specimen to tolerate high quenching beam powers may be the 
major resolution determining factor in the present system microscopy or 
for microchip fabrication. Furthermore, it will be appreciated that as the 
total power for the quenching beam increases, so does the intensity of the 
central point of the central minimum, I.sub.b, and consequently the 
effective sensitivity of the process is reduced. Therefore another design 
objective in the present system is to reduce the intensity of the central 
point of the central minimum of the quenching beam to the lowest practical 
level, in order to preserve the sensitivity of the process, while 
achieving good resolution improvement. 
FIG. 4 also shows that only a small part of the energy of the quenching 
beam, in the central part where the intensity is lowest, is involved in 
resolution enhancement. This means that for each milliwatt of laser energy 
needed in the crucial central part of the quenching laser beam, a total 
beam power of perhaps hundreds of milliwatts may be required. However such 
powers are easily attainable with available lasers. Furthermore, high 
intensities of the quenching beam in the bright ring surrounding the 
central minimum do not degrade the image because the final excitation 
probability of the fluorescent molecule can never be lower than zero, so 
high quenching intensities will saturate at zero net excitation. 
Therefore, from the point of view of image quality, the intensity of the 
quenching beam can be adjusted for optimal sharpening at the center of the 
intensity minimum, without worry about the high intensities surrounding 
the central minimum. By choice of a quenching wavelength where there is 
negligible absorption by the specimen except by excited fluorescent 
molecules, thermal effects on the specimen of the quenching beam are 
minimized. Thermal effects might also be reduced by use of diamond or 
other high thermal conductivity material as a support for the specimen. 
Another possible concern about the high intensity of quenching radiation is 
that some stray radiation could enter the photodetector, degrading image 
contrast. However both filter 27 and dichroic mirror 15 block such 
quenching radiation from entering the detector. Additionally, in forms of 
the invention using pulsed radiation, quenching light can be eliminated by 
gating off the detector sensitivity during times the quenching light is 
on. From the point of view of specimen and fluorophore damage, in the 
presence of excited fluorophores capable of producing photodynamic damage, 
quenching radiation can reduce such damage by deexciting the excited 
species. Therefore, by choice of a quenching wavelength where there is 
negligible absorption by the specimen except by excited fluorophore 
molecules, high quenching intensities are not simply tolerable, but can 
actually be beneficial. 
Under same the conditions described above to produce the minimum in the 
focal plane, which is perpendicular to the optical axis, the intensity 
distribution measured along the optical axis also has a minimum sharing 
the same central point. With microscope objectives generally, the width of 
the central maximum of the point diffraction image, measured in the focal 
plane perpendicular to the optical axis, is smaller than the width 
measured along the optical axis resulting in a better lateral resolution 
than longitudinal resolution. This same elongation along the optical axis 
occurs with the central minimum of the diffraction image of the annular 
aperture 21, so that in general, following resolution enhancement by the 
apparatus shown in FIG. 1, longitudinal resolution will also be improved 
by quench sharpening, but the lateral resolution will still be better than 
the longitudinal resolution. 
Because (at least near the focal point) excitation of the fluorescent 
molecule due to the cone of rays converging to the focal point and the 
cone of rays diverging from the focal point is eliminated by quenching, 
only the in-focus rays at the focal point remain for production of the 
fluorescent image. Therefore the present system (in common with confocal 
microscopes) permits extending the practical depth-of-focus, by scanning 
in depth in addition to scanning laterally. This is particularly useful in 
more parallel forms of image formation discussed below, where the same 
image would be formed at many different closely spaced focus settings, to 
produce a composite image which is sharp at every depth of the specimen. 
Additionally, by increasing the intensity of the image for deeper layers, 
it is possible to compensate for absorption of the excitation light by 
superficial layers of the specimen. 
FIG. 5 shows how the resolution of the present invention might be increased 
by combining the field plane annular aperture used in the present 
invention to create in the specimen a quenching beam pattern with a 
central minimum, with an aperture plane annular aperture to improve the 
lateral resolution of that quenching beam pattern. (Born and White, 
Principles of Optics, 3rd edition, p. 416). A relay lens system consisting 
of lenses 45 and 46 with annular aperture 47 in an aperture plane, images 
annular aperture 21 onto a real image plane 48 which is conjugate to 
pinhole 13 relative to the beam splitting mirror 14 (i.e., real image 
plane 48 is in the same plane as annular aperture 21 in FIG. 1 before 
being displaced to the position shown in FIG. 5). The diameter of annular 
aperture 21 is readjusted to cause the central minimum in its image in the 
specimen to have a minimum intensity. The use of an aperture plane annular 
aperture 47 in addition to the field plane annular aperture 21, causes the 
central minimum in the specimen to have a smaller width, to improve 
lateral resolution, and at the same time to have a reduced resolution in 
the axial dimension for improved depth-of-field. Instead of the annular 
aperture 47 in the aperture plane of the relay lens system, there can be a 
complex aperture of different annuli, each with specified phase 
retardation and opacity, for further decreases in the width of the central 
maximum, as shown originally by Toraldo di Francia (Nuovo Cimento, Suppl. 
9:426 (1952)). It should be noted that in the design of such an aperture 
plane aperture 47, it is more important to maximize the sharpness of the 
first minimum of the point spread function rather than the usual design 
criterion of maximizing the sharpness of the central maximum. (The central 
minimum might also be "sculpted" to optimize, for example, a particular 
desired tradeoff between image brightness and resolution, by replacing the 
uniform ring of aperture 21 with a series of annuli of independently 
controllable phase retardation and absorption, in addition to the choice 
of phase retardations and absorptions for annular rings in the aperture 
plane aperture 47.) It should also be appreciated that a major problem 
with Toraldo type aperture plane apertures, namely the defection of beam 
power away from the central maximum, causing problems of reduced contrast 
and increased photobleaching and photodynamic specimen damage, are 
overcome in the present invention, because, as described above, light from 
the quenching beam outside the central minimum, does not degrade image 
contrast or increase photobleaching or photodynamic damage. 
FIG. 6 shows an embodiment of the present invention where the pinhole 
aperture 13 of FIG. 1 is replaced by a slit aperture 60, which is 
illuminated by the same excitation laser 10 as in FIG. 1, but where the 
beam emanating therefrom is focused by cylindrical lens 61 to a line 
coincident with the slit in aperture 60. Slit aperture 60 in the 
cross-sectional view of FIG. 6 appears identical to the cross-sectional 
view of pinhole aperture 13 in FIG. 1. The beam emanating from aperture 60 
is reflected successively by dichroic mirror beam splitters 14 and 15, 
identical to those shown in the device of FIG. 1, and is passed through 
beam scanning means 62, which differs from the scanning means 16 of FIG. 1 
because it is required to scan in just one dimension. The scanned beam is 
focused successively by eyepiece 17 and objective 18 lenses onto specimen 
19, identical to those of FIG. 1, however the image of the excitation beam 
in the specimen 19 is an illuminated strip, with a central maximum which 
is elongate in the dimension parallel to the strip. The nominal width of 
such an elongate central maximum is defined herein as the distance between 
the lines where the intensity is half-maximal. The quenching laser 11 is 
directed on two parallel slits 63 and 64, by means of two parallel 
cylindrical lenses 65 and 66. The spacing between slits 63 and 64 is such 
that the first minima of their diffraction images in the specimen 19 
coincide to produce a central minimum, made to coincide with the central 
maximum of the diffraction image in the specimen of slit 60, such that the 
central line of the central maximum coincides with the central line of the 
central minimum of the focused quenching light. Fluorescent emission from 
specimen 19 is focused by successive objective 18 and eyepiece 17 lenses 
to focus light from the central maximum in the specimen onto a linear 
photodiode array 67, oriented perpendicular to the plane of FIG. 6. The 
output from array 67 is stored in video frame store 24 which is 
synchronized by the output of the scan drive circuit 68 which drives the 
one dimensional beam scanner 62. 
The advantage of a one-dimensionally scanned strip arrangement as in FIG. 6 
compared with a two dimensionally scanned spot arrangement as in FIG. 1, 
is that one less dimension in scanning is required, so much faster scans 
at a higher scan frequency can be produced, and the apparatus is simpler. 
The disadvantage is that the resolution gain of the present invention is 
secured only in one dimension. However for many applications a gain in 
resolution in just one dimension is sufficient, and the simplicity and 
scanning speed of the slit arrangement are preferred. It should be noted 
that instead scanning produced by beam scanning means 16 or 62, the 
required relative movement between specimen and the image of the focused 
light beams in the specimen can be produced by movement of the specimen, 
or an optical element in the light path between the light source and the 
specimen, synchronized with the image acquisition process of frame store 
24. 
FIG. 7 illustrates an embodiment of the present invention employing 
synchronized ultrashort-pulse (shorter than a few picoseconds), 
repetitively pulsing lasers for excitation and quenching. In particular, 
the pulse output from the lasers is adjusted so that the quenching beam is 
turned on within picoseconds of the offset of the excitation beam, before 
there has been any time for significant fluorescent emission, so that 
virtually all such emission will follow the offset of the quenching beam. 
There are significant advantages of such a pulsed laser embodiment of the 
present invention compared to embodiments wherein the excitation and 
quenching beams are continuously on. Most importantly, there is more 
efficient quenching per watt of average quenching beam power. This may be 
understood from a specific example where the excitation pulse frequency is 
assumed to be 100 MHz, and the fluorophore is assumed to have a 1 ns 
half-life for the excited state. (For the purpose of this example it is 
assumed that the fluorophore is very efficient, so in the absence of 
optical quenching, the excited state decays almost exclusively by 
fluorescent emission.) If it is assumed that each quenching photon 
incident on the fluorophore has a 20% probability of quenching it, then 10 
incident photons together would have about a 90% chance of producing 
quenching. If these 10 quenching photons were delivered within several 
picoseconds following each excitation, then substantially all the 
quenching would take place before there was an opportunity for 
fluorescence so the resulting quenching would be 90%. However if these 10 
quenching photons were emitted by a continuous wave laser, so they arrived 
spaced over the 10 ns interval between excitation pulses, after the first 
nanosecond, there would have been roughly a 50% likelihood of fluorescent 
emission, but only roughly a 20% likelihood of quenching, and obviously 
any quenching photons which arrive after the fluorescent emission have 
zero effect. In other words, bunching the photons in the interval 
immediately after excitation greatly improves the quenching efficiency. 
The quenching efficiency can be further increased in a pulsed system by 
making the excitation and quenching lasers have the same polarization, so 
there is insufficient time for a significant change in direction of 
polarization by rotation of the fluorophore between excitation and 
quenching, hence the quenching laser will be optimally aligned with the 
excited molecules. 
The pulsed laser embodiment of the present invention shown in FIG. 7 has 
additional advantages. The ultrafast laser excitation makes it convenient 
to excite fluorescence by two-photon absorption (Denk, et al Science 
248:73 (1990), Denk, et al U.S. Pat No. 5,034,613 (1991)), which 
substantially confines light induced damage and photobleaching to the 
plane of focus, and provides illumination with light of a relatively long 
wavelength which can penetrate to greater depths of tissue. Unfortunately, 
with state of the art two-photon microscopy, the advantage of limitation 
of excitation to the plane of focus is gained (for a given fluorophore) 
only with some loss of lateral resolution (Sheppard and Gu, Optik 86:104 
(1990)). However in the present invention, the quenching beam rather than 
the excitation beam is the principal determiner of lateral resolution, so 
that two-photon excitation can be used for excitation, with the resulting 
confinement in excitation, and the use of single-photon absorption for 
quenching insures high lateral resolution. Still an additional advantage 
of the pulsed embodiment shown in FIG. 7 is that it allows laser dyes and 
their local environments to be characterized by fluorescence lifetime 
measurements, with minimal additional equipment costs. Furthermore, with a 
photodetector 23 that is not blinded by direct reflections from the laser 
pulses, time discrimination can replace the wavelength discriminating 
blocking filter 27, thus avoiding one source of potential light wastage. 
In FIG. 7, excitation laser 70 and quenching laser 71 are ultrashort pulse 
mode-locked lasers, using, for example, optically pumped Ti-sapphire, dye 
or Cr-forsterite as the active medium. These types of lasers result in a 
repetitive train of pulses of durations from about 100 femtoseconds to 
several picoseconds, and at a frequency of about 50 to 100 MHz which 
depends on the length of the laser cavity. Lasers 70 and 71 are 
synchronized by means of a phased locked loop synchronizing circuit 73, 
which, by means of phase detector 74 detects phase difference between the 
amplified and filtered electrical outputs of high frequency response 
photodetectors 75 and 76, which receive a portion of the output beams of 
lasers 70 and 71 respectively by means of beam splitters 77 and 78. The 
output 79 of circuit 73, representing a correction signal to stabilize the 
desired phase difference between the pulse trains from the two lasers, is 
applied to a piezoelectric actuator 80 which controls the longitudinal 
position of one of the end mirrors 81 of the cavity of laser 70, thereby 
adjusting the laser pulse frequency, and thus stabilizing this phase 
difference. The desired phase difference, where the pulse from laser 71 
follows the offset of the pulse from laser 70 by an interval from zero to 
several picoseconds can be adjusted either electrically in circuit 74, for 
example by adding a controlled phase shift to one of the inputs of phase 
detector 74, or optically by means of adjusting the optical path 
difference between the outputs of the two lasers. A commercially available 
unit to implement circuit 73 is the Model 3930 Lok-to-Clock Tm Electronics 
Control from Spectra-Physics Lasers, Inc., Mountain View, Calif., which 
can be used when the lasers 70 and 71 are Spectra-Physics Model 3960C 
Tsunami.TM. Ti-sapphire lasers. FIG. 7 also shows a frequency doubling 
crystal 82, which can be optionally placed in the beam path of the 
quenching laser to halve the output wavelength. This doubling crystal 82 
is representative of such frequency multiplying means which can be placed 
in the path of either laser to change the output wavelength. Both lasers 
70 and 71 may be pumped by a common argon ion laser (unillustrated) with a 
divided output. Apart from the lasers 70 and 71 and their synchronizing 
apparatus, all the elements in the embodiment shown in FIG. 7 are 
substantially identical to and serve substantially the identical function 
to the respective elements shown in FIG. 1, so they are numbered with the 
same numerals as FIG. 1, and are described by the text corresponding to 
FIG. 1. 
Many alternative methods of producing two beams of synchronized ultrashort 
pulses are known in the art. The synchronization between lasers 70 and 71 
could be by means of a purely optical coupling, for example by having both 
lasers optically pumped by the pulsed output of a synchronous pumping 
laser (Moritz, N. et al, Optics Comm. 103:461(1993)). Still another 
possibility is for a portion of the light output of the excitation laser 
70 to be used as a synchronous pump energy source for the quenching laser 
71 (or vice versa). Another possibility is to provide a single laser which 
emits ultrashort pulses in the near i.r., for example the 1.3 .mu.m output 
of a Cr-forsterite laser in a self-modelock configuration, or the 1.5 
.mu.m, 2 ps, 27 MHz output of a Er, Yb doped fiber laser (Laser Focus 
World, July 1993 p. 15) and split the output into a portion directed 
through a frequency doubler crystal and a frequency tripler crystal to 
derive the quenching and excitation beams respectively. Furthermore, the 
recently commercially available optical parametric oscillator coherent 
light sources intrinsically produce simultaneous outputs at several 
wavelengths, which, if necessary, can be frequency multiplied to the range 
required for a broad range of fluorescent dyes. Finally it has been 
possible to produce two color pulses from the same laser(de Barros M. R. 
X. and Pecker, P. C., Optics Lett. 18:631(1993)), and the outputs could be 
separated by wavelength for use as the excitation and quenching beams. 
The quenching radiation emitted by laser 11 or 71 must be of a wavelength 
adapted to induce stimulated emission from the fluorescent dye molecules 
in specimen 19, and consequently must be of a wavelength where there is 
significant fluorescent emission. Furthermore, it is important that this 
quenching radiation not itself fluorescently excite ground state 
fluorescent molecules. These simultaneous requirements may be met in 
several ways. In dyes such as the coumerin derivatives with relatively 
large Stokes shifts, the excitation spectrum has dropped to a negligible 
level in the long wavelength portion of the fluorescent emission spectrum. 
Alternatively, in a fluorescent dye with a large probability for 
transition between the ground vibrational level of the first 
electronically excited singlet (i. e., the fluorescently excited state) to 
the second vibrational level of the ground state, such that at the 
wavelength corresponding to this transition, there is still a significant 
emission likelihood, and hence a significant stimulated emission 
cross-section, but the wavelength is long enough that the absorption of 
the quenching light by the ground state fluorophores has dropped to 
essentially zero. When used for biological microscopy, additional desired 
attributes for a dye in the present application which are also generally 
desirable for any fluorescent dye in a biological microscopy application 
are that it have a high fluorescent efficiency, that it be commercially 
available in forms such as antibody and dextran conjugates, and that it 
have a low intersystem crossing probability for production of triplet 
states (or be self quenching for triplet excitation). A large two-photon 
excitation cross-section leaves open the possibility of excitation by 
two-photon excitation. Finally, the excitation and quenching wavelengths 
must be chosen with respect to cost and availability limitations of the 
excitation and quenching lasers. 
The choice of wavelength of excitation and quenching is also subject to a 
tradeoff since shorter wavelengths lead to increased resolution by the 
classical resolution criteria, whereas longer wavelengths, especially 
above 630 nm (Puppels, G. J., et al, Exptl. Cell. Res. 195:361(1991)) are 
reported to be less toxic to biological tissue at high power densities and 
can penetrate biological tissue with less scattering. In fact, as 
discussed, the lowering of scattering may be more critical to achieving 
good resolution in the present invention than the resolution performance 
as predicted by diffraction theory for a non-scattering medium, because it 
may allow a lower intensity at the central minimum of the quenching beam 
focus. In case it is necessary to use a quenching beam in a part of the 
spectrum which can be injurious to the specimen, these quenching photons 
can be used most efficiently by insuring that the just preceding 
excitation pulse was of sufficient intensity to nearly saturate the 
fluorescent excitation. For relatively inefficient fluorescent dyes this 
may require reducing the frequency of the pulse output of laser 70, so 
that for a given time averaged power output, the power per pulse 
increases. On the other hand, with an efficient fluorophore, there might 
be near saturation with each pulse, even with a few milliwatts average 
beam power and a frequency of about 80 MHz. (see Tsien and Waggoner, 
Fluorophores for Confocal Microscopy, in Handbook of Confocal Microscopy, 
James B. Pawley, ed., Plenum, N.Y., 1990). 
The recently developed cyanine dye, Cy5, has been reported to be easily 
conjugated to antibodies, avidin, DNA and other molecules important in 
fluorescence biomicroscopy and, in addition, possesses the desirable 
qualities of a high quantum efficiency, stability and long wavelength 
excitation (Majumdar, et al, Bioconjugate Chem. 4:105 (1993)). The 
embodiment illustrated in FIG. 7, when outfitted with Ti-sapphire lasers 
could excite Cy5 by setting the excitation laser 70 at the 680 nm low 
wavelength end of the laser's tuning range, and quench by setting laser 71 
at about 740 nm. The coumarin dyes are commercially available conjugated 
to molecules useful in fluorescence microscopy (Molecular Probes, Inc. 
Eugene, Oreg.), and have the advantage of a large Stokes shift to minimize 
unwanted fluorescence excitation by the quenching beam. Furthermore they 
have a large stimulated emission cross section, as evidenced by their 
widespread use in dye lasers, and they also have been successfully used in 
two-photon excitation microscopy (Denk et al, Science 248:78 (1990)). The 
dye coumarin 1 (7-diethylamine-4-methylcoumarin) can be excited by two 
photon excitation by a Ti-sapphire laser set to about 700 nm. Quenching 
can be by the frequency doubled 950 nm output of the Ti-sapphire laser, to 
produce pulses at 475 nm. The widely used dye, lucifer yellow, has the 
advantage of a very large Stokes shift, and can be two-photon excited by 
the output of a Ti-sapphire laser at 850 nm or single photon excited by 
the frequency doubled 850 nm output (i.e., 425 nm), and the quenching beam 
can be the frequency doubled 1080 nm output of the Ti-sapphire laser 
(i.e., at 540 nm). Because of lucifer yellow's large Stokes shift, it is 
possible to quench by tuning the quenching laser in the optimum of the 
emission band, which is also at the long wavelength cutoff region of the 
frequency doubled Ti-sapphire laser. It may desirable to lengthen the 
pulse width for the quenching beam to, say, 10 picoseconds, both to 
eliminate two-photon fluorescence excitation in the UV portion of the 
lucifer yellow excitation spectrum, and also to sharpen the spectral 
spread so that a narrow band rejection beam filter 27 can eliminate 
unwanted direct and scattered light from the quenching laser from adding 
noise to the fluorescence signal recorded by the photodetector 23. 
Alternatively, or in addition to spectral filtering of this direct and 
scattered quenching laser light, the output of detector 23 can be gated to 
be unresponsive during the time such direct scattered light from the 
quenching laser is falling on it. Yet another means to reduce quenching 
beam photons from reaching detector 23 is to replace dichroic beam 
splitter 15 with a polarizing beam splitter which reflects plane polarized 
light from lasers 70 and 71 and transmits the opposite plane of 
polarization. To the extent that the fluorescent emission is depolarized, 
it will be able to be partially transmitted through the polarizing beam 
splitter. (In case the fluorescent emission is polarized, a quarter wave 
plate between the polarizing beam splitter 15 and the specimen will rotate 
the plane of polarization by 90 degrees, so it will pass through the beam 
splitter and reach the detector.) 
These examples of fluorescent dyes have been discussed principally because 
they can be excited and quenched with the wavelengths available from 
Ti-sapphire lasers, which unfortunately have a wavelength gap from about 
540 nm to about 680 nm, which is in the region of excitation or quenching 
of some dyes which otherwise would be good candidates for the present 
invention. The use of optical parametric oscillators, or lasers able to 
operate within this wavelength gap of the Ti-sapphire laser, will permit 
the use of such dyes. A particularly promising class of dye for use in the 
present invention are the inclusion compounds of the cyclodextrin molecule 
and various laser dyes occupying its hydrophobic central cavity. The 
extensive search for laser dyes has found dyes which in many aspect are 
ideal for the present invention, having a high quantum efficiency, a high 
stimulated emission cross section and a low ground state absorption at the 
wavelength of stimulated emission. The use of a cyclodextrin host allows 
hydrophobic dyes which ordinarily are not suitable for aqueous 
environments to operate in a hydrophobic microenvironment within an 
aqueous environment. 
In the embodiments described so far, the mechanism focusing laser light on 
annular aperture 21 insures that the light leaves this aperture generally 
coherently and in-phase. However such illumination is not optimal for 
reducing the width of the central minimum. The reason is that, starting 
from a point on the first minimum ring of an Airy disc pattern, movement 
towards the central maximum or movement away from it (towards the first 
bright ring of the Airy disc) both lead to an increase in intensity, 
however in the two directions the oscillating electric field vector of the 
light is opposite. The problem this opposite electric field causes may be 
seen by considering just the contribution of two small segments of 
aperture 21 on opposite sides from the center. Each of these segments 
projects its own Airy disc in the specimen, positioned so that the first 
dark ring of both of these Airy discs passes through the central minimum. 
However a small distance from the central minimum, the area between the 
central maximum of one of the Airy discs and the common central minimum 
coincides with the area between the central minimum and the first bright 
ring of the second Airy disc. Since the two light sources are coherent and 
in phase, these areas will have opposite electrical vectors, and therefore 
there will be destructive interference. The result of this cancellation is 
that the net light intensity grows relatively slowly with distance from 
the central minimum. One solution to this problem is to employ as 
quenching laser 11 or 71, a laser with inherently low coherency. The 
excimer laser has the right coherency properties, but unfortunately is a 
pulsed laser with too low a frequency to be practical in a scanned laser 
device such as the device of FIG. 1, however it might be usable in the 
more parallel embodiments of the invention. 
FIG. 8 shows a solution to this problem an embodiment of the invention 
where diametrically opposite sources of quenching light, which are 
positioned so as to summate in the specimen to create the central minimum, 
produce light which is 180 degrees out-of-phase. Therefore in the areas 
near the central minimum, light from the two sources will constructively 
interfere, causing the intensity to rise sharply with distance from the 
central minimum, thereby decreasing the width of the central minimum. 
Measured in the plane of focus, the sharpening due to just two 
out-of-phase sources is in just one dimension. Unfortunately to try to 
extend each source into a semicircle, so that together they encompass the 
entire annular aperture, will still produce a central minimum in which 
only one dimension is narrowed by the process. However in arraying two or 
more pairs of out-of-phase sources at the corners of a regular polygon, 
such that coherence between different pairs is minimized, will solve the 
problem by approximating a radially symmetrical central minimum, narrow in 
two orthogonal dimensions. 
In the device of FIG. 8, the annular aperture 21 of FIG. 1 is replaced by a 
hexagon of illuminated optical fibers. Light from excitation laser 10 is 
focused by lens 12 onto one end of optical fiber 91, and light emerging 
from the other end 98 of fiber 91 is reflected from dichroic mirror 14, 
and directed through beam scanning means 16 to lens 18, which focuses this 
excitation light to a spot on the specimen 19. Three lasers 11, 11' and 
11", each within the band of effective quenching, but of wavelengths far 
enough from each other that they are mutually incoherent, have their 
output beams focused by lenses 92, 92' and 92" onto three pairs of 
phase-preserving optical fibers, one pair containing fibers 94 and 95 
being illustrated along their full length. The non-illuminated ends of 
these fibers, for example end 96 and 97, are in the plane 99, that is 
conjugate to the plane of focus of specimen 19, and consequently is the 
same plane occupied by annular aperture 21 in FIG. 1. By mechanically 
adjusting fiber end 96 with respect to fiber end 97 (the means for such 
adjustment is not illustrated) the quenching light emerging from these 
ends on plane 99 is 180 degrees out-of-phase. As shown in FIG. 9, which 
shows a cross section through plane 99, the ends 96 and 97 are at 
diametrically opposite vertices of a hexagon, and the separation between 
ends 96 and 97 is such that at the wavelength of laser 11, the Airy discs 
of these fiber ends projected into the specimen have their first minima 
passing through the central point of the central maximum of the Airy disc 
projected by lens 18 on the specimen 19 from light emerging from the end 
98 of fiber 91. Dichroic mirror 14 makes the end 98 of fiber 91, 
conducting excitation light, conjugate to the central point of the hexagon 
of non-illuminated ends 96, 96', 96", 97, 97' and 97", but it is also 
possible to locate the end 98 physically within that hexagon, so that 
dichroic mirror 14 is unnecessary. (FIG. 8, it will be realized has 
described just the system for illuminating the specimen, and the viewing 
system, using a dichroic mirror, is the same as in other embodiments.) 
Another way to provide the necessary incoherence between the various pairs 
of out-of-phase sources, is for them to have the same wavelength, but to 
be on at different times, so that interference is impossible between one 
pair and another pair. The device of FIG. 10, which is the preferred 
microscope embodiment of the present invention, uses a pulsed laser 71 
directed by lens 92 into four phase-preserving optical fibers 100, 101, 
102 and 103 (six or any even number of fibers greater than two could have 
been used instead of four), so that a single coherent pulse simultaneously 
enters all four fibers. (Though these four fibers have been shown 
receiving the focused beam from laser 71 arranged in a line, more likely 
they would be arrayed in a compact square, or the laser would be directed 
into just one fiber, the output of which would be split twice by well 
known methods in the art of fiber optics.) Two of the fibers 100 and 101 
are short, and have a difference in length of one half the wavelength of 
the laser light, in order to make their outputs out-of-phase. (Or they 
have the same length and the phase difference is adjusted by mechanically 
adjusting their ends,) The two remaining fibers 102 and 103 (illustrated 
for just part of their lengths) are long (also with a small difference in 
length to insure that their output is out-of-phase), and their length is 
such that by the time the light pulse emerges from them, the light has 
stopped exiting the short fibers (that is to say that the length 
difference between the short and the long fibers is equal or greater than 
the laser pulse duration times the speed of light). The exciting light 
from laser 70 emerges from the end 98 of fiber 91, end 98 being in the 
center of the square formed by the non-illuminated ends 104 to 107 of the 
fibers 100 to 103. A diagram of the ends of the fibers through the plane 
of these ends is shown in FIG. 11. 
As in the embodiment of the invention shown in FIG. 7 excitation laser 70 
of FIG. 10 emits a pulse first, followed by quenching laser 71. The 
fluorescent emission from the specimen is reflected by dichroic mirror 108 
then passes through pinhole 22 onto a photodetector 23, the output of 
which modulates either the spot of a display cathode ray tube, raster 
scanned in synchrony with the scanning of the superimposed excitation and 
quenching beams across specimen 19, by scanning means 16, or scans the 
write address in an image frame store. As in earlier embodiments, there is 
assumed to be a blocking filter (unillustrated) in front of the detector 
23, to eliminate both excitation and quenching photons from entering the 
detector, and furthermore, the detector may be time gated to be 
insensitive during the times the excitation and quenching lasers are on, 
and their light may be directly reflected back by the specimen. The convex 
lens 18 here symbolizes the complex imaging optics of a compound 
microscope, which may include in addition to an objective, an eyepiece 
lens and perhaps other lenses and other optical elements as well. 
FIG. 12 shows the form of the present invention adapted to the field of 
photolithography, which enjoys the benefits of quench sharpening, while at 
the same time applying an image to the wafer with massive parallelism, to 
achieve a high throughput. When the present invention is adapted to 
microlithography, the excitation laser excites a photoactive molecule in a 
photoresist layer of a wafer to be made into microchips. The photoactive 
molecule then enters a transient excited state analogous to the excited 
state of a fluorescent molecule, which at least in many cases should be 
susceptible to optical quenching, though the period of vulnerability of 
the state to quenching is far shorter than the case of the fluorescent 
molecule. When quenched the photoactive molecule reverts to its ground 
state. However if not quenched it has a probability of causing a lasting 
change, constituting the "latent image," which will change the 
vulnerability of the photoresist layer to some development process, where 
for example exposed areas become insoluble and remain on the wafer, 
protecting it, while non-exposed areas can wash away, leaving parts of the 
wafer vulnerable to some etching process. 
In the microlithographic embodiment, excitation pulsed laser 70 is focused 
by lens 12 to a pinhole 13 and the beam emerging from pinhole 13 is 
collimated by lens 115 and the emerging plane wave 116 is directed on 
microlens array 117, which projects focused spots from laser 70 on a 
projection mask 118. The lenses of microlens array 117 are arranged in a 
regular hexagonal array, so that the spots projected onto mask 118 are at 
the centers of the hexagons of such an array. Where a spot of excitation 
light is imaged by any of the microlenses on a transparent region of mask 
118, the light emerging, for example ray cone 119, passes through dichroic 
beam splitter 14, and is focused by lens 18 to the photoresist layer 120 
on wafer 121. Because array 117 can simultaneously focus thousands of 
spots, an image can be transferred from the mask 118 to the photoresist 
layer 120 on wafer 121 with an enormous degree of parallelism. Light from 
ultrafast pulsed quenching laser 71 (synchronized with laser 70 as 
described above) is imaged by lens 122 onto a bundle of 32 phase 
preserving optical fibers, arranged into four bundles of eight fibers 
each, two bundles 123 and 124 of which are illustrated, each with only 
four out of the eight fibers, to simplify the illustration. Each bundle 
has a different length, so that the pulses from the laser emerge at 
different times from each of the bundles, and therefore light emerging 
from fibers in one of the bundles cannot interfere with light emerging 
from another bundle. The rectangle 131 in the lower right corner of FIG. 
13 shows the ends of each of the 32 fibers in the plane 125 of FIG. 12. 
The fibers in bundle 123 are schematically shown in rectangle 131 as 
circles with either a lower case "a" or a capital "A," those in the lower 
case "a" having light which emerges 180 degrees out-of-phase with respect 
to light emerging from the circles with capital "A." (Note that the 
diameters of the circles in FIG. 13 are not drawn to scale, in 
relationship to the dimensions of the hexagons.) Similarly, fibers in 
bundle 124 terminate in the circles with a lower case "b" or a capital 
"B," with a similar out-of-phase relationship between the lower case "b" 
and the capital "B" fibers, and so on for the unillustrated fibers ending 
in the circles with c's and d's. Light emerging from the unilluminated 
ends of the fibers terminating in plane 125 (which has been drawn 
separated from the ends of the fibers for clarity, but is actually assumed 
to be in the same plane with these ends), is focused to infinity by lens 
126, and the light passing through lens 126 is directed onto microlens 
array 127, which is a rectangular array with the same aspect ratio shown 
in FIG. 13, and which is of the appropriate lens spacing to image the ends 
of the fibers at plane 125 to the repeating hexagonal pattern at plane 128 
shown in FIG. 12. (One typical hexagon is labeled 132 in FIG. 13.) The 
scale of the pattern at plane 128 of the focused images of the fibers at 
plane 125 is such that when the light leaving plane 128 is reflected from 
dichroic mirror 14, and imaged by lens 18 onto the photoresist layer 120, 
there is an image of each spot from the plane of the mask 118, where the 
mask is transparent, projected into the center of one of the hexagons of 
the array of fibers conducting the quenching radiation. It should be noted 
that each hexagon has the same coherency and phase relationship as the 
device shown in FIGS. 8 and 9, namely quenching light imaged on opposite 
sides of the hexagon is coherent and 180 degrees out-of-phase while light 
imaged on adjacent vertices is mutually incoherent, in this case because 
the imaging occurs at different times due to the different time delays in 
the different fiber bundles such as bundle 123 and 124. It is assumed that 
the pulses from the lasers 70 and 71 are short enough, e.g., some hundreds 
of femtoseconds or less, that it is possible to have four different delays 
for the fiber bundles conducting the quenching light, so that quenching 
still arrives at the photoresist layer 120 before the excited state 
induced in the photoactive molecules by the light pulse from laser 70 has 
decayed either to a non-quenchable triplet excited state, or has initiated 
the latent image local chemical change. Therefore, provided sufficient 
accuracy can be provided in the fabrication of the two microlens arrays 
117 and 127, and the relative phases can be maintained through the 
successive imaging steps for the quenching radiation, the projection in 
the photoresist of each hexagon provides satisfactory quench sharpening 
for the spot of excitation in its center. 
The array of spots projected onto the photoresist layer 120 is converted 
into a continuous two dimensional image of the mask 118 by laterally 
translating the mask by motor means 129 and laterally translating the 
wafer 121 in opposite direction by motor means 130, with a ratio of 
velocities equal to the magnification of lens 18, so that during scanning, 
the image of mask 118 maintains a stable relationship with the photoresist 
layer. 
As shown diagrammatically in FIG. 14, the direction of translation of the 
mask (shown by arrow 140) is such that it is at a small angle 141 with 
respect to one of the major axes 142 of the hexagonal array of excitation 
spots projected by microlens array 117, so that the trajectory of two 
adjacent spots in the array form two parallel lines separated by less than 
the post-quenching resolution distance when referred to the photoresist 
layer, and such that by the time the entire mask has been scanned in front 
of the microlens array 117, the trajectories of all the excitation spots 
form a continuous grating pattern on the photoresist, with no gaps or 
regions of overlap. As is evident from FIG. 14, the angle 141 would be 
much smaller as the number of hexagons per row is increased from the five 
in the figure, to perhaps a thousand or more, as imagined for the device 
of FIG. 12. It is also possible to have an angle 141 which is a small 
multiple of the angle required for no overlap, in order to have the same 
point on the resist exposed by different sets of excitation/quenching 
microlens combinations, so that any defects in a particular microlens can 
be compensated by proper exposure from another microlens. 
Unlike the apparatus shown in FIGS. 8 and 9, where only the contributions 
from six points must be considered when computing the shape of the central 
minimum for the quenching beam on the photoresist, in an array such as in 
the embodiment of FIG. 12, contributions from farther points must also be 
considered. Fortunately, in the case of a hexagonal array, neither the six 
second nearest neighboring points, nor the 12 third nearest neighboring 
points contribute substantially to the intensity at the central point of 
the central minima, because at their particular distances, they are near 
minima in the Airy disc distribution. Interestingly this is not the case 
for square arrays. 
As the technology for making large matrices of shutters evolves, it would 
probably be preferable to implement the technology of the parallel device 
of FIG. 12, by having the information about mask 118 encoded in a digital 
memory, which would be read out with massive parallelism to a shutter 
array positioned at the plane of the mask 118, so there would be no need 
to move the mask. 
It is therefore believed that the present invention, and in particular the 
preferred embodiment shown in FIG. 12, may allow microchips of exceedingly 
fine critical dimensions to be fabricated economically, for the most part, 
using tool and processing components currently in place at chip 
fabricating facilities. Because these chips would not have to be exposed 
to ionizing radiation in the exposure of the photoresist, annealing steps 
required in chip manufacturing processes requiring ionizing radiation 
could be eliminated, and the resulting absence of any defects which might 
escape the annealing process would lead to chips of greater reliability. 
The same resolution enhancement techniques described above for 
microlithography are also applicable to the microfabrication of small 
parts. Here the ability to manipulate a block of optically writeable 
material in three dimensions, would allow the production of microminiature 
parts which may not be available by any other technique, as described in 
more detail in the parent application of this application, U.S. patent 
application Ser. No. 08/275,967, now abandoned, and which is incorporated 
herein by reference. Similarly the present technique could be invaluable 
in the optical storage of information, both in the writing and the reading 
phases of such storage, again as described more fully in U.S. patent 
application Ser. No. 08/275,967. 
While in the embodiments of the present invention described in this 
specification, both excitation and quenching were carried out by focused 
beams of light, either or both of these roles might be implemented by 
focused beams of other types of radiation, for example by X-rays, by 
focused electron or other particle beams or by focused ultrasonic 
radiation. In the examples given, the radiationally quenchable excited 
states have been electronically excited states, however any other types of 
excited state, including nuclear excited states, excited states involving 
macroscopic quantum structures, molecular isomerizations, or crystal 
lattice phenomena, for example, would also fall within the scope of the 
present invention. In the examples given, focusing of the exciting and 
quenching radiation is provided by lenses, however other devices for 
focusing are known, including concave mirrors, tapered light pipes and 
optical fibers, and these can be used for focusing in the present 
invention. The examples given have considered a specimen or target 
material with just one radiationally excitable species, however it is 
often useful in fluorescent microscopy of biological material to employ 
two or more contrasting fluorescent stains, and the present invention 
could be used in such applications, fo r example by choosing two 
flourophores which have the same excitation and quenching wavelengths but 
differ in fluorescent lifetimes or emission spectra. All the examples 
given have employed scanning of a spot or line, but the present invention 
is also applicable to applications requiring selective illumination of 
just a single unscanned spot. In the examples given, just one point or 
line in a specimen is scanned at each moment, however it is possible to 
simultaneously scan multiple points, with the use of Nipkow discs for 
example, as in the microscope of Petran (U.S. Pat. No. 3,517,980 (1970)) 
and in such devices, each scanned point is individually subject to the 
resolution enhancement of the present invention. Thus the scope of the 
invention should be determined by the appended claims and their legal 
equivalents, rather than by the examples given.