Pulsed and gated multi-mode microspectrophotometry device and method

A short pulse of laser excitation and a synchronized gating time control of a fluorescence spectrograph detector are combined with a microscope, precise focusing and delivery optics, multi-mode illumination means, cooling, and temperature controls to form a new microspectrophotometry system. The gating control can block detection of any unwanted excitation radiation, such as nearly instantaneous (within 10.sup.-12 seconds) emissions after the sample is pulse excited and delayed emissions (after 10.sup.-7 seconds) after the sample is pulse excited. The gating also allows delayed emissions to be separately isolated. The microscopic focusing, pulse excitation and time segregation of detected emissions achieves a new level of precision in the detected structural spectra and property measurements not generally achievable for composite samples by prior art methods and devices.

FIELD OF THE INVENTION 
This invention relates to spectral radiation analysis of emissions from 
irradiated samples. More specifically, the invention relates to 
microscopic devices and methods for the combined optical and spectral 
emission analysis of geological, biological and other organic polymeric 
samples composed of different types of microscopic particles or interest, 
such as kerogen contained in petroleum source rocks. 
BACKGROUND OF THE INVENTION 
Many geological, biological and other solid materials are heterogeneous 
composite structures formed from interrelated, but microscopically and 
chemically discrete particles or cells. The commercial use of these 
materials can require information related to the chemical and physical 
properties of the material's individual microscopic particles. However, 
conventional laboratory analysis of a sample of these composite materials, 
such as a wellbore core sample is performed on a bulk basis. Analysis of 
these samples is commonly accomplished by a variety of methods, including 
fluorescent spectroscopy, fluorometry and fluorescent microscopy. 
Fluorescent techniques use an energy source, such as incident continuous 
wave ultraviolet (UV) irradiation, to excite a sample to cause fluorescent 
emissions from the sample. Fluorescence is the emission of radiant energy 
(such as light) as the excited electrons of an atom or molecule within the 
sample return to a lower or ground state after being promoted to a higher 
energy state by absorption of the exciting energy. Fluorescent radiations 
are normally distinct from light absorption, transmission and reflection 
with respect to time (from absorption of incident irradiation), direction, 
intensity and wavelength. 
Molecules contained in the materials can possess ground state and many 
excited electron states. Electron transitions between the many electron 
states cause fluorescent emissions to be at several different intensities, 
wavelengths, and times after absorption, the emissions forming a spectral 
structure. The fluorescent spectral structure can be quite complex for 
materials having heterogeneous chemical structures. 
Conventional fluorescence spectroscopy and photometry are commonly used to 
detect the composition and concentration of organic compounds in diluted 
solutions. The emission spectra of these compounds, which normally possess 
de-localized electrons, can display vibrational bands as a result of 
transitions between the different electron states. However, for a majority 
of solid composite samples, these techniques may not yield molecularly 
resolved spectral or intensity information. 
If a fluorescent analysis of a composite sample having many constituents is 
desired, the analysis method and device becomes much more complex. 
Conventional fluorescence analysis devices typically use continuous wave 
mercury emission as illumination and excitation source. These are 
primarily designed for homogeneous samples (typically dissolved in a 
diluted solution) and are not normally used for composite solid samples 
without process and/or apparatus changes. These devices bulk illuminate 
(i.e., energize many particles within the entire sample) and measure bulk 
fluorescence (i.e., the intensity and spectral emissions of the entire 
sample), producing potentially overlapping spectral structures from a 
composite sample. 
One composite sample analysis technique involves splitting the sample. A 
small sample portion is prepared and isolated for optical analysis 
(microscopic examination) and the other portion of the sample is then used 
for a separate bulk chemical analysis. This two step process tends to be 
slow, complex, and unreliable. 
Another composite sample analysis technique is fluorescence microscopy. In 
one embodiment of the fluorescence microscopy method, bulk illumination of 
several particles of the sample is accomplished, typically by a high 
pressure mercury arc lamp. The integration of a microscope, a scanning 
monochromator, and a photomultiplier detector, forms a microscopic 
detector which can be directed to a relatively small area of interest. 
With the use of a measuring diaphragm, the focused detection of an 
illuminated microscopic particle within the sample allows 
microspectrophotometry to be performed on the fluorescent emissions. 
Quantitative measurement of the detected fluorescence intensity and 
spectral distribution provides information regarding one or more 
fluorescing particles. 
However, because several particles of the sample are illuminated, emissions 
from unwanted particles or portions of the system cannot always be totally 
excluded. Other sample portions can produce significant fluorescence 
within the sample which may be emitted towards the focused detector. It 
may not be possible to accurately segregate the contribution(s) of each 
type of particle from the mixed detection information generated by this 
method. The detector focus area of interest may also not be able to be 
reduced to isolate a single particle's emissions. 
Secondly, as a consequence of the broad excitation and absorption bands, 
continuous-wave excitation, and long exposure time (generally in the order 
of several milliseconds to several seconds), the fluorescence spectrum 
obtained by conventional fluorescence microscopy may not be completely 
resolved, and the spectrum typically provides little chemical information 
regarding the microscopic particles of interest. 
As an alternative to this fluorescence microspectrophotometry method, a 
continuous wave laser beam can be used as a source of irradiation energy. 
This is illustrated in U. S. Pat. Nos. 4,616,133. The laser irradiation 
process essentially irradiates an area of interest within a sample with 
ultraviolet (UV) radiation from a helium-cadmium or nitrogen laser, 
separates the resulting emission spectrum into wavelength segments, and 
measures the intensities within each wavelength segment. 
The laser selectively excites certain constituents or particles in the 
sample. The monochromaticity of the laser also tends to limit the 
excitation states of specific molecules whose absorption bands coincides 
with the laser emission. A Xenon lamp coupled with a scanning 
monochromator provides a tunable continuous wave source which can also be 
used to selectively excite constituents or selectively achieve certain 
excitation states. Measured emissions (intensity in a given direction 
within a wavelength segment) are compared to one or more reference 
emissions to identify properties of particles in the area of interest. 
Prior work indicates that the fluorescent emissions are not always 
constant, but may change over extended exposure time. Although most 
fluorescence is relatively rapid, delayed fluorescence or phosphorescence 
is also common. Exciting irradiation can thus produce overlapping rapid 
and delayed emissions over time. In addition, other changes in emission 
intensity and wavelength appear to be caused by absorption of laser energy 
which is not emitted as fluorescence. Temperature increases, oxidation or 
other reactions are some of the results of the absorption of laser energy. 
These time dependent factors can cause significant changes to the spectral 
structure over time, such that unique spectral time changes can also be 
used for identification purposes. 
Even with microspectrophotometry and the use of a continuous wave laser, 
the composite samples create spectral analysis problems. First, the use of 
a continuous wave laser cannot separate fluorescence from slower 
emissions. The temporal behavior of fluorescence cannot be precisely 
examined, especially in the first hundred nanoseconds. Second, the 
continuous excitation causes photochemical reactions of the composite 
samples which may give rise to chemical information unrelated to the 
original chemistries. Third, a continuous-wave UV laser produces 
fluorescence emission with little chemical information, since the 
fluorescence spectra are almost as broad and unstructured as induced by 
mercury excitation. 
SUMMARY OF THE INVENTION 
A fluorescent microspectrophotometry apparatus and method for obtaining a 
much more distinct spectral structure from particles within a composite 
sample is presented. The apparatus and method also now allows time 
isolation of a microscopic particle's fluorescent emissions as well as 
intensity and wavelength information. This is accomplished by providing a 
short burst of pulsed and tunable laser excitation and a synchronized 
gating control of a fluorescence microspectrograph detector. The pulsed 
laser, and synchronized and gated control components are combined with a 
microscope, precise laser microbeam and emission focusing and delivery 
optics, multi-mode illumination means, cooling, and temperature controls 
to complete the system. The synchronized gating blocks detection of 
unwanted emissions. The gating also allows delayed emissions, such as 
delayed fluorescence and phosphorescence, to be isolated and measured. The 
short pulse of excitation energy also minimizes unwanted temperature and 
spectral changes over time. 
The synchronization of single pulse excitation with the gatable 
microspectrograph/detector and time isolation of spectral emissions allows 
the detector system to block detection of, for example, delayed emissions, 
and to detect the desired emissions within certain time segments after the 
sample is excited. The gated system opens to detect only the normally 
rapid fluorescent emissions, i.e., emissions that most frequently take 
place after 10.sup.-12 seconds and before 10.sup.-7 seconds (100 
nanoseconds) as measured from (i.e., synchronized with) the leading edge 
of the excitation pulse. The gated system can also separately detect 
delayed fluorescence or phosphorescence, most of which occurs after 
10.sup.-7 seconds from pulsed excitation. 
The microscopic beam focusing, laser pulse delivery and excitation, and 
time synchronization and segregation of detected emissions achieves a new 
level of spectral and properties determination precision for microscopic 
particles in composite samples. Although time and area ranges are 
theoretically unlimited, the preferred system can irradiate a microscopic 
particle/area of interest as small as 2.times.10.sup.-7 cm.sup.2 with a 
pulse as short as 15 nanoseconds, detect emissions from the particle in a 
gated time window as little as 100 nanoseconds long beginning anywhere up 
to 10 milliseconds after the initiation of the excitation pulse and can 
detect emissions from an irradiated area as small as 2.times.10.sup.-7 
cm.sup.2. The improved apparatus and method should also minimize the cost 
and time required for sample preparation and analysis, sample positioning, 
separate focusing, and minimize analysis errors by being tolerant of 
off-design conditions. 
The time isolation capabilities coupled with a pulsed laser and a 
continuous-wave source can be used to study the photochemistry of 
composite materials comprised of microscopically and chemically discrete 
particles. The system can permit time-resolved photochemical alteration 
with time of fluorescence with one nanosecond resolution. 
The tunability of the laser also allows selection of different wavelengths. 
Since different molecular structures possess different absorption spectra, 
this tunability further permits excitation of selective molecular species. 
Moreover, the short but intense laser excitation and microbeam delivery 
and focusing optics allow fluorescence analyses of some microscopic 
particles which cannot be induced by conventional means to produce 
sufficient fluorescence for detection. One example of this is sample 
containing gas-prone and non-productive kerogens. 
The apparatus can also be used to create new spectral data opportunities, 
such as multi-pulsing and separate gated times for fluorescent emissions 
in different directions, such as polarized fluorescence. At the same time, 
the system retains the more conventional illuminations, non-pulsed laser 
excitation, and non-gated detector analysis capabilities. The combination 
of synchronized pulsed micro-illumination/micro-detection and more 
conventional illumination/detection provides a powerfully improved 
microspectrophotometry tool, especially useful for complex constituents of 
particles within composite samples.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 shows a schematic of a new microspectrophotometry system. The system 
uses a pulsed beam output of an excimer (or exciplex) laser 2 controlled 
by controller 1. The excimer laser 2 generates a short burst of 
irradiation or a short laser pulse to pump or excite a tunable dye laser 
10. An excimer laser obtains its name from a process where lasing occurs 
from an excited state diatomic complex, such as XeCl complex. The complex 
is generally formed by a chemical reaction between rare gas (Xe.sup.+) and 
halide (Cl.sup.-) ions produced by a rapid electrical discharge in a 
buffer and inert gas carrier. Since the XeCl complex has a very short 
lifetime, it decays rapidly and produces lasing photon emissions. 
Although lasers of other wavelengths are available, typically below 400 nm, 
the preferred XeCl excimer laser 2 produces a pulsed coherent emission at 
about 308 nm wavelength. The single excimer pulse duration is 
approximately 17 nanoseconds. Although infinitely shorter and longer pulse 
durations are theoretically possible, a pulse duration of less than 20 
nanoseconds is preferred, most preferably less than one nanosecond. A 
train of excimer pulses is also preferred for some sample compositions. 
Although higher and lower repetition rates and energies can also be used, 
the preferred adjustable repetition pulse rate ranging from 1 to 100 Hz. A 
train of pulses each having relatively constant energies is preferred, but 
a range of pulse energies within a train is also possible. The preferred 
single excimer pulse energy nominally 200 mj or more. As a safety measure, 
the controller 1 is interlocked with a personnel access means, such as a 
door, shutting off the excimer laser when the door is opened. 
The preferred excimer laser requires a supply of rare, inert, buffer and 
halogen containing gases. An exhaust conduit 4 is used to pump air (and 
other gases) from the laser head which may contain leaked gases used 
inside the laser reservoir. A vacuum pump 3 and the exhaust conduit 4 are 
also used to purge reservoir in the excimer laser 2 after the mixture has 
been used. The preferred halogen containing gas (5% HCl, 1% H and 94% He) 
is a corrosive mixture supplied by a halogen gas source 5 comprising a 
cylinder and regulator. Corrosion resistant materials, such as stainless 
steels, are used for halogen gas carrying tubing, cylinder, and regulator 
construction. The rare gas source (cylinder and regulator) 6 supplies 
laser grade Xe (99.99% purity). The buffer gas (Ne) source 7 (cylinder and 
regulator) and inert gas (He) source 8 cylinder and regulator supply Ne 
and He gases, respectively, at 99.999% purity. 
The output pulses from the excimer laser are deflected by two high UV 
mirrors 26 mounted at 45 degree angles to direct the excimer pulsed pump 
energy to dye laser 10. The dye within dye laser 10 is a molecular dye, 
such as polyphenyl 1, dissolved in a solvent such as ethyl glycol and 
methanol. The solvent serves as a diluting and cooling medium, reducing 
thermal decay and quenching effects of the dye molecules. 
After the dye molecules absorb the pump excitation energy from the excimer 
laser beam, they are raised to the excited states. The excited molecules 
can then release the absorbed energy in several different processes. 
Radiationless energy release or transition dissipates some of the absorbed 
energy into thermal vibration, but the energy release portion of primary 
interest is released as stimulated emissions. There are two forms of 
emissions, non-coherent spontaneous emission and coherent emission. 
Non-coherent emission is random in direction and phase, and is generally 
lost from the system. The stimulated or coherent emission emits photons 
with the same phase and direction, amplifying the light signals 
coherently. As the coherent light beam passes through the dye mixture, it 
stimulates emission of more photons of the same energy, phase and 
direction. 
As the excimer pump beam enters into the dye laser 10, it is split toward a 
pre-amplifier cuvette and toward an amplifier cuvette (not shown for 
clarity) within the dye laser 10. The pre-amplifier dye circulator 11 and 
amplifier dye circulator 12 pump dye solutions through respective dye flow 
cuvettes in the dye laser 10. The output dye laser beam is collimated and 
shaped into a round beam 2-3 mm in diameter. 
At least a few hundred laser dyes are currently commercially available. 
Table 1 lists tuning ranges and solvents for some Lambdachrome.RTM. dyes, 
supplied by Lambda Physik GmbH, D-3400, West Germany. 
TABLE 1 
______________________________________ 
LAMBDACHROME TUNING RANGES AND SOLVENT 
Dye Number and Title 
Tuning Range (nm) 
Solvent 
______________________________________ 
LC 3300 BM-Terphenyl 
312-343 CH 
LC 3400 p-Terphenyl 
332-350 DI 
LC 3590 DMQ 346-377 DI 
LC 3600 QUI 368-402 DI 
LC 3860 BiBuQ 367-405 DI 
LC 4000 PBBQ 386-420 DI 
LC 4090 DPS 399-415 DI 
LC 4100 Stilbene 
405-428 EG 
LC 4200 Stilbene 3 
412-442 ME 
LC 4400 Courmarin 120 
423-462 ME 
LC 4500 Courmarin 2 
432-475 ME 
LC 4700 Courmarin 47 
440-484 ME 
LC 4800 Courmarin 102 
460-510 ME 
LC 5000 Courmarin 307 
479-553 ME 
LC 5210 Courmarin 334 
506-537 ME 
LC 5400 Courmarin 153 
522-600 ME 
LC 5900 Rhodamine 6G 
569-608 ME 
LC 6100 Rhodamine B 
588-644 ME 
LC 6200 Sulforhodamine B 
594-642 ME 
LC 6400 Rhodamine 101 
614-672 ME 
LC 6500 DCM 632-690 DMSO 
LC 7000 Rhodamine 700 
701-768 ME 
LC 7100 Pyridine 1 
670-760 DSMO 
LC 7270 Oxazine 750 
735-796 DSMO 
LC 7300 Pyridine 2 
695-790 DSMO 
LC 8000 Rhodamine 800 
776-823 DSMO 
LC 8400 Styryl 9 
810-875 DSMO 
LC 8500 HITC 837-905 DSMO 
LC 8800 IR 144 + IR 125 
842-965 DSMO 
LC 9210 IR 125 890-960 DSMO 
LC 9300 IR 140 882-985 DSMO 
______________________________________ 
Lasing in the dye laser 10 is accomplished by using an optical cavity or 
resonator which defines a self-repeating or amplified optical path through 
the active medium (i.e., the dye solution) between a mirror and grating 
(not shown for clarity). A standing wave of essentially one wavelength can 
be generated when the optical cavity distance between the grating and 
mirror is an integer multiple of 1/2 the standing wavelength. Tuning of 
the dye laser 10 is accomplished by moving (e.g., turning) the grating 
which changes the grating to mirror cavity dimensions and therefore the 
wavelength of amplified emissions. 
The dye used in dye laser 10 may be one of a series depending upon the 
particle types, sample, sample preparation, and spectral structures 
desired. Although the maximum number of dyes that can be used is 
essentially unlimited, usually no more than 3 dyes are needed to precisely 
define properties of a geological sample. 
The dye laser output pulse is passed through an optical attenuator 13 which 
adjusts the level of excitation energy suitable for microscopic excitation 
and optics. The attenuated dye laser beam pulse then travels through a 
beam expander and collimating optics 14. The expansion and collimating of 
the dye laser beam makes it easier to manipulate and later concentrate the 
beam to form a microbeam spot on the sample surface portion which is of 
interest, e.g., the particle of interest. 
An optical attenuator 13 is preferred in order to adjust the laser pulse 
energy so that the microscope and laser beam delivery optics are not 
destroyed by the short, but very intense laser energy. Normally, a 
circular linear-wege neutral density filter or a polarizer can be used, 
for example, the Melles Griot 03FDC 001/E. 
The beam expander portion of optics 14 is desired because the laser beam 
can be expanded for uniform illumination when it is recondensed by the 
microscope objective lenses. Secondly, the expanded beam is easier to 
manipulate within the microscope. Both mounted and unmounted beam 
expanders can be used. An expansion ratio of at least 2.times. is 
preferred, typically 10.times., although other ratios may be used. The 
beam expander and collimating optics 14 can consist to two plano-convex 
lenses such as the Melles Griot 01 LQF023 and 01LQF244 synthetic fused 
silica lenses. 
The collimated dye laser beam then passes through a beam splitter 15 where 
a small portion (&lt;10%) of the beam energy is extracted towards an energy 
probe 16 and joulemeter 17 for measuring the energy of the irradiating 
beam. The energy probe 16 and joule-meter 17 allow the system user to 
monitored the system while the main portion of the pulsed beam is directed 
to the microscope body 24 and associated internal reflection mirrors 18. 
Since the fluorescence intensity and possibly spectral structure can be 
dependent upon the energy levels of the impinging laser pulses, a single 
pulse or pulse-to-pulse energy level monitoring is desirable. For example, 
in determining petroleum source rocks or coal ranks, the intensity emitted 
from kerogens and macerals can be used to evaluate the maturity of the 
rocks or coals. Secondly, time-resolved fluorescence yield and spectral 
distributions may also need to be corrected for the energy level 
differences of the impinging laser pulses. Although other means for 
measuring pulse energy are possible, the preferred embodiment uses the 
beam splitter 15 to extract a small portion of the laser pulse and reflect 
the extracted portion into a coupled energy probe 16. An example of a beam 
splitter and energy probe is a Molectron JS50Q beam splitter coupled with 
a J50 energy probe and JD500 Joulemeter. 
After extracting and measuring the collimated beam's energy, the dye laser 
beam is directed to a microscope body 24. In the preferred embodiment a 
modified Leitz MPV 3 microscope is used, allowing a variety of 
illuminations and excitation options. Several lamp housings (one shown for 
clarity) 19 allow white light, UV and blue light illumination and 
excitation of a sample located on the microscope stage 20, for example 
using a high pressure mercury arc lamp with a HBO 100 lamp housing and a 
tungsten lamp for both UV and white light illumination. For pulsed 
excitation, the major portion of the laser beam from the splitter 15 
enters the body 24 from a back port coaxial to the microscope optical axis 
(shown as a line between an eyepiece 25 and a pair of objective lenses 
21). Rotation (as shown by arrow) of the first internal mirror 18 
proximate to the beam splitter 15 allows choosing between a transmitted 
beam mode (where the position of the first internal mirror 18 is as shown, 
reflecting irradiating beam initially downward) and a reflected beam mode 
(as shown by alternative path of the irradiating beam initially directed 
upward from first internal mirror 18). 
When the system is in a reflected beam mode, the upwardly reflected beam is 
reflected again by another one of the internal mirrors 18 towards a 
dichroic mirror 22. The dichroic mirror 22 is efficient in reflecting 
certain radiation frequencies such as UV light (i.e., reflecting the 
excitation laser beam) while transmissive to other radiation frequencies 
such as visible light (from the sample). The dichroic mirror 22 therefore 
reflects the pulsed UV laser beam through the upper objective lens 21 to 
the area of interest (e.g., a particle) on the surface of a sample 
supported by the microscope stage 20. 
When the transmitted mode is desired, the first of the internal mirrors 18 
system reflects the incoming pulsed beam downward. Other of the internal 
mirrors 18 then reflect the UV irradiating beam towards the sample on the 
microscope stage 20 from below. Fluorescent emissions from the sample 
which are directed towards the phototube 26 pass through the objective 
lens 21, dichroic mirror 22, and barrier filter 23. The dichroic mirror 22 
may also be removed from the optical axis of the microscope body 24 in 
this mode. 
The dye laser beam optics (expander, collimator, attenuator, and focus 
devices) allow an area within the boundaries of a microscopic particle to 
be irradiated. The surface area irradiated is typically less than a square 
millimeter, and as small as 2.times.10.sup.-7 cm.sup.2. Even smaller areas 
are also theoretically possible. 
Fluorescent emissions from the sample which emanate in the direction of the 
eyepiece 25 are transmitted through the upper objective lens 21, the 
dichroic mirror 22, and a barrier filter 23 to a phototube 26. The barrier 
filter 23 is a long pass filter, allowing only the desired fluorescence 
emission frequencies to pass through and blocking any reflected excitation 
from reaching the detector or eyes at eyepiece 25. 
Filters are conventionally desirable to isolate and reflect excitation 
bands, and to block excitation light from reaching the detector 28. For 
conventional mercury excitation, at least three filters are desirable, an 
excitation filter, a dichroic mirror 22 and a barrier filter 23. Although 
other combinations may be used, examples of conventional filter 
combinations for visual examination and identification of microscopic 
particles include a Leitz Ploemopak Filter Block H2 combination 
(consisting of a band pass filter BP 390-490 nm, a dichroic mirror RKP 510 
nm, and a barrier filter LP 515nm), and an H3 combination (consisting of a 
band pass filter BP 420-490 nm, a dichroic mirror RKP 510 nm, and a 
barrier filter LP 515 nm). For mercury-induced spectral fluorescence 
analysis, Filter Block A (consisting of a band pass filter BP 340-380 nm, 
a dichroic mirror 400 nm and a barrier filter LP 430 nm) is used. 
For a pulsed laser excitation, the lasing emission is already monochromatic 
and also may by blocked out by the gated detector, therefore no excitation 
or other filters are necessary. Only a dichroic mirror to efficiently 
reflect the laser beam (in the reflected mode) and a barrier filter to 
block reflected laser excitation are typically used. 
The filtered emissions then pass through the phototube 26 to the entrance 
slit of the spectrograph 27. At the exit of the preferred phototube is a 
small condensing lens (not shown). The emitted fluorescence microbeam is 
focused by the condensing lens into the entrance slit of the spectrograph 
27, enabling efficient and rapid detection of fluorescence. 
The emissions entering the entrance slit of spectrograph 27 are isolated 
according to wavelength by a grating (not shown) after reflection and 
collimating inside the spectrograph 27. After emissions within the 
isolated range of wavelengths emerges from the exit slit of the 
spectrograph 27, they enter and are detected by an intensified photodiode 
array in detector 28. The preferred photodiode array detector is an EG&G 
C 1455B-700-HQ intensified and blue enhanced. This array detector has 
1024 elements, 700 of which are active. The detector has a gatable, 
proximity focussed microchannel plate (MCP) image intensifier. The 
intensifier generates more than one analog-to-digital conversion (ADC) 
count for each detected photon. 
The synchronization of the firing of the excimer laser 2, excitation pulse 
from dye laser 10, and gating of the spectrograph detector 28 is 
accomplished by the electronic controller 1, a pulse generator 30 for 
generating a control pulse, a pulse amplifier 29 for amplifying the 
control pulse, a detector interface 31, and a computer 32. The 
synchronized sequence is programmed so that the exciting dye laser pulse 
is synchronized with the gating window of the detector 28. 
The logic flow for control of synchronization and detector gating is shown 
schematically in FIG. 2. Synchronization and gating is accomplished by 
control or triggering Transistor-Transistor-Logic (TTL) pulses. When the 
controller 1 is activated to initiate a dye excitation pulse from excimer 
laser 2, it also generates (as shown from SYN OUT) a control or logic 1 
TTL pulse to trigger the pulse generator 30 (as shown at TRIG IN). Upon 
receiving the leading edge of the logic 1 TTL pulse, the pulse generator 
30 begins a delay period initiated through the detector interface 31 and 
computer 32. 
The delay period is computer program controlled. It was experimentally 
determined that the preferred dye laser pulse begins approximately 570 
nanoseconds after the leading edge of the TTL logic 1 pulse. This 
information is entered in the computer program. The delayed control pulse 
triggering of the pulse amplifier 29 and diode array within the detector 
28 allows detector 28 to be synchronized with the exciting pulse from the 
dye laser 10. Thus, a delay of 570 nanoseconds was programmed so that only 
fluorescence emissions induced by the dye laser 10 were detected by the 
gate controlled detector 28. 
The control pulse from the pulse generator 30 (a Delay Trig Out terminal) 
is also sent to the detector interface 31 to prepare for active scanning 
and reading of the signals. This is accomplished by having a read and 
reset command in the data acquisition program before a scan is executed, 
as shown in this case as Trig In. Upon receiving this pulse, the charged 
diode arrays in detector 28 are read and reset. The analog to digital 
converted data are then sent to the computer 32 for processing. 
An additional delay can also be programmed. If only delayed fluorescence 
and phosphorescence is desired to be detected, a program delay of 
approximately 670 nanoseconds is programmed since a majority of fast 
fluorescence occurs within 100 nanoseconds of the excitation irradiation. 
If only fluorescence detection after termination of most of the excitation 
irradiation is desired, a program delay of approximately 585 nanoseconds 
is programmed since the dye laser pulse lasts approximately 15 
nanoseconds. Although time delay (or advance) adjustment is theoretically 
unlimited, the preferred programmed delay is adjustable from about 500 
nanoseconds to 3 milliseconds. 
The controls also set the time window or gate width, i.e., the length of 
time the detector 28 is detecting. For example, a gate width of 100 
nanoseconds (after an approximately 570 nanosecond delay) would detect a 
majority of the fast fluorescence from the irradiated sample. Although the 
control pulse width is theoretically unlimited, the preferred programmed 
pulse width was adjustable from approximately 100 nanoseconds to 10 ms. 
Several units in the system typically need to be cooled and temperature 
controlled while in operation, specifically the excimer laser 2, the 
pre-amplifier and amplifier dye circulators of the dye laser 10, and the 
photodiode array detector 28. Although the preferred embodiment 
temperature controls are described, many conventional alternative 
temperature controls may also be used. Preferably, cooling is accomplished 
by circulating chilled water (not shown) at about 13.degree. C. to these 
units. The excimer laser 2 was typically temperature controlled by a 
chilled water flow rate of between 4 and 5 liters/minute, the dye 
circulators 11 and 12 by a flow rate of between 1 and 2 liters/minute, and 
the diode array detector 28 by a flow rate of approximately 1 
liter/minute. It is especially advantageous to cool and control the diode 
array detector head of detector 28. The detector head was typically 
controlled to -25.degree. C. by use of a thermoelectric cooler discharging 
heat into the 13.degree. C. circulating chilled water. It may also be 
advantageous to cool the sample on stage 20 to cryogenic temperature. 
The invention satisfies the need to obtain a more precise spectral 
structure of fluorescent emissions and to provide new composite sample 
analysis capabilities. It achieves these precision and added capabilities 
primarily by: 1) micro-focusing of short laser pulses of excitation 
irradiation tunable to different wavelengths within narrow bands of 
frequencies; 2) a gated and micro-detecting fluorescent emission detector; 
and 3) a means for synchronizing the gating and excitation pulses. The 
resulting emission spectral structure avoids intensity and wavelength 
errors caused by extraneous emissions and can now be segregated as to 
time. The invention minimizes spectral overlapping due to illumination and 
excitation of multiple components. The invention permits fast fluorescence 
analysis of a microscopic particle with very short exposure times, e.g., 
100 nanoseconds. The invention allows separate measurement of delayed 
fluorescence and phosphorescence, fluorescent emissions in different 
directions and modes, and coordination of conventional optical and 
fluorescent analysis. Further advantages of the invention include: safety 
(e.g., interlocked excitation), reliability (e.g., avoids unwanted 
emission errors); and reduced cost (integrated one step analysis). 
Although the preferred electronic controls are described, other 
configurations of control pulses, gating, reset, read, and delay controls 
may also be used. Still other alternative embodiments are also possible, 
including: alternative sources of UV sources instead of the excimer laser, 
such as flashlamps, frequency-doubled Nd:YAG and ion lasers; a plurality 
of detectors for different emission directions or wavelength; extending 
the computer controls to excitation beam focusing or scanning and sample 
cooling; combining more components within a single housing; and separating 
the excitation beam to excite different samples or portions of a sample. 
The invention is further described by the following examples which are 
illustrative of specific results when using the invention and are not 
intended as limiting the scope of the invention as defined by the appended 
claims. 
EXAMPLE 1 
Example 1 is derived from testing samples formed by molding particles 
composed of one of several polycyclic aromatic hydrocarbon (PAH) compounds 
in an epoxy resin binder. A surface of the sample was polished for 
microscopic and fluorescence analysis. A solid PAH sample composed of 
particles having varying dimensions from approximately several square 
microns to over one hundred square microns was placed on a microscope 
stage in a system similar to that illustrated in FIG. 1. All measurements 
of solid particles were performed within the boundaries of a microscopic 
particle of interest so that fluorescent contributions from the epoxy 
resin binder were virtually eliminated. 
The particle of interest was irradiated with a series of excitation pulses 
from the excimer laser and dye laser combination similar to as previously 
discussed. The detector was gated to detect fast fluorescent emissions (a 
delay of approximately 570 nanoseconds and a gate width of approximately 
100 nanoseconds). The spectral results were compared to samples of liquid 
PAH compounds in a dilute solution contained within a quartz cuvette when 
irradiated by more conventional continuous wave mercury lamps. 
A sample of the spectral results is shown in FIG. 3. The top (a) portion of 
FIG. 3 shows the spectral results (as solid line) when analyzing a liquid 
perylene (a PAH) sample dissolved in toluene. This is compared to similar 
published data from perylene dissolved in benzene. Comparison to published 
data shows that the three vibronic bands of the published data (Berlman, 
1965) were consistently detected by the system and confirmed general 
system operation. 
The lower (b) portion of FIG. 3 shows one of the solid sample results from 
a single excitation pulse. The three vibronic bands are again detectable 
and small peaks on top of the three broad bands are now also resolved and 
detectable. The magnitude of these peaks appears to also be three to four 
times stronger than random background noise. This result can be compared 
to spectral results produced by a tunable dye laser having greatly reduced 
bandwidths when a PAH compound was vapor deposited in an alkane matrix 
(Maple et al., 1980). 
A minor difference can be noted that the 0-2 transition vibronic band is 
less distinct for the solid than the liquid solution data. Although the 
cause of this difference is not certain, the less distinct transition 
results may be due to the solid form and gated detector, i.e., the 
invention results may be a more accurate spectral structure of the sample. 
FIG. 4 shows the fluorescence spectrum of perylene sample deposited in a 
solvent matrix (Wehry and Mumantor, 1981) and the spectrum of a solid 
perylene particle sample. Spectrum (a) was excited by a dye laser at 405.9 
nm and spectrum (b) at 370 nm. Precise peaks were obtained from the 
solvent deposited perylene as shown in the top (a) portion of FIG. 4. The 
broad vibronic bands have been subtracted from the spectrum shown in the 
lower (b) portion of FIG. 4. The baseline subtracted spectra appears to 
correspond roughly to published measured small peaks from perylene in a 
heptane matrix. The high resolution achieved provides a basis for 
confidence in the ability to selectively excite and detect individual 
compounds in multi-component samples. 
Additional resolution may be obtained if the sample is also temperature 
controlled and cooled to cryogenic temperatures. The resolution is 
generally expected to improve as the temperature declines, but some shift 
in wavelength may be possible due to the thermal changes. 
FIG. 5 shows the fluorescence and phosphorescence of solid chrysene 
(another PAH) measured in a polished sample pellet. The known vibronic 
bands are well resolved as shown in the upper (a) portion of FIG. 5. Ten 
gated measurements (20 ms each) were made of the phosphorescence of 
chrysene after continuous wave exciting by a mercury lamp ceased, as shown 
in the lower (b) portion of FIG. 5. The intensity of phosphorescence 
decreases rapidly over time, but the vibronic bands are still detectable. 
However, the vibronic bands do not seem to have shifts in wavelength, 
although the relative intensity changes over time. Although the cause of 
this lack of spectral shift is unknown, the gated spectral system results 
may again be a more accurate representation of the actual spectral 
structure over time. 
EXAMPLE 2 
FIGS. 6-8 are the results of using the system to analyze kerogen pellet 
samples. The dye in the dye laser 10 was polyphenyl 1 dissolved in 
ethylene glycol in a concentration of 0.18 gram/liter. The repetition rate 
was set at 5 hertz using an exciting wavelength of 370 nanometers. Each 
spectrum was induced by a single laser pulse of approximately 15 
nanoseconds long immediately prior to opening of the detector gate. Gate 
widths were 100 nanoseconds and the array detector was temperature locked 
at -25.degree. C. 
The solid samples were composite geological samples. Particles varied 
between 5-10 microns in size. FIG. 6 depicts the cryogenic (a), low 
temperature (b) and room temperature (c) results from a filamentous 
alginite contained in a Green River Shale sample. FIG. 7 shows the results 
of different areas of interest in a Kishenehn Formation sample. FIG. 7 (a) 
is the graphical results from an alginite portion, FIG. 7 (b) a cutinite 
portion, and FIG. 7 (c) a resinite portion. FIG. 8 shows the results when 
broad baselines are subtracted from the acquired spectra of the 
aforementioned kerogen sample. The kerogen sample results show precise 
spectral structure and a new ability to differentiate between different 
samples and microscopic structures. 
While the preferred embodiment of the invention has been shown and 
described, and some alternative embodiments also shown and/or described, 
changes and modifications may be made thereto without departing from the 
invention. Accordingly, it is intended to embrace within the invention all 
such changes, modifications and alternative embodiments as fall within the 
spirit and scope of the appended claims.