Two-Photon Laser Microscopy
United States Patent |
|
Denk, et al. |
July 23, 1991 |
Abstract
A laser scanning microscope produces molecular excitation in a target material by simultaneous absorption of two photons to thereby provide intrinsic three-dimensional resolution. Fluorophores having single photon absorption in the short (ultraviolet or visible) wavelength range are excited by a stream of strongly focused subpicosecond pulses of laser light of relatively long (red or infrared) wavelength range. The fluorophores absorb at about one half the laser wavelength to produce fluorescent images of living cells and other microscopic objects. The fluorescent emission from the fluorophores increases quadratically with the excitation intensity so that by strongly focusing the laser light, fluorescence as well as photobleaching are confined to the vicinity of the focal plane. This feature provides depth of field resolution comparable to that produced by confocal laser scanning microscopes, and in addition reduces photobleaching. Scanning of the laser beam, by a laser scanning microscope, allows construction of images by collecting two- photon excited fluorescence from each point in the scanned object while still satisfying the requirement for very high excitation intensity obtained by focusing the laser beam and by pulse time compressing the beam. The focused pulses also provide three-dimensional spatially resolved photochemistry which is particularly useful in photolytic release of caged effector molecules. This invention was made with Government support under Grant Nos. P41RR04224 awarded by the National Institute of Health; NSF-BBS-8714069 awarded by the National Science Foundation, and NSF-DMB-8609084 awarded by the National Science Foundation. The Government has certain rights in the invention.
Inventors: |
Denk; Winfried (Zurich, CH); Strickler; James P. (Ithaca, NY); Webb; Watt W. (Ithaca, NY) |
Assignee: | Cornell Research Foundation, Inc. (Ithaca, NY) |
Appl. No.: | 436045 |
| Filed: | November 14, 1989 |
U.S. Class: |
250/458.1; 250/459.1; 250/461.1; 250/462.1; 356/318 |
Intern'l Class: |
G01N 021/39; G01J 003/00 |
Field of Search: |
250/458.1,461.1,462.1,423 P,459.1 356/318 365/127,106 |
References Cited [Referenced By]
U.S. Patent Documents
Sep., 1983 |
Illanuccia et al. |
365/301. |
|
Sep., 1983 |
Schmidt et al. |
358/93. |
|
Aug., 1984 |
Swainson et al. |
365/106. |
|
Sep., 1984 |
Swainson et al. |
365/127. |
|
Dec., 1986 |
Carlsson |
358/93. |
|
Mar., 1988 |
Horikawa |
250/234. |
|
Nov., 1988 |
Groebler |
356/318. |
|
Dec., 1988 |
Honig et al. |
250/458. |
|
Nov., 1988 |
Kozikowski et al. |
8/103. |
|
May., 1989 |
Goldstein |
250/234. |
|
Jun., 1989 |
Bille |
351/205. |
|
Sep., 1989 |
Houpt et al. |
350/6. |
|
Oct., 1989 |
Dandliker et al. |
250/458. |
|
Dec., 1989 |
Martin et al. |
209/579. |
|
Primary Examiner: Hannaher; Constantine
Assistant Examiner: Glick; Edward J.
Attorney, Agent or Firm: Jones, Tullar & Cooper
Claims
1. A laser scanning microscope comprising:
stage means for receiving target material to be imaged, the target material
including fluorescent means responsive to excitation by photons in a short
wavelength spectral range to produce characteristic fluorescence;
lens means positioned to direct light toward said stage and having an
object plane in target material at said stage means;
a source of subpicosecond monochromatic coherent light pulses of high
instantaneous energy intensity comprised of photons in a long wavelength
spectral range to which target material at said stage does not respond by
single photon excitation to produce its characteristic fluorescence, said
pulses having a high repetition rate;
detector means;
means directing said coherent light pulses along an optical path including
said lens means to impinge on target material at said stage means, said
lens means focusing said light pulses at said object plane so that said
long wavelength light pulses provide sufficient instantaneous intensity to
produce in target material at said object plane simultaneous absorption of
two incident photons to thereby excite characteristic fluorescence in
target material at said stage means, said fluorescence providing output
light which travels on said optical path to detector means.
2. The microscope of claim 1, wherein said light source produces
subpicosecond pulses of sufficient instantaneous intensity and repetition
rate that target material at said stage means will absorb energy
simultaneously from at least two incident long wavelength photons
substantially only in said object plane.
3. The microscope of claim 1, wherein said lens means is located with
respect to said stage means to focus said long wavelength light from said
source to a submicron diameter at said object plane to produce a
sufficiently high intensity at said object plane to produce fluorescence
in target material at said stage means and insufficient intensity outside
said focal plane to produce such fluorescence.
4. The microscope of claim 1, wherein said lens means is arranged to focus
said long wavelength light into a conical configuration to produce
converging and diverging light on opposite sides of said object plane,
whereby said long wavelength light is concentrated at a focal point on
said plane.
5. The microscope of claim 1, further including target material carried by
said stage means, and wherein said long wavelength light from said source
is in the red wavelength spectral range, and wherein said lens means
focuses said light at a focal point in said target material at said stage
means, said target material being a fluorophore having a single photon
absorption peak in the ultraviolet wavelength spectral range and being
capable of absorbing two photons in the red wavelength spectral range to
produce fluorescence.
6. The microscope of claim 5, wherein said lens means is located to focus
said long wavelength light at a focal point in said target material to
produce a light intensity which excites fluorescence in a limited
ellipsoidal volume around said focal point.
7. A laser scanning microscope, comprising
stage means for receiving a target material having an absorption energy
level peak responsive to single photon excitation by light of a
predetermined wavelength;
lens means positioned to direct light to said stage means and having an
object plane in target material at said stage means;
a laser source of subpicosecond a laser light pulses, said laser light
having a wavelength about twice said predetermined wavelength;
mirror means directing said laser light pulses along an optical path
including said lens means to cause said pulses to impinge on target
material at said object plane, said lens means focusing said laser light
pulses on a focal point in the target material, the intensity of said
pulses producing in the region of said focal point a two-photon excitation
energy level equivalent to the single-photon excitation energy level which
corresponds to said single photon absorption peak.
8. The microscope of claim 7, wherein said long wavelength light is in the
red wavelength spectral range, and wherein said lens means focuses said
light at a focal point in target material at said stage means, said target
material including a fluorophore having said absorption peak.
9. The microscope of claim 7, wherein said long wavelength impinging light
pulses provide photons of light energy in the red wavelength spectral
range to target material at said stage, and wherein the combined energy of
two photons of said impinging light is required to produce fluorescence
therein.
10. The microscope of claim 9, wherein said lens means is adjustable to
select focal points at different depths within target material at said
stage means.
11. The microscope of claim 10, further including scanning means to move
said focal point with respect to target material at said stage means.
12. The microscope of claim 11, further including detector means responsive
to light in said optical path for detecting fluorescence produced by
target material at said stage means.
13. The microscope of claim 12, wherein said detector means is a
photosensitive array which responds to said fluorescence.
14. The microscope of claim 7, further including target material at said
object stage, wherein said target material is a biological cell responsive
to said two-photon excitation energy level produced at said focal point by
said light pulses.
15. The microscope of claim 14, wherein said target material at said stage
means includes means responsive to said light pulses at said focal point
to produce localized release of biologically active chemicals.
16. The microscope of claim 7, further including target material at said
object stage, wherein said target material is a photon-activatable
reagent.
17. A method of fluorescence microscopy by a two-photon excitation
technique, comprising:
providing a sample containing fluorescent molecules which radiate photons
of a first characteristic energy;
illuminating said sample with a beam of rapidly repeating, intense,
subpicosecond pulses of laser light comprising photons of a second
characteristic energy, wherein said second characteristic energy is about
one-half said first characteristic energy;
focusing said illumination to a focal point having a submicron diameter
with said sample to produce an illumination intensity sufficiently high at
said focal point to produce molecular excitation and fluorescence of said
sample by simultaneous absorption of two incident photons;
scanning the submicron diameter focal point of said beam in a raster
pattern through said sample; and
detecting the fluorescence produced by said sample.
18. The method of claim 17, wherein said step of illuminating includes
directing a beam of light having an illumination intensity sufficient to
produce molecular excitation substantially only at said focal point to
thereby suppress background fluorescence.
19. The method of claim 17, wherein said step of illuminating includes
directing a beam of light having an illumination intensity sufficient to
produce molecular excitation substantially only at said focal point to
thereby suppress photobleaching of said sample material at locations
outside the focal plane.
20. The method of claim 17, wherein the step of providing a sample includes
providing a sample of a living biological specimen.
21. A method for producing localized photolytic release of caged
biologically active compounds by a two-photon excitation technique,
comprising:
providing a sample containing caged biologically active molecules which are
excitable by photons of a first characteristic energy;
illuminating said sample with a beam of rapidly repeating, intense,
subpicosecond pulses of laser light comprising photons of a second
characteristic energy wherein said second characteristic energy is about
one-half said first characteristic energy;
focusing said illumination to a focal point within said sample to produce
an illumination intensity sufficiently high only at said focal point to
produce molecular excitation and consequent release of caged biologically
active compounds by simultaneous absorption of two incident photons of
said second characteristic energy.
Description
BACKGROUND OF THE INVENTION
Although the principle of a flying spot scanner has been known for many
years, its application in microscopy has prospered only in the last few
years as the necessary technology has been developed. Stable laser light
sources and fast electronic image acquisition and storage technology are
necessary ingredients for a scanning microscope. While the imaging
properties of a non-confocal scanning microscope are very similar to those
of conventional microscopes, a new domain is opened by confocal scanning
microscopes. The resolution provided by such devices is only moderately
increased, but the vastly improved depth discrimination they provide
allows the generation of three dimensional images without complicated
deconvolution algorithms. The depth discrimination reduces background, and
this, together with the use of a single high quality detector such as a
photomultiplier, allows quantitative studies with high spatial resolution.
The resolution along the optical axis of a confocal scanning microscope
provides useful discrimination against background scattering or
fluorescence arising above and below the plane of focus in a transparent
object. It is also very helpful in constructing three dimensional
fluorescent images from a series of sections and for the use of
quantitative fluorescence indicators or for mapping of fluorescent markers
of cell surface receptors on non-planar surfaces. Such devices provide
slightly better lateral resolution, much better depth field
discrimination, and orders of magnitude better background discrimination
under ideal conditions than was available with prior devices, under ideal
conditions.
Scanning can be carried out either by moving the specimen stage under a
stationary beam or by precisely synchronized optical scanning of both the
illumination and the fluorescent response signals. Although the moving
stage solution is preferable from an optical point of view, it puts limits
on sample access and mounting, the use of environmental chambers, and
electrical recording with microelectrodes. Accordingly, the moving spot
approach is often favored. Such a moving spot may be produced by the use
of mirrors mounted on galvonometer scanners, although this limits the
obtainable frame frequency. The use of accousto-optical deflectors
interferes with the confocal spatial filtering in fluorescence microscopy
because of their strong dispersion. Although polygonal mirrors are faster
than galvonometer scanners, one alone does not allow a vector mode of
operation.
A conventional arc light source can be used for many applications of a
confocal scanning microscope which utilizes a rotating disc illuminator,
but apparently inescapable intensity modulations limit its use for
quantitative applications. In such devices, the image is formed either
through a dual set of confocal pin holes in the disc, or, in recent
versions, through the illumination pinholes themselves.
Confocal scanning microscopes in which a single point illuminated by a
laser is scanned across the moving object work quite well at slow scanning
speeds, and good laser scanning micrographs have been obtained using
fluorescence markers that absorb and emit visible light. However, confocal
scanning images with fluorophores and fluorescent chemical indicators that
are excited by the ultraviolet part of the spectrum have not been
available, largely because of the lack of suitable microscope lenses,
which must be chromatically corrected and transparent for both absorption
and emission wavelengths, but also because of the damage done to living
cells by ultraviolet light. Furthermore, the limitations of ultraviolet
lasers have inhibited such usage.
Fluorescence microscopy is further limited, in all of its manifestations,
by the photobleaching of fluorophores in the target material, for the
exciting light slowly photobleaches the fluorophores while it is exciting
fluorescence. Even in laser scanning confocal fluorescence microscopy,
essentially the same photobleaching is incurred as happens in wide field
microscopy, because the focused exciting light still illuminates the full
depth of the target specimen uniformly, in a time average, as it scans the
plane of focus. Photobleaching is particularly troublesome in a
three-dimensional image reconstruction because many two-dimensional images
are required for this purpose, and the acquisition of each two-dimensional
image produces photobleaching throughout the specimen.
SUMMARY OF THE INVENTION
The foregoing difficulties are overcome, in accordance with the present
invention, by the use of two-photon molecular excitation of fluorescence
in laser scanning microscopy. Two-photon excitation is made possible, in
accordance with the present invention, by the combination of (a) the very
high, local, instantaneous intensity provided by the tight focusing
available in a laser scanning microscope, wherein the laser can be focused
to diffraction-limited waist of less than 1 micron in diameter, and (b)
the temporal concentration of a pulsed laser. A high intensity, long
wavelength, monochromatic light source which is focusable to the
diffraction limit such as a colliding-pulse, mode-locked dye laser,
produces a stream of pulses, with each pulse having a duration of about
100 femtoseconds (100.times.10.sup.-15 seconds) at a repetition rate of
about 80 MHz. These subpicosecond pulses are supplied to the microscope,
for example by way of a dichroic mirror, and are directed through the
microscope optics to a specimen, or target material, located at the object
plane of the microscope. Because of the high instantaneous power provided
by the very short duration intense pulses focused to the diffraction
limit, there is an appreciable probability that a fluorophore (a
fluorescent dye), contained in the target material, and normally excitable
bY a single high energy photon having a short wavelength, typically
ultraviolet, will absorb two long wavelength photons from the laser source
simultaneously. This absorption combines the energy of the two photons in
the fluorophore molecule, thereby raising the fluorophore to its excited
state. When the fluorophore returns to its normal state, it emits light,
and this light then passes back through the microscope optics to a
suitable detector.
The two-photon excitation of fluorophores by highly intense, short pulses
of light constitutes a general fluorescence technique for microscopy which
provides improved background discrimination, reduces photobleaching of the
fluorophores, and minimizes the photo damage to living cell specimens.
This is because the focused illumination produced in the microscope fills
a converging cone as it passes into the specimen. All of the light which
reaches the plane of focus at the apex of the converging cone, except the
tiny fraction which is absorbed in the fluorophore, then passes out the
opposite side of the specimen through a diverging cone. Only in the region
of the focal point on the object plane at the waist formed by the
converging and diverging cones is the intensity sufficiently high to
produce two photon absorption in the specimen fluorophore, and this
intensity dependence enables long wavelength light to provide the effect
of short wavelength excitation only in the small local volume of the
specimen surrounding the focal point. This absorption is produced by means
of a stream of fast, high intensity, femtosecond pulses of relatively long
wavelength which retains a moderate average illumination intensity of long
wavelength light throughout the remainder of the specimen outside the
region of the focal point. As a result, photobleaching of the fluorophore
outside the plane of focus is virtually eliminated. One-photon absorption
of the long wavelength light is negligible, and outside the plane of focus
the instantaneous intensity is too low for appreciable two-photon
absorption and excitation, even though the time average illumination is in
reality nearly uniform throughout the depth of the specimen. This effect
also significantly reduces the damage to living cells.
The two-photon excitation of the present invention allows accurate spatial
discrimination and permits quantification of fluorescence from small
volumes whose locations are defined in three dimensions, and thus provides
a depth of field resolution comparable to that produced in confocal laser
scanning microscopes without the disadvantages of confocal microscopes
previously described. This is especially important in cases where thicker
layers of cells are to be studied. Furthermore, the two-photon excitation
greatly reduces the background fluorescence.
The two-photon absorption technique discussed above can also be used to
excite selected locations in a three-dimensional optical memory device of
the type described by Dimitri A. Parthenopoulos et al in an article
entitled "Three-dimensional Optical Storage Memory", Science, Vol. 245,
pages 843-845, Aug. 25, 1989. In that device, selected locations in a
matrix are illuminated by two beams of different wavelengths which overlap
in time and space to produce absorption. In accordance with the present
invention, extremely short, high intensity pulses of relatively long
wavelength light from a single laser source, or from coaxial multiple
sources, are directed through a scanning microscope into a storage medium
which may be a photochromic or a photolyzable fluorescent material such as
crystals, composites, or chomophores embedded in a polymer matrix. The
incident light beam is highly focused onto any one of many layers in the
matrix, and its intensity is modulated as it is scanned or stepped across
the selected layer. The beam excites selected locations in the matrix so
that coded information represented by the beam is stored in a binary
format within the medium. The highly focused beam provides the spatial
resolution required for accurate storage. The femtosecond, high intensity
pulses induce two-photon absorption in the matrix material to write
information into the material, which normally requires excitation by light
in the ultraviolet range. The excitation level of the written points in
the matrix can be detected, or read, by a "read" laser of long wavelength
which will produce fluorescence in the previously written molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing, and additional objects, features and advantages of the
present invention will become apparent from the following detailed
description of preferred embodiments thereof, taken in conjunction with
the accompanying drawings, in which:
FIG. 1 is a diagrammatic illustration of a laser scanning confocal
microscope utilized in accordance with the present invention;
FIG. 1A is an enlarged partial view of the region of the object plane of
the device of FIG. 1;
FIG. 2 is a synthesized stereo image pair showing blue fluorescence excited
by two-photon absorption of red light;
FIG. 3 is a plot of the average intensity from an area inside a fluorescent
latex bead versus the applied average laser power;
FIG. 4 is a two-photon excited fluorescence image of chromosomes of live
cultured pig kidney cells stained with a DNA stain;
FIG. 5 is an image of a latex bead, showing two-photon photobleaching
confined to the plane of focus; and
FIG. 6 is an image of a two-photon bleached pattern inside a fluorescently
stained latex bead.
DESCRIPTION OF PREFERRED EMBODIMENT
Turning now to a more detailed description of the present invention, there
is illustrated in FIG. 1 in diagrammatic form a conventional laser
scanning microscope 10 which includes an objective lens 12 for focusing
incident light 14 from a source 16 such as a laser onto an object plane
18. As illustrated in FIG. 1A, the object plane may lie on, or in, a
specimen or target material 20 which may be carried on a movable stage 22.
The illumination provided by incident light beam 14 fills a converging
cone generally indicated at 24, the cone passing into the specimen 20 to
reach the plane of focus at object plane 18 and, except for the tiny
fraction of light absorbed by the specimen, passing out through a
diverging cone 25. The incident light forms a waist, or focal point, 26 on
the object plane 18. The diameter of the focal point 26 is limited by
diffraction in the optical path, but preferably is less than 1 micron. As
is known, by adjustment of the microscope optics, the vertical location of
the focal point in the specimen 20 can be selected. Additionally, the
stage 22 may be movable in a horizontal plane, as in a raster motion along
X and Y axes, to position the incident light at selected locations in the
specimen in the horizontal plane, so that three-dimensional scanning of
the specimen can be obtained. However, since mechanically scanned stages
present difficulties, it is preferred to use a stationary stage, and to
scan the incident beam in the X-Y plane optically, as by means of scanning
mirrors in the optical path of the microscope.
The optical path from laser 16 to the object plane 18 includes a dichroic
mirror 28 onto which the light from the laser 16 is directed. As will be
explained in greater detail below, in accordance with the present
invention the output from the laser consists of short intense pulses of
light having a relatively long wavelength, preferably in the visible red
or near infrared spectral range. The mirror 28 deflects this long
wavelength light downwardly to a mirror 30 which in turn directs the light
to a pair of scanning mirrors 32 and 34 by way of curved mirrors 36 and
38. The mirrors 32 and 34 are rotatable about mutually perpendicular axes
in order to move the incident light 14 along perpendicular X and Y axes on
the object plane so that the stationary specimen is scanned by the
incident beam. The light from the scanning mirrors passes through eyepiece
40 and is focused through the objective lens 12 to the object plane 18.
Fluorescence produced in the specimen 20, indicated by dotted arrows 42 in
FIG. IA, travels back through the microscope 10, retracing the optical
path of the incident beam 14, and thus passes through objective lens 12
and eyepiece 40, the scanning mirrors 34 and 32 and the curved mirrors 38
and 36, and is reflected by mirror 30 back to the dichroic mirror 28. The
light emitted by fluorescent material in the specimen is at a wavelength
that is specific to the fluorophore contained in the specimen, and thus is
a different wavelength than the incident light 14. This fluorescent light
is able to pass through the dichroic mirror 28, rather than being
reflected back toward the laser 16, and follows the light path indicated
generally at 44. The fluorescent light 42 thus passes through a barrier
filter 46 and is reflected by flat mirrors 48, 50 and 52 to a suitable
detector such as a photomultiplier tube 54. In accordance with the present
invention, a confocal laser scanning microscope is preferred, and
accordingly such a microscope is illustrated in the drawings. However, it
will be understood that other laser scanning microscopes may be used. In
the confocal microscope 10, an adjustable confocal pin hole 56 is provided
in the collection optics 44 to minimize background fluorescence excited in
the converging and diverging cones 24 and 25 above and below the plane of
focus. This confocal pinhole is useful, but is not necessary in the two
photon fluorescence excitation of the present invention, since excitation
is essentially limited to the region of the focal point 26 on the object
plane.
With prior fluorescence microscopes the visible light fluorescence photons
42 are produced by molecules that are excited by absorbing a single photon
from incident light 14 that has higher energy; that is, a shorter
wavelength, than the fluorescence 42 generated during relaxation of the
molecule from its excited state. The number of fluorescence photons
released per molecule in such prior devices is ordinarily linearly
proportional to the number of exciting photons absorbed. Because only a
single photon need be absorbed in such devices, photolysis of molecules
that absorb the exciting light 14 can occur all along the double cone beam
24 and 25 within the specimen 20, although this process is not necessarily
linear with intensity. Because fluorescence is generated all along the
double cone beam, the amount of fluorescence released from each plane in
the specimen above, below and within the plane of focus of the exciting
light 14 tends to be the same, and three dimensional resolution is
difficult to obtain. As a result, the high energy of the incident light
throughout the specimen tends to damage the specimens and this is
particularly undesirable when living cells are being viewed.
In order to obtain three dimensional resolution in scanning microscopy and
to reduce damage to the specimen in regions outside the focal point of the
microscope, the present invention utilizes two-photon excitation of a
fluorophore which has a one-photon absorption peak at a wavelength which
overlaps one-half that of the exciting light. To accomplish this, the
laser 16 produces a very short pulsed laser beam of high instantaneous
power and of a relatively long wavelength, for example in the visible red
or the infrared range. This light is directed to a specimen containing a
fluorophore normally excited by a single photon in the short wavelength,
for example ultraviolet, range so that two low energy (red) photons must
combine their energy to provide the same excitation of the specimen that
would be provided by a single high energy (ultraviolet) photon. Both the
excitation and hence the fluorescence rates in the specimen are
proportional to the square of the intensity of the incident light. In the
focused excitation laser beam 14, the intensity of the long wavelength
incident light becomes high enough to excite the fluorophores in the
specimen only in the region of the focal point 26 of the microscope
optics. This focal point may be adjustably positioned within the specimen,
so that fluorescence and/or photolysis of the specimen are produced only
in a selected ellipsoidal volume around the focus. Thus, in accordance
with the invention, only long wavelength excitation light has to pass
through the specimen, and this long wavelength light is focused to produce
sufficient intensity to excite fluorescence only in a very small region.
This fluorescence is produced even if the fluorophore normally absorbs
only in the ultraviolet. Since the focal point can be selectively
positioned in the specimen, three-dimensional resolution is provided in
both scanning fluorescence microscopy and in photolysis, including
photolysis of photon-activatable reagents which ca be released by
photolysis.
In accordance with the present invention, the necessary excitation
intensity is provided at the focal point of the microscope 10 from a light
source 16 which may be, for example, a colliding pulse, mode-locked dye
laser generating pulses of light having a wavelength in the red region of
the spectrum, for example about 630 nm, with the pulses having less than
100 fsec. duration at about 80 MHz repetition rate. Other bright pulsed
lasers may also be used to produce light at different relatively long
wavelengths in the infrared or visible red region of the spectrum, for
example, to generate the necessary excitation photon energies which will
add up to the appropriate absorption energy band required by the
fluorophores in the specimen which normally would be excited by absorption
of a single photon in the spectral region having wavelengths about
one-half the wavelength of the incident light. Thus, for example, two
photons in the visible red region at 630 nm would combine to excite a
fluorophore which normally absorbs light in the ultraviolet region at 315
nm, while two photons in the infrared region of, for example, 1070 nm,
would excite a fluorophore which absorbs at 535 nm in the visible light
region.
In a modified form of the invention, the single wavelength light source 16
can be replaced by two different long wavelength laser sources so that the
incident light beam 14 consists of two superimposed pulsed light beams of
high instantaneous power and of different wavelengths. The wavelengths of
the incident beam are selected to excite a fluorophore which is absorbent
at a short wavelength which may be described as:
1/.lambda..sub.abs =1/.lambda..sub.1 +1/.lambda..sub.2
where .lambda..sub.abs is the short wavelength of the absorber, and
.lambda..sub.1, .lambda..sub.2 are the laser incident beam wavelengths..
In two-photon excitation, with a typical two-photon cross section .delta.
of:
.delta.=10.sup.-58 m.sup.4 s/photon (eq. 1)
with the pulse parameters given above (100 fsec. pulses at a repetition rate of 80 MHz), and with the beam focused by a lens of numerical aperture A=1.4, the average incident laser power (P.sub.0) of approximately 50 mW saturates the fluorescence output of a fluorophore at the limit of one absorbed photon per pulse per fluorophore. The number n.sub.a of photons absorbed per fluorophore per pulse depends on the following relationship: ##EQU1## where .tau. is the pulse duration;
f is the repetition rate;
P.sub.0 is the average incident laser power;
.delta. is the photon absorption cross section;
h is the Planck quantum of action;
c is the speed of light; and
A is the numerical aperture of the focusing lens.
The fluorescence emission could be increased, however, by increasing the
pulse repetition frequency up to the inverse fluorescence lifetime, which
typically is:
##EQU2##
For comparison, one-photon fluorescence saturation occurs at incident
powers of about 3 mW.
FIG. 2 illustrates the depth discrimination achieved by the two photon
technique of the present invention. A stereo pair of images 60 and 62 was
generated from a stack of images of a cluster of fluorescent 9 micrometer
diameter latex beads which are normally excited by ultraviolet light
having a wavelength of about 365 nm. These images were obtained using a
standard laser scanning microscope, but with its continuous-wave argon-ion
laser illuminator 16 replaced by a 25 mw colliding-pulse mode-locked
dyelaser producing output pulses at a wavelength of about 630 nm.
Measurements made on the microscope 10 indicated that about 3 mw reached
the object plane. An emission filter, passing wavelengths from 380 to 445
nm, was provided at the barrier filter 46, and the detector aperture 54
was opened to its limit in order to reduce the optical sectioning effect
that would result from a small confocal aperture.
The intensity of the incident beam 14 from laser 16 was adjusted by placing
neutral density filters in the excitation beam between laser 16 and the
dichroic mirror 28 and the blue fluorescence produced by the individual
latex beads was measured. As illustrated in FIG. 3 by the graph 64, the
detected intensity of fluorescence from the latex beads making up the
specimen increased with the square of the excitation laser power, clearly
indicating two-photon excitation in the beads. The excitation cross
section of the beads, which were "fluoresbrite BB" beads produced by
Polysciences Corporation, was estimated to be 5.times.10.sup.-58 m
s/photon, accurate within a factor of 3, by taking into account the dye
concentration in the beads, the optical throughput of the laser scanning
microscope, the pulse duration, the repetition rate, the numerical
aperture and the incident power. This value was found to be comparable to
previously measured values for similar dyes.
FIG. 4 is a scanned image of chromosomes in dividing cells (LLC-PK1; ATTC),
using cellular DNA labeling with an ultraviolet excitable fluorescent
stain (33258; Hoechst) the image acquisition time of 13 seconds was short
compared to the bleaching time of several minutes. Furthermore, no
degradation was apparent in these live cells even after illumination by
the scanning laser for several minutes.
Photobleaching during protracted scanning of a fluorescent bead occurred
only in a slice about 2 micrometers thick around the focal plane, as
demonstrated by the horizontal section 70 of reduced brightness bleached
out of the bead 72 illustrated in FIG. 5. This bead was scanned for six
minutes at a constant focal plane position. Similar localization of
bleaching was observed in the fluorescently stained cell nuclei. This
localization illustrates a distinct advantage over the use of
single-photon excitation, where the entire specimen is bleached even when
only a single plane is imaged. This is because for one-photon excitation,
bleaching in both scanning and broad field microscopy depends on the time
averaged excitation intensity, which does not vary along the axial, or
Z-direction indicated in FIG. I. For two-photon excitation, on the other
hand, bleaching depends on the time averaged square of the intensity,
which falls off strongly above and below the focal plane.
The dependence of the fluorescent signal on the square of the excitation
intensity is responsible for another advantage of two-photon excitation;
that is, such excitation provides an optical sectioning effect through the
specimen, even when using a detector, such as a CCD array, which views the
whole field, without a pinhole being used as a spatial filter. This
sectioning effect, which is illustrated in FIG. 5, avoids the serious
problems associated with chromatic aberration in the objective lens and
some of the throughput losses in conventional confocal laser scanning
microscopes.
Two-photon photolysis can also be used for fast and localized release of
biologically active chemicals such as caged Ca++, H+, nucleotides and
neurotransmitters. For example, when caged neurotransmitters are released
by a scanning beam, the whole-cell transmembrane current so produced is
usable as the contrast-generating mechanism to map the distribution of
receptor activity for those transmitters on the cell surface. The
feasibility of two-photon cage photolysis was demonstrated, in accordance
with the present invention, by irradiating DMNPE caged ATP (33mM) [from
Molecular Probes, Eugene Oregon], by the colliding pulse mode locked
dyelaser 16 focused to a beam waist diameter at the object plane of about
10 micrometers. Photolysis yields of about 10.sup.-11 moles of ATP were
measured using a luciferin bioluminescence assay from Calbiochem, San
Diego, CA. Typically, about 10% of the caged ATP in an aliquot volume of
about 10.sup.7 (.mu.m).sup.3 was photolyzed in the illumination volume of
about 10.sup.4 (.mu.m).sup.3 during about 600 seconds.
Since two-photon excitation in accordance with the present invention
provides access by visible light to excitation energies corresponding to
single-ultraviolet-photon excitation, a whole new class of fluorophores
and fluorescent indicators becomes accessible to three-dimensionally
resolved laser scanning microscopy. Such indicators may be Indo-1 for
Ca.sup.+, Mag-Indo-1 for Mg.sup.+2, ABF1 for Na.sup.+ and PBFI for
K.sup.+. Although two-photon cross sections are not yet known for many of
these compounds, and different selection rules apply to two-photon
absorption, molecular asymmetry often allows both one photon and
two-photon transitions into the same excited state. Visible fluorescence
was observed from 10mM solutions of Indo-1, FURA-2, Hoechst 33258, Hoechst
33342, DANSYL hydrazine [Molecular Probes], Stilbene 420 [Exciton Chem.
Co., Dayton, OH], and several Coumarin dyes upon excitation by a CMP
weakly focused to a 25 .mu.m diameter waist, and two-photon excited LSM
fluorescence images of microcrystals of DANSYL and Coumarin 440 were
recorded.
Another application of the present invention may be in three-dimensional
optical memory devices which rely on multi-photon processes in two
intersecting beams for writing and reading operations. A single beam would
be simpler than the two intersecting beams, and would permit maximal
information packing density. The multi-photon processes would be localized
to the high intensity region at the focus, as illustrated in FIG. 5 where
the bleaching of microscopic patterns inside fluorescent beads constitutes
a high density write once memory which is readable about 10.sup.3 times
with present fluorophores.
Thus there has been described and illustrated a practical two-photon laser
scanning fluorescence microscope for biological and other applications.
The two-photon excited fluorescence microscope provides inherent
three-dimensional resolution with a depth of field comparable to that
produced by confocal laser scanning microscopes. The use of a confocal
pinhole in conjunction with this two-photon excitation further improves
resolution along all three axes. Background fluorescence can be eliminated
by scaled subtraction of images which are recorded at different input
powers. With the present technique, photobleaching, as well as
photodynamic damage, can be confined to the vicinity of the focal plane,
thereby providing a considerable advantage over both confocal laser
scanning microscopy and area detector imaging for the acquisition of data
for three dimensional reconstruction, since ultraviolet damage to cells
and fluorophores would be confined to the volume from which information is
actually collected. This also allows sharp localization of photochemical
processes such as photolysis and photoactivation within the focal volume.
The invention is principally described as utilizing two photons from a
single laser, but it should be understood that excitation of the target
material can also be accomplished by two photons from two sources, as long
as the two different wavelengths add up to the excitation wavelength of
the target material. Thus, for example, two different laser sources could
be used, with their output beam being directed coaxially into the optical
path of the microscope. Alternatively, two different wavelengths could be
derived from a single source, as by means of a frequency doubler.
Although the present invention has been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations and modifications may be made without departing from the true
spirit and scope thereof as set forth in the accompanying claims.