GB2479012A

GB2479012A - Aligning a collection fibre with a sampling region in an emission measurement system

Info

Publication number: GB2479012A
Application number: GB1010206A
Authority: GB
Inventors: Andrew Colm Dennis; David Mark Thompson
Original assignee: Avalon Instruments Ltd
Current assignee: Avalon Instruments Ltd
Priority date: 2010-03-25
Filing date: 2010-06-17
Publication date: 2011-09-28
Also published as: WO2011117565A3; GB201010206D0; GB201005075D0; WO2011117565A2

Abstract

Devices and methods for aligning a collection fibre with a sampling region in particular for a Raman-scattering measurement system. An illumination source 725 projects an illumination signal onto a sample space. A collection fibre 765 receives a signal emitted from the sample space. An alignment fibre 715 projects an alignment signal onto the sample space. The ends of the alignment and collection fibres are positioned in a known configuration. For example, the collection fibre may be a central optical fibre (210, Fig 2A) within an optical fibre bundle (200; Fig 2A) surrounded by six alignment fibres. A camera detects the position of the projected alignment and illumination signals in the sample space. An adjuster adjusts the position of at least one of the projected illumination or alignment signal so that the signals can be positioned with a selected configuration in the sample space. Alternatively the ends of the collection fibre and illumination fibre may be positioned adjacent one another with a known offset for example positioned concentrically within a fibre bundle.

Description

ALIGNMENT METHODS AND SYSTEMS AND DEVICES USING ThEM

TECHNOLOGICAL FIELD

Certain features, aspect and embodiments are directed to methods, devices and systems to align optical fiber based collection devices. In particular, certain embodiments are directed to methods and devices that can be used to align an illumination source and a sampling region.

BACKGROUND

When light passes through a transparent medium, the light may be scattered in all directions. Two common light scattering phenomena are Rayleigh scattering and Raman scattering. In Rayleigh scattering, the light is scattered by molecules whose dimensions are smaller than the wavelength of radiation. The blueness of the sky, which results from the increased scattering of shorter wavelengths of the visible spectrum, is an example of Rayleigh scattering.

In Raman scattering, the wavelength of the scattered light is shifted from the wavelength of the incident light. The exact shifts in wavelength depend on the chemical structure of the medium or sample scattering the light. Raman lines having wavelengths higher than the incident wavelength are referred to as Stokes lines and those having wavelengths lower than the incident wavelengths are referred to as anti-Stokes lines. The intensities of Raman lines can be 0.001% or less when compared to the intensity of the incident light.

Thus, detection of Raman scattering remains difficult.

Known apparatus for performing measurement of Raman scattering involve the use of optical fibers for illuminating a sample and collecting scattered light. Collected light is generally delivered along a collection fiber to a spectrometer for further analysis.

Typically a laser illumination source provides a spot of light on a sample that has a diameter in the range of microns. Raman scattering occurs from the illuminated region of the sample and the collection fiber must be accurately aligned with the illuminated region if it is to capture the scattered light efficiently. Typically, therefore, an alignment method ) is performed to position the collection fiber before any spectroscopic measurements are performed.

In one known alignment method a collection fiber is roughly aligned with an illumination region by eye. A scanning operation is then performed in which measurements are taken at a plurality of points in a two-dimensional grid. The scanning results are collated and the position at which the collection fiber is aligned is presumed to be the position at which peak scattering results are achieved. The collection fiber is then moved to the aligned position so that further measurements, such as spectroscopic measurements, can be performed. This alignment method is considered to be undesirably slow and inefficient.

In another alignment method the collection fiber is disconnected from its associated spectrometer and an alignment signal is delivered into the fiber by an alignment source.

The alignment signal is then projected onto the sample from the end of the collection fiber. Thus, two spots appear on the sample: an illumination spot from the illumination source and an alignment spot projected from the end of the collection fiber. These spots can be examined using a high resolution camera and their positions can be adjusted until they lie on top of one another. At this point the collection fiber is taken to be aligned with the alignment spot. The collection fiber can then be disconnected from the alignment source and re-connected to the spectrometer so that the scattered light can be analysed. This alignment method is considered to be inefficient because it is not feasible to disconnect a collection fiber from a spectrometer in many environments. In addition, any intervention with sensitive spectrometer equipment can lead to errors in results.

An object of the present invention is to provide a method for aligning a collection fiber, and a corresponding apparatus, that allows rapid alignment without interfering with any of the sensitive spectroscopic equipment.

SUMMARY

According to an aspect of the present invention there is provided an optical system comprising: an illumination source capable of projecting an illumination signal onto a sample space; a collection fiber having an end that is configured to receive a signal emitted from the sample space; an alignment fiber having an end that is configured to project an alignment signal onto the sample space, wherein the end of the collection fiber and the end of the alignment fiber are positioned in a known configuration; a camera capable of detecting the position of a projected alignment signal and a projected illumination signal in the sample space; and an adjuster, capable of adjusting the signal position of at least one of the projected illumination signal and the projected alignment signal in the sample space, as detected by the camera, so that the projected illumination signal and the projected alignment signal can be positioned with a selected configuration in the sample space.

In this way, the end of the collection fiber can be aligned with the illumination signal in the sample space. This can be achieved because the alignment signal and the illumination signal can be given positions in the sample space that correspond to the configuration of the ends of the collection fiber and the alignment fiber. Thus, the position of the illumination signal can be directly aligned with the end of the collection fiber. Equally the position of the illumination signal can be offset from the end of the collection fiber by a predetermined amount so that there is a precise misalignment.

Preferably there is a plurality of fibers in a group consisting of one of alignment fibers and collection fibers and the ends of all of the fibers are preferably arranged in a known configuration. In this way one can easily infer the position to which a collection fiber would project a notional signal onto the sample space. The alignment signal that is projected onto the sample space would be in the same configuration as the ends of the alignment fiber(s). However, the configuration of spots in the sample space would differ from the known configuration of fiber ends because there would be an absence of signal in a position in which a collection fiber points. Thus, the collection fiber can be easily aligned with an illumination signal by adjusting the position of the illumination signal to a position in the sample space corresponding to the collection fiber.

Preferably there is a plurality of alignment fibers, the ends of which are configured in a known geometric configuration with respect to the end of the collection fiber.

The alignment fiber ends can be arranged in a predetermined geometric configuration along with a single collection fiber. For example, a plurality of alignment fibers may be arranged in a ring around a single collection fiber. The ring may be formed from a large number of alignment fibers in an optical fiber bundle, or could be formed with only three or four alignment fibers configured as a triangle or a rectangle. In a simple arrangement there may only be two alignment fibers that are positioned adjacent a single collection fiber.

In many embodiments the collection fiber may be positioned centrally within a plurality of alignment fibers. In an alternative arrangement the collection fiber could be a single fiber in a geometric arrangement of fiber ends, otherwise formed of alignment fibers. For example, the geometrical arrangement may be a circle of fibers and the collection fiber may be a single fiber in the ring of fiber ends forming the circle. Thus, the position corresponding to the end of the collection fiber can be inferred in the sample space by a dark spot in a circle of alignment signals.

The alignment optical fiber may be configured as an optical fiber bundle. Also, the collection optical fiber may be a central optical fiber positioned within the optical fiber bundle.

In one arrangement there may be a plurality of collection fibers, the ends of which are configured in a known configuration with respect to the ends of one or more alignment fibers. This arrangement may be most useful when a specific misalignment is desired between the collection fiber and the illumination signal. For instance, a single alignment fiber may be surrounded by a plurality of collection fibers, and each collection fiber may be offset equally from the central alignment fiber. In this arrangement, the collection fibers would each have the same degree of misalignment from the illumination signal when the alignment signal and the illumination signal are collocated in the sample space. )

This may be a useful arrangement when performing SORS (Spatially Offset Raman Scattering).

Preferably the adjuster comprises an optical element positioned in the path of one of the illumination signal and the alignment signal. The optical element may have an adjustable mounting so that its inclination can be modified with one or more degrees of freedom.

By adjusting the inclination of the optical element it may be possible to adjust the position of the signal in the sample space.

In many arrangements it would be equally convenient to adjust the path of the alignment signal and path of the illumination signal. In some arrangements it would be possible to adjust the positions of both signals simultaneously; such arrangements may involve the use of two adjustable optical elements, one in the path of the illumination signal and one in the path of the alignment signal.

Preferably the optical element comprises a mirror, but one alternative would be a lens.

The adjuster may be capable of adjusting the position of the end of the alignment fiber.

By translating the alignment fiber it may be possible to adjust the position of the alignment signal in the sample space. The collection fiber would normally be physically bound to the alignment fiber so that a translation of the alignment fiber would cause an equal translation of the collection fiber. Preferably the adjuster includes a motor.

The illumination source may be coupled to the alignment fiber so that the illumination source provides the illumination signal and the alignment signal. In one configuration there may be an alignment source coupled to the alignment fiber and capable of providing the alignment signal.

The camera may be one of a high resolution CCD camera, CMOS camera and CCTV camera.

Preferably the camera is arranged to determine the respective positions of the alignment signal and the illumination signal in the sample space. This information may be communicated in real-time to the adjuster so that movement in the positions of the signals can be recorded and controlled.

Optionally the camera could be connected to a display so that a user can monitor the respective positions of the illumination signal and the alignment signal in the sample space. Thus, a user may be able to observe the signals move when their positions are adjusted. In some arrangements the user may be able to control the positions of the signals and therefore it would be useful for the user to be able to observe the effect of their actions.

The optical system may include a processor configured to: receive the detected position of the projected alignment signal and the projected illumination signal from the camera; and calculate an adjustment to the position of at least one of the signals in order to position the signals in a selected configuration, wherein the selected configuration of signals in the sample space is related to the known configuration of the ends of the alignment fiber and the collection fiber.

Thus, the processor can automatically adjust the positions of the signals in order to align the illumination signal with the end of the collection fiber. Precise alignment can be achieved when the selected configuration of the signals in the sample space is the same as the known configuration of the ends of the alignment fiber and the collection fiber. A precise misalignment can be achieved when the selected configuration of the signals differs slightly from the known configuration of fiber ends.

Preferably the optical system further comprises a detector coupled to the collection fiber for receiving and analyzing a signal emitted from the sample space. The detector may be a spectrophotometer that is capable of analyzing a signal received by Raman scattering.

The collection fiber is preferably dedicated to the detector so that it does not need to be disconnected during the course of the alignment or measurement process. This is ) advantageous because any intervention in a sensitive spectrophotometer can lead to measurement errors. The detector may be an imaging spectrometer in some embodiments; for example, the detector may be embodied as a photomultiplier tube, a charge-coupled device, a photovoltaic cell, a phototube, a photoconductivity detector, a silicon diode detector, a linear photodiode array, a vidicon, CCD detector, video camera.

CMOS video camera or lnGaAs array.

According to another aspect of the present invention there is provided a method for configuring an optical system comprising the steps of: projecting an illumination signal onto a sample space; configuring an end of a collection fiber to receive a signal emitted from the sample space; projecting an alignment signal onto the sample space from an end of an alignment fiber that is positioned in a known configuration with the end of the collection fiber; detecting the position of a projected alignment signal and a projected illumination signal in the sample space; and adjusting the position of at least one of the projected illumination signal and the projected alignment signal in the sample space, as detected, so that the projected illumination signal and the projected alignment signal can be positioned with a selected configuration in the sample space.

According to another aspect of the present invention there is provided an optical system comprising: an illumination fiber having an end that is configured to project an illumination signal onto a sample space; and a collection fiber having an end that is configured to receive a signal emitted from the sample space, wherein the end of the collection fiber and the end of the illumination fiber are positioned adjacent one another and with a known offset from one another so that the collection fiber is aligned with a point in the sample space that is substantially adjacent the projected illumination signal.

Thus, there is a slight mis-alignment between the collection fiber and the illumination signal built into the apparatus, as would be useful in a spatially offset Raman collection system. The signals may be described as "substantially" aligned because they point at the same region of the sample (often within a few microns of one another), but in general the signals in this set are aligned with a built-in offset.

Preferably the collection fiber is coupled to a detector such as a spectrophotometer that is capable of analyzing a signal received by Raman scattering. In the self-aligning system there is no need for an alignment process because the illumination fiber and the collection fiber already point at the same region of the sample space.

Preferably one of the illumination optical fiber and the collection optical fiber is configured as an optical fiber bundle, and the other is a central optical fiber positioned within the bundle. In such a system it may be important to ensure that there is an equal offset between each col1ection fiber and each illumination fiber. This set up will ensure that the same spatially offset Raman conditions are present for each collection fiber/illumination fiber pair.

According to yet another aspect of the present invention there is provided a method for configuring an optical system comprising the steps of: projecting an illumination signal onto a sample space from an end of an illumination optical fiber; and receiving a signal emitted from the sample space with an end of a collection optical fiber, wherein the end of the collection fiber and the end of the illumination fiber are positioned adjacent one another and with a known offset from one another so that the collection fiber is aligned with a point in the sample space that is substantially adjacent the projected illumination signal.

Any apparatus features may be provided as method features and vice-versa.

Additional aspects, embodiments, example and features are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

Certain illustrative embodiments are described in detail below with reference to the accompanying figures in which: FIG. 1 is a schematic of an optical device, in accordance with certain examples; FIGS. 2A and 2B are illustrations showing alignment of an alignment signal and a sample signal, in accordance with certain examples; FIG. 3 is an illustration showing the different optical fibers used to provide an alignment signal and to collect emitted light, in accordance with certain examples; FIG. 4A shows signals representing of non-aligned optical paths and FIG. 4B shows signal representing aligned optical paths, in accordance with certain examples; FIG. 5A-5C show various geometry configuration that can be used in the methods described herein, in accordance with certain examples; FIGS. 6A-6C show the various optical paths to be aligned, in accordance with certain

examples;

FIG. 7 is an illustration of an optical device that includes a separate light source for alignment, in accordance with certain examples; FIG. 8 is an illustration of an optical device that includes a single light source for alignment and illumination, in accordance with certain examples; FIG. 9 is one embodiment of an optical steering unit, in accordance with certain

examples;

FIG. 10 is another embodiment of an optical steering unit, in accordance with certain

examples;

FIGS. 1IA-llC schematically show alignment of an alignment signal and a sample signal on a screen, in accordance with certain examples; and FIGS. 12A and 12B show arrangements for steering a collection fiber / alignment bundle.

It will be understood by the person of ordinary skill in the art, given the benefit of this disclosure, that the exact size and arrangement of the various components shown in the figures can be altered, e.g., enlarged, stretched, reduced, rearranged or otherwise configured differently to provide a desired result or a desired mode of operation. In addition, the particular placement of one component, e.g., a laser, relative to another component, e.g., an optical fiber bundle, should not be construed as limiting.

Additionally, other components such as, for example, lenses and other optical elements may be included in the devices even though they may be omitted in the figures to provide

a more user friendly description.

DETAILED DESCRIPTION

The following description is intended to demonstrate some of the useful, novel and non-obvious subject matter provided by the technology described herein. Such description is not intended to be limiting but rather illustrative of the many configurations, embodiments and uses of the alignment methods and devices described herein and the components and uses thereof. The exact shape, size and other dimensions of the components shown in the figures can vary depending on the intended use of the device, the desired form factor and other user factors that will be selected by the person of ordinary skill in the art, given the benefit of this disclosure. Moreover, the number of optical fibers in a particular bundle may vary and the number shown in the figures and described below is provided for illustration purposes only.

Certain embodiments described herein are particularly suited for use with multi-well plates such as, for example, 96-well plates, 384-well plates or larger multi-well plates. In some examples, slides, cuvettes or other types of sample holders may be used. In addition, the devices may be used to measure light emission from fluorescence or phosphorescence emission, Raman scattering or other suitable light emission and/or scattering processes. Reference is made herein to an "emitted light signal." Such term is used broadly and is intended to encompass light emission, light scattering and other optical phenomenon and processes that may result after the absorption of light by a sample. For example, where the device is configured for fluorescence or phosphorescence, light emission from a sample can be measured. Where the device is configured for Raman measurements, scattered light can be measured.

Certain examples described herein refer to optical paths or optical pathways. Such terms refer to the path along which light travels from one particular device or region to another particular device or region and may include intervening optical elements or other devices or may consist of open space between the particular devices or regions. The optical paths or pathways need not be continuous but instead may be angled or altered using suitable

N

optical elements such as, for example, lenses, gratings and the like. In addition, reference is made herein to a light source or Light sources. However, it will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that any electromagnetic energy, including that provided by a Light source, may be used to illuminate or excite a sample and that the source is not necessarily limited to light. Also, where reference is made to an optical fiber, it will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that an optical fiber bundle may be used in place of a single optical fiber. Similarly, where an optical fiber bundle is described, it may be desirable to replace the optical fiber bundle with a single optical fiber.

In certain embodiments described herein, the components of the devices described herein may be moved or positioned manually during assembly, may be moved or positioned manually during setup of the instrument, may be moved or positioned manually by an end-user, may be moved or positioned using automated methods either during assembly, setup or by the end-user or may generally be moved or positioned using other suitable means arid at other suitable times. Periodic checking of the alignment may reoccur during operation of the instrument according to a fixed or selected schedule to ensure accurate measurements. In certain examples, real-time measurements may be performed to ensure that alignment is substantially constant during measurements of data from a sample. In some examples, alignment may be verified prior to each measurement, whereas in other examples alignment may be verified after a certain number of measurements.

In certain embodiments, many analytical spectroscopic methods provide excitation energy to a sample region and attempt to collect energy from that region. It is desirable that the region illuminated by the light source and the sampling region coincide. The term excitation volume or illumination volume is often used to refer to the sample volume in x-y-z space that is illuminated by the illumination source. Similarly, the term illumination region refers to the sample region in x-y space that is illuminated by the light source. The smaller the excitation region/volume becomes and the more matched the excitation and collection regions/volumes are, the more desirable it is to provide proper alignment. When using optical fibers to couple light into a spectrometer, it is difficult to use a back illumination method to ensure the position of the focal point associated with that optical fiber. It is possible to provide a method of coupling light into the optical fiber which resides in the spectrometer, but this configuration increases spectrometer complexity. Also, it is not always practical to remove the optical fiber from the spectrometer to attempt back illumination. Additionally, providing a different optical fiber in lieu of the spectrometer's optical fiber may not desirable as there is uncertainty in the centering between optical fibers and also the centering after disconnection and reconnection. In conventional systems, the optical fibers are aligned in a blind manner by moving the collection optical fiber in relative motion to maximize a signal on a spectrometer.

Certain embodiments described herein are directed to methods that provide for alignment of an illumination region of a sample and a collection device, e.g., an optical fiber or a receiver that includes, for example, an optical fiber. For example, in many instances the optical path of incident light on a sample may not be aligned with the optical path of a receiver that collects light from a sample. In such instances, there is a decrease in the energy and amount of light received by the collection device, measurements may be imprecise, or the collection device may be receiving light outside of a desired collection region, which could result in the measurement of incorrect data. It is desirable to have the illumination source and the collection device pointing at the same sample region in space so that the region of sample excited or illuminated by the illumination source is substantially the same as the region of sample that provides emitted light to the collection device. In this manner, more accurate and precise measurements may be performed. In certain examples, the collection device may be aligned with the center of the illumination spot or region in the sample such that collected light is representative of the particular region of the sample of interest. In addition, the illumination and collection diameters may be matched, e.g., at 100 microns each, if desired, such as in the case of confocal Raman measurements, to increase the number of photons incident on the collection device. In certain instances, the collection diameter may be less than the illumination -12 -diameter, and the collection device can be centered on the illumination signal to receive emitted light from only that portion of the sample.

In certain embodiments, one illustration of an optical device is shown in FIG. 1. The device 100 includes a light source 110, an optical element 120, an optical steering unit 130, a sample holder or sample space 140, a camera 142, and a collection device or receiver 150. In operation, light or excitation energy is provided along optical pathway to optical element 120 which is operative to transmit the light to the optical steering unit 130 along an optical pathway 125. The optical steering unit 130 can provide the light to the sample holder 140 along an optical pathway 135. Light that is emitted by the sample can be received by the optical steering unit 130 along an optical pathway 135.

The optical steering unit 130 can provide light to the receiver 150 along an optical pathway 145. Illustrative configurations for each of the components of FIG. I are described in more detail below. When aligned, the receiver 150 receives emitted light from substantially the same region of the sample as the region that is illuminated by the light source 110.

In some examples, the light source 110 may be, for example, a laser, pulsed laser, arc lamp, cathode lamp or other suitable light sources. Where it is desirable to pulse the light source, choppers or other suitable devices may be implemented in the optical pathway or in other optical pathways between the light source 110 and the sample holder 140.

The exact wavelength and intensity of the light provided by the light source 110 may also be varied depending on the particular sample to be measured. Desirable wavelengths are those in the ultraviolet, visible and infrared spectrum including, but not limited to, near infrared light. In certain examples, the wavelength of light may be between 300 nm and 1000 nm, more particularly between 400 nm and 800 nm, for example about 785 rim.

The exact power of the light provided can vary and desirably not so much power is used that sample degradation occurs during the measurements. In some embodiments, the light can be provided at a power of about 200 milliWatts to about 1 Watt, more particularly about 300 milliWatts to about 800 milliWatts, for example about 350 milliWatts. The power delivered to the sample is typically less than the power outputted -13 -near the light source. For example, about 50 milliWatts to about 200 milli Watts of power may be delivered to the sample, e.g., about 100 milli Watts of power may be delivered to the sample.

In certain examples, one or more other optical elements (not shown) may be placed in the optical pathway 115 to provide a desired output of the light. For example, where it is desirable to provide polarized light, a polarizer may be placed between the light source and the optical element 120. Similarly, band pass filters such as high-band pass filters, low-band pass filters or combinations thereof may be used to provide light of a selected wavelength to the optical element 120. As discussed in certain embodiments herein, one or more mirrors, switches or splitters can be used to split the light into two different directions.

In certain examples, the optical element 120 may be a mirror and may include suitable films or coatings to filter the light. For example, the optical element 120 may be a mirror that is operative to redirect the light along the optical pathway 125 to the optical steering unit. One or more choppers or other devices may be placed within the optical pathway to provide pulsed light to the optical steering unit 130. The exact positioning of the optical element 120 may vary and desirably the angle of the optical element 120 is selected such that a sufficient amount of light can be provided from the light source 110 to the optical steering unit 130 and on to the sample holder or space 140.

In certain embodiments, the optical steering unit 130 may include suitable components to provide light from the optical element 120 to a sample in the sample holder or space 140.

The optical steering unit 130 may also include suitable components to receive emitted light from the sample and provide the emitted light to the receiver 150. Illustrative components that can be used in the optical steering components are described in more detail below. In certain examples, the sample holder 140 may be configured to retain a sample and to orient the sample at a suitable angle to receive light from the optical steering unit 130. The exact configuration of the sample holder or space 140 may vary depending on the type of sample to be analyzed. In particular, depending on the nature of -14 -the sample, the sample holder or space 140 may be configured to receive microplates, slides, live or fixed cells, solutions, crystals, solids and other materials and forms that are amenable to analysis using emitted or scattered light measurements.

In certain embodiments, the receiver 150 may be configured to receive emitted light from the optical steering unit 130 and provide that light to a detector (not shown) that is optically coupled to the receiver 150. In certain examples, the receiver 150 may include an optical fiber that can be used to receive the emitted light and transmit it by an optical cable to the detector. The optical fiber may be a single optical fiber or may be an optical fiber bundle as described herein, and where an optical fiber bundle is used, the exact number of optical fibers in the bundle is not critical. The detector is generally a combination of a spectrometer and a scientific CCD camera (or some other spectrophotometer). The detector is used to analyse the scattered light from the sample.

Generally, the detector (not shown) is not used in the alignment process. The camera 142 is used in the alignment process to image any laser spots that are projected onto the sample. A high resolution is typically required if the camera is to image laser spots that have dimensions in the micron range. The high resolution camera 142 is independent of the optical fibers that are used to collect light from the sample.

One or more additional optical fibers can be optically coupled to the receiver 150 and used to align the optical paths. In particular, it is desirable to align the collection optical fiber of the receiver 150 with the optical path of the scattered light such that the illumination region of the sample and the region of the sample "seen" by the receiver are substantially the same. The exact number and arrangement of these additional optical fibers can vary and illustrative arrangements and numbers are described herein. One arrangement is shown in FIG. 2A. In this "6 around 1" or "6+1" arrangement, an optical fiber bundle 200 includes a central optical fiber 210 which is optically coupled to the optical steering unit 130 to receive light emitted by the sample. The six optical fibers arranged around the central optical fiber 210 can be used to provide light for alignment of the collection optical fiber with the optical path of the scattered light from the sample.

-15 -j By aligning the central optical fiber with the illuminated region of the sample, overall accuracy and precision can be increased and detection limits can be lowered.

By way of illustration only and referring to FIG. 2B, an alignment signal shown as a ring of light 220 may be provided by an alignment optical fiber bundle and projected onto the sample space. The ring of light 220 projected on the sample is typically imaged using the high resolution camera and can be visualized on a screen in the alignment process.

During the alignment process, the illumination signal on the sample 230 desirably is centralized within the ring of light 220 to align the illumination region with the sampling region. In operation, the various components of the optical device can be moved until the illumination signal 230 is centralized within the ring of light 220. Such movement may be accomplished manually or using motors coupled to stages where the various components are mounted. The particular device or methods to move the components in the optical device are not critical, and in certain instances, a calibration routine may be used such that the instrument itself automatically moves the various components to centralize the illumination signal 230 within the ring of light 220. Once aligned, the illumination light and the collection optical fiber or fibers are directed at substantially the same region of the sample to provide for more accurate and precise measurements.

While the term centralized is used in reference to the alignment process, the sample signal 230 need not be exactly within the center of the ring of light 220. Small offsets from the center may be used advantageously, as described herein, to provide spatially offset the receiver such that additional information about the sample can be measured.

In one configuration as shown in FIG. 3, a collection optical fiber 310 and an alignment optical fiber bundle 320 are each optically coupled to a receiver 330. In operation, the collection optical fiber 310 receives light from a sample along an optical path. Along substantially the same optical path, the alignment optical fiber bundle 320 can provide an alignment signal from a light source optically coupled to the alignment optical fiber bundle 320 so that the signal from a sample that is received by the collection optical fiber 310 is contained substantially within a central portion of the light pattern provided by the alignment optical fiber bundle 320.

-16 -Another example of the alignment process is shown in FIGS. 4A and 4B. Referring to FIG. 4A, an illumination signal 410, which is representative of the emitted light from a sample, is shown with an alignment signal 420, which is representative of a signal from an alignment optical fiber bundle. While FIG. 4A shows a signal from an alignment bundle, a single optical fiber can be used to provide an alignment signal, as discussed herein. As shown in FIG. 4A, the illumination signal 410 and alignment signal 420 are spatially offset, which is indicative of misalignment of the illumination source and the collection optical fiber with respect to the illumination region of the sample. In this instance, emitted light from the sample may not be collected as efficiently as is possible.

In FIG. 4B, the illumination signal 410 is shown as being centralized with the alignment signal 420, which results in alignment of the collection optical fiber with the region of the sample that is illuminated by the illumination device. As discussed in more detail below, to align the spots, the receiver, collection device or other components of the optical device can be moved or translated in one or more dimensions, e.g., x-, y-or i until the signal from the sample is substantially surrounded by the alignment signal, e.g., centralized or within an alignment signal. The movement of the signals in the sample space can be monitored by the camera (not shown) which can be configured as a high resolution camera pointing at the sample space.

In certain examples, it may be desirable to reverse the collection fiber and the alignment fiber bundle. Thus, an optical fiber bundle can be used to collect emitted light from a sample and a single spot or alignment signal can be projected onto the sample space. The illumination spot and the alignment signal can be positioned on top of one another using the camera to monitor their positions. In this arrangement the collection fiber bundle may be offset from the illumination spot by a known amount, which would be useful for analyses of spatially offset Raman scattering. Generally it would be transparent to the user if the collection fiber and alignment fibers were reversed as the end result to accomplish alignment would appear the same on the screen or camera once the signals are aligned. Thus, it is not critical whether the alignment signal is central or outside of -17 -the scattered light signal provided the two signals are aligned with each other in a suitable manner as described herein.

In many embodiments it is desirable to achieve the highest possible degree of alignment between the alignment signal and the sample signal. In certain embodiments, however, the spot(s) or signal provided by the alignment optical fibers and the light from the sample need not be perfectly aligned. That is, the signal from light emitted by the sample does not necessarily need to be contained entirely within a central cavity or core formed by the signal from the alignment optical fibers. In particular, there can be some overlap of the spots provided that the illumination region and the sampling region are aligned to a sufficient degree. In other embodiments it may actually be desirable for a misalignment between the alignment signal and the sample signal; this may be the case for spatially offset Raman scattering (SORS) where 0% alignment may be desirable.

In certain embodiments, the exact configuration of the alignment fiber optical bundle and the collection optical fiber can vary. For example, it is possible to use a signal from an alignment optical fiber bundle having three optical fibers 505, 510 and 515, as shown in FIG. 5A, to surround a collection fiber 520. Referring to FIG. 5B, an alignment optical fiber bundle having four optical fibers 525, 530, 535 and 540 can be used to surround a collection fiber 545. Referring to FIG. 5C, an alignment optical fiber bundle having eight optical fibers 550-585 can be used to surround a collection fiber 590. The exact number of optical fibers and geometry of the signal created by the optical fibers is not critical and any particular number of optical fibers and geometry may be used. Desirable numbers of alignment optical fibers vary from about three to about ten, more particularly from about four to about nine, for example, from about five to about eight, e.g., six or seven.

One illustration of the particular optical pathways that become aligned using the alignment methods described herein are shown in FIGS. 6A-6C. In these schematics, the optical pathways are first shown as separate (FIGS. 6A and 6B) and then shown as superimposed (FIG. 6C). Referring to FIG. 6A, an optical device has a detection area or region 605 through which light may be received from a sample and provided to a detector.

-18 -Emitted light outside of this detection area 605 is not provided to or "seen" by the detector and remains undetected. The emitted light 610 from a sample is shown as falling within the detection area 605; the emitted light 610 emanates from a position which is the same as the position of a spot of an illumination signal on the sample. The emitted light 610 can be focused on a collector or receiver 630 using optical elements 615 and 620, which are typically lenses though they may each independently be, for example, other focusing, defocusing or collimating optical elements. The light 610 scattered from a sample is first incident on the optical element 620 which passes the light 610 to the optical element 615. The optical element 615 passes and focuses the light on a receiver 630, which in this embodiment includes a single collection optical fiber. The exact positioning of the optical elements 615 and 620 relative to each other and relative to the receiver 630 may vary depending on the optical properties of the optical elements.

Referring to FIG. 6B, the optical path of a ring of light from an alignment signal is shown.

An alignment signal 635 is provided by the receiver 630 to the optical elements 615 and 620. In a typical setup, an alignment optical fiber bundle is optically coupled to the receiver 630, and the collection optical fiber can be a central fiber within the alignment optical fiber bundle, as described herein. The alignment signal 635 is shown as being substantially circular as a result of the circular arrangement of the optical fibers used to provide the alignment signal. The alignment optical fibers are optically coupled to a light source (not shown), which as discussed below, may be the same light source used to excite the sample or may be a separate light source. When the illumination signal optical path and the alignment signal optical path are aligned, the illumination signal 610 is positioned substantially central within the open space of the alignment signal 635 as shown in FIG. 6C, and both signals are within the detection area 605. Such alignment can provide, for example, a substantially similar illumination region and sampling region to increase the overall accuracy of the measurements and to provide for increased repeatability from measurement to measurement.

In certain embodiments, the exact configuration and components used to provide an alignment signal may vary depending on the configuration of the instrument or device -19 -where alignment of the collection optical fibers with the optical path of the emitted light is desired. One embodiment of an optical device that uses an alignment optical fiber optically coupled to its own light source is shown in FIG. 7. The device 700 includes a first light source 710 optically coupled to an alignment optical fiber bundle 715 which itself is coupled to a receiver 720. The first light source 710 provides light of a suitable wavelength for use in the alignment process. In some examples the wavelength of light provided by the first light source 710 can be the same, a shorter wavelength or a longer wavelength than the wavelength of light used to illuminate the sample. The device 700 also includes a light source 725 that is configured to provide light to illuminate or excite a sample. Energy from the light source 725 travels along an optical path 730 to an optical element 735 which may be, for example, a mirror. The optical element 735 is effective to redirect the energy to an optical steering unit 745 along an optical path 740. The optical steering unit 745 can provide the light to a sample (not shown) along an optical path 750.

The light energy can excite the sample and cause the sample to emit or scatter the light, which typically is collected by the optical steering unit 745 along optical path 750; the optical path of the illumination signal need not be the same as the path taken by scattered light, despite the fact both are denoted with numeral 750. The optical steering unit 745 can provide the emitted or scattered light to the receiver 720 along an optical path 760.

In this embodiment, a lens 755 is shown in optical path 760 to provide focused light to the receiver 720. The emitted light received by the receiver 720 can be provided to a detector (not shown) through an optical fiber 765, which is optically coupled to the detector.

Prior to any measurements using the illustrative device of FIG. 7, an alignment operation can be performed to ensure that the collection optical fiber of the receiver 720 is aligned with the illumination region. This alignment operation may take a form similar to that described in reference to FIGS. 2A-6C. In particular, the various components of the optical device 700 can be moved or manipulated until the illumination signal (or the signal from the emitted light) aligns with the signal or signals from the alignment fibers.

For example, the illumination signal can be centered within an alignment signal to provide for overall alignment of the device. The movement of the signals can be -20 -monitored using a high resolution camera (not shown) until a desired alignment is achieved.

In one configuration, light may be introduced through the light source 710, which can be a laser, pulsed laser, cathode lamp or other suitable light source. The exact wavelength provided by the light source 710 can vary, and, in certain examples, the wavelength is desirably shorter or longer than the wavelength provided by the light source 725 to be able to differentiate the various light sources. A high bandpass filter, low bandpass filter, combinations thereof or the like may be present in the optical steering unit 745 to filter out any light from the light source 725 such that light from the light source 725 does not reach the receiver 720. In other examples, the wavelength of the two light sources can be the same and no filtering is needed. A user can switch on the light source 710, e.g., manually or through software or other control, and the various components of the optical device 700 including, for example, the receiver 720, the optical steering unit 745 and/or the sample holder or space (not shown) can be moved until the alignment signal aligns with the sample signal. During operation of the device 700, the alignment may be periodically rechecked, if desired, or alternatively, prior to recording of any scattered light signal from the sample, a pre-measurement alignment check may be performed each time, as discussed below. Once the collection optical fiber is properly aligned, light from the light source 710 may be switched off to avoid any interference with the measurements, or the light may remain on during operation of the device 700. The signal that is provided to the optical fiber 765 may be detected using conventional detectors, such as those described herein, that are optically coupled to the optical fiber 765. In certain examples, light from the alignment light source may remain on during measurement such that alignment can be continuously monitored in real time.

While the light source 710 and alignment optical fiber bundle 715 are shown as being integral to optical device 700, the light source 710 and alignment optical fiber bundle 715 may be configured as an accessory module that can couple or plug into existing instruments. Where such an accessory module is used, the device 700 may include an optical port that can optically couple the alignment optical fiber bundle of the accessory -21 -to the receiver 720 of the device 700. Such a module would permit the use of the alignment devices and methods with more than a single instrument and can reduce the overall cost of the instrument. In addition, the module may be removed after alignment or may include optical stops that prevent operation of the module after alignment to avoid any interferences with the measurements.

Another configuration of an optical device is shown in FIG. 8. In this configuration, the device 800 uses the same light source to provide an alignment signal and to illuminate the sample. Referring to FIG. 8, an optical device 800 includes a light source 810 that is optically coupled to both an alignment optical fiber bundle 840 and an optical steering unit 860. The light source 810 provides light along an optical path 815 to a splitter, mirror, switch or other optical element that can split the light. A portion of the light is provided to the alignment optical fiber bundle 840 along an optical path 830. An optical element 825 such as, for example, a lens, can be used to focus the light into an aperture of the alignment optical fiber 840. The remaining light is provided to an optical element 850 along an optical path 835. The optical element 850 may be a mirror or other optical devices that can redirect the light at a desired angle. The optical element 850 redirects the light along an optical path 855 to an optical steering unit 860. The optical steering unit 860 provides the light to a sample (not shown) along an optical path 865. Scattered or emitted light from the sample is collected along an optical path 865 by the optical steering unit 860. The optical steering unit 860 provides the collected light along an optical path 875 to a receiver 845. In this illustration, an optical element 870 is used to focus the light provided to the receiver 845 along the optical path 875. The light collected by the receiver 845 is provided to a detector (not shown) optically coupled to the receiver 845 through an optical fiber 880.

In certain embodiments, an alignment operation or step can be performed using the device of FIG. 8 to ensure that the collection optical fiber is aligned with the illumination region of the sample. As discussed herein, such alignment ensures that the illumination light and the collection optical fiber are pointed at substantially the same region of the sample space. This alignment operation may take a form similar to that described in -22 -reference to FIGS. 2A-7. In particular, the position of the various components of the optical device 800 can be moved or manipulated until the illumination signal aligns with the signal from the alignment optical fibers. In one example, light may be introduced through the light source 810, which can be a laser, pulsed laser, cathode lamp or other suitable light source. The exact wavelength provided by the light source 810 can vary. A user can switch on the light source 810, e.g., manually or through software or other control, and the various components of the optical device 800 including, for example, the receiver 845, the optical steering unit 860 andlor the sample holder (not shown) can be moved until the aligiunent signal or signals align with the illumination signal or signals.

During operation of the device 800, the alignment may be periodically rechecked, if desired, or alternatively, prior to the measurement of any data from the sample, a pre-measurement alignment check may be performed each time, as discussed below. Once the illumination region and the collection optical fiber are properly aligned, light provided to the alignment optical fiber 840 may be blocked by turning the switch 820 off or otherwise changing the angle of the switch 820 so that substantially no light is provided along an optical path 830. In other examples, the alignment signal may remain present during operation. The signal that is provided through the optical fiber 880 may be detected and analysed using conventional detectors, such as spectroscopic detectors described herein, that are optically coupled to the optical fiber 880.

While the alignment optical fiber 840 is shown as being integral to optical device 800, the alignment optical fiber may be configured as an accessory module that can couple or plug into existing instruments. For example, the alignment optical fiber may couple to optical ports on the device such that the optical fiber is optically coupled to the light source 810 and the receiver 845. Where such an accessory module is used, the device 700 may include an optical port that can optically couple the alignment optical fiber bundle of the accessory module to the receiver 845 of the device 800. Such a module would permit the use of the alignment devices and methods with more than a single instrument. In addition, the module may be removed after alignment or may include optical stops that prevent operation of the module after alignment to avoid any interferences with the measurements.

-23 -In certain embodiments, the optical steering unit that can be used in the optical devices disclosed herein may include suitable optics to provide light from a light source to a sample and to provide emitted light from the sample to the collection optics. One illustration of an optical steering unit is shown in FIG. 9 in an arrangement that can steer an illumination beam only. Referring to FIG. 9, the optical steering unit 910 includes one or more devices or optical elements 920 for manipulation of the laser light or other light source when non-laser light is used. Such manipulations include, but are not limited to, expanding, contracting, shaping, attenuating, polarizing, depolarizing, redirecting, filtering, displacing, temporally stretching or temporally compressing the laser light. The manipulations are optional and may be replaced by lenses, mirrors or other optical elements that can direct the light at a desired angle and along a desired optical path.

Because the optical path between the optical elements 930 and 935 only includes laser light, manipulation of the laser light does not affect the Raman scattered radiation.

Similarly, the system may also include one or more devices or optical elements 950 for manipulation of the emitted light, e.g., Raman scattered light. Such manipulations include, but are not limited to, expanding, contracting, shaping, attenuating, polarizing, depolarizing, redirecting, filtering, displacing, temporally stretching or temporally compressing the Raman scattered radiation. Similar to manipulation of the laser light, manipulation of the emitted light signal is optional. Because the optical path between the optical elements 960 and 955, respectively, only includes the emitted light signal and not the laser light, manipulation of the emitted light signal does not affect the laser light. The configuration shown in FIG. 9 permits individual tuning andlor manipulation of the laser light and the emitted light signal, if desired, to provide increased flexibility for performing measurements.

In operation. the optical steering unit 910 can receive light from a laser source and output the light to a sample holder or sample space along an optical path 970. Emitted light from the sample can be received along the optical path 970 and manipulated using an optical element 950. The alignment signal (not shown) may be directed along the optical path 970 such that signal from the scattered radiation can be aligned with the alignment -24 -signal (or vice versa) to provide proper alignment of the collection optical fiber with the illumination signal optical path.

While the laser light and the scattered radiation signals are shown in FIG. 9 as having a common optical pathway between the optical steering unit and the sample, such common optical pathway is not required. Instead, the laser light (or non-laser light where sources other than a laser are used) can be provided along a different optical pathway than the optical pathway used to receive the scattered radiation. One configuration of such an optical steering device is shown in FIG. 10. The optical steering device 1000 includes separate optical pathways where the excitation light is not recombined along a common optical pathway after its separation from the common optical pathway. Light 1005 from the laser or other light source can be provided along a common optical pathway 1010 to a Iirst optical element 1015. The first optical element 1015 may be configured, for example, as a beam separator to separate laser light from the common optical pathway and provide it to a second optical element 1020. The optical steering 1000 may optionally include a device 1025 for manipulating the laser light, as discussed below.

The second optical element 1025 may provide light to a sample (not shown) optionally using a lens 1030 or other suitable optical element. Lenses 1030 and 1040 are optional and may be omitted if desired. In the configuration shown in FIG. 10, the laser light is not recombined into the common optical pathway as shown in the embodiment of FIG. 9.

Instead, the laser light is provided to the sample and emitted light may be received by the optical element 1015, optionally through one or more additional optical elements (not shown), along an optical path 1045. The emitted light may be provided to a detector (not shown) along the common optical path 1010. In the configuration shown in FIG. 10, the exact angle that the emitted light is received by the optical element 1015 is not critical, as emissionlscatterrng occurs generally in all directions. Thus, the optical paths may be separated and not recombined, which permits individual manipulation of the laser light and the emitted light, and simplifies the overall production of the system.

In certain examples, an alignment signal may be introduced generally along the common optical pathway 1015. To align the collection optical fiber with the region of the sample -25 -that is illuminated, the receiver, one or more components in the optical steering unit or other optical elements can be moved, e.g., in one, two or three dimensions, until the spot(s) or signal(s) provided by the alignment optical fiber or fibers are aligned with or surround the spot(s) or signal(s) provided by the emitted light (or vice versa).

In certain embodiments, one or more motors may be coupled to the collection optical fiber, the components of the optical steering unit and/or the sample space. For example, the components of the optical devices and systems can be coupled or mounted to moveable stages or fixtures that can be moved by actuation of a motor coupled to the stage or fixture. The motors can be configured to permit movement in one, two or three dimensions depending on the particular configuration of the device. Suitable motors and stages are known in the art. Illustrative motors include piezoelectric motors, stepper motors, AC or DC Servo motors, AC or DC motors, voice-coil motors, and linear actuators. All motors could be used with and without encoders.

FIGS 1 2A and I 2B show arrangements for steering the collection fiber / alignment bundle; these arrangements may comprise components in optical steering units. In FIG 12A an arrangement is shown in which there is reflection from Cartesian 2-dimensional (for example gimbal or kinematic mount) reflective surfaces, each reflective surface having one or more degrees of freedom. The arrangement of FIG 1 2A could be implemented using the first and second optical elements 1015, 1020 shown in FIG 10. In FIG 12B an arrangement is shown in which a receiver can be translated in three dimensions, such that the receiver has two or more degrees of freedom. By translating the receiver it may be possible to adjust the position of a projected alignment signal on a sample.

It will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that the motor may be directly coupled to the element to be moved or may be indirectly coupled to the element to be moved through one or more linkages, gears, mounts or other mechanical devices or fittings.

-26 -In other embodiments, the user may move the components manually by turning or twisting knobs or controls to alter the position of one of the components of the device and systems. Such knobs can include gross and fine control to provide accurate alignment of the signals. Once the desired components are moved and the collection optical fiber is aligned, the controls can be locked into place to prevent further movement. Other configurations to move the components in an automated fashion or manually will be readily selected by the person of ordinary skill in the art, given the benefit of this

disclosure.

In certain embodiments, the alignment methods and devices disclosed herein are particularly suited for use with Raman spectroscopic devices. Raman spectroscopy can be used, for example, with liquids, solids, gases, polymers, gels, slurries, powders, crystals, suspensions and many other types of samples including mixtures of different components. Raman spectroscopy also requires no or little sample preparation and thus permits analyses in situ, if desired. Raman spectroscopy may also be used to monitor chemical reactions or to provide process feedback.

In certain examples, the exact configuration and form factor of the device may vary depending on the particular sample or material to be analyzed. In some examples, the device may be configured as a microscope. For example, the devices and methods may be implemented with one or more of the RamanMicro family of microscopes available commercially from PerkinElmer, Inc. (Waltham, MA). In other examples, the methods and devices described herein may be implemented in a table top instrument such as, for example, the RamanStation family of instruments or the RamanFlex family of instruments, each of which are also commercially available from PerkinElmer, Inc. The methods and devices may be permanently integrated into the instrument or microscope or may be included as an accessory device or add-on probe or module that can be used, when desired, by an end-user. Whether a microscope or a larger instrument is selected can depend on many factors including sample properties and dimensions, e.g., size or thickness, sample type, desired sampling region, etc. In some examples, the instrument may be portable for field-based analyses or may be fixed to a bench top.

-27 -In certain embodiments, the particular camera used in the optical devices and systems may vary depending on the sample to be analyzed and/or the particular type of measurement being performed. Illustrative cameras include, but are not limited to, a photomultiplier tube, a charge-coupled device, a photovoltaic cell, a phototube, a photoconductivity detector, a silicon diode detector, a linear photodiode array, or a vidicon.

In certain examples, a high resolution CCD camera may be used to provide an image corresponding to the alignment signal and/or the emitted radiation. The CCD camera is provided to observe the sample surface, independently of the arrangement of fibers; this can be achieved via dielectric mirrors in certain instances. One such illustrative image from a CCD camera is shown in FIG. 11 A, where an alignment signal 1120 and a Raman signal 1110 from a sample are shown. The signals 1110 and 1120 are shown as being separated spatially, which reflects poor alignment of the illumination region of the sample and the collection optical fiber. The emitted light signal 1110 may be driven or moved onto the alignment signal 1120 through movement of one or more of the components of the device. For example, it may be desirable to change the angle of a component in the optical steering unit with respect to the position of the collection optical fiber. The collection optical fiber itself may also be moved. The position of the sample can be moved. In some examples, two or more components within the optical device can be moved to provide alignment of the signals. This may be achieved by a two-plane rotation of two planar reflective surfaces such as the adjustment of two mirror mounts (typically gimbal or kinematic mounts). In one embodiment, the collection optical fiber may be moved in one, two or three dimensions to move the emitted light signal 1110 within and central to the alignment signal 1120, as shown in FIG. 1 lB. Once the signals are aligned, the collection optical fiber may be fixed in space by stopping any further movement, and camera cross-hairs 1130 (or some other type of referencing element) may be aligned on the central portion of the emitted light signal 1110 (see FIG 11C) so that measurements may commence. Alignment of the camera on the central portion of the emitted light signal 1110 is not required, but it can provide more accurate measurements and reduce the likelihood of detecting a signal not representative of the emitted light from the sample.

While the signals shown in FIGS. hA-i IC are described in reference to a CCD camera, similar methods may be used with other cameras/detectors. In one arrangement the S optical device can be coupled to a first detector, e.g., a CCD detector, video camera, CMOS video camera or other device, for alignment and then, optionally, a different type of detector, e.g., a CCD detector, video camera, CMOS video camera or other device, could be trained on the sample during actual measurements; of course, there may be no need for a secondary camera during the measurements, but such a camera could be a desirable feature so that alignment can be monitored continuously. Such additional cameras may be configured as part of the accessory module to interface with the device or instrument during the alignment process. The accessory module may remain in place after alignment or may be removed subsequent to alignment.

In certain embodiments and depending on the exact configuration of the alignment devices, it may include one or more suitable interfaces, mechanical couplers, fittings or other suitable components so that the module may be physically coupled to the instrument housing andlor any printed circuit boards of the instrument. Where the device is configured as an alignment module, the module may include anti-reflective coatings on external surfaces such that stray light may be absorbed or otherwise not interfere with any measurements. In some examples, the module may be sized and arranged such that it can be drop fitted or dropped into an existing instrument after removal of suitable pre-existing components from the instrument. The couplers or fittings on the housing of the module may be selected such that the optical paths of the existing components of the instrument (such as, for example, the optical path of the light source, the optical path of the detector or other components of the system) may be generally aligned with the optical components of the modules simply by coupling the fittings in suitable holes or apertures in the housings of the instrument. Final alignment of the optical pathways prior to use may be accomplished using the methods described herein. For example, the module may be designed such that it is operative with a RamanStationlM 400 Raman spectrometer, a RamanFlexTM 400F Fiber Optic Analyzer, a RamanMicrolM 200 Raman Microscope, -29 -each of which are commercially available from PerkinElmer, Inc. (Waltham, MA), or other Ranian devices commercially available from PerkinElmer, Inc. or other suitable suppliers.

In certain embodiments, the systems disclosed herein may be used for optical analyses other than Ranian measurements. For example, light emissions from fluorescence or phosphorescence may also be monitored using the configurations described herein. En addition, the configuration shown and described herein permits the use of a single instrument for a variety of different Raman techniques. For example, the configurations may be used for polarized Raman measurements, where typically a laser polarizer and Raman polarization analyzer are placed in the main optical part of the instrument in the laser and Raman paths, respectively. In other examples, the configurations may also be used for Spatially Offset Raman Spectroscopy (SORS), which may include a bespoke optical configuration. The configuration may also be used for transmission Raman, which allows the illuminating laser to be sent into the sample and the Raman spectrum to be acquired from the opposite side, or at some other desired angle.

In certain embodiments, the alignment methods disclosed herein may advantageously be used to ascertain bow far from a particular region of interest that a collection device is receiving data. For example, an alignment procedure may be performed as described herein, and the collection device may then be moved a desired distance from the alignment position to receive data in a spatially offset position. The alignment methods described herein permits spatial offsetting in a more accurate manner than previously performed. En one example, the spatial offsetting may be used to look at Ranian spectra of bone. For example, the skin may be illuminated, and where light is collected after alignment, the spectrum would be representative of the skin. The skin may then be illuminated in the same place, but Raman light may be collected at desired distance from the illumination region to increase the bone component of the overall Raman spectrum.

In this manner, various layers or regions of a sample, at known distances from the illumination region, may be measured or imaged with good precision and accuracy. The -30 -collection fiber can be directly positioned at a desired offset position by positioning the illumination signal at a selected offset position relative to the alignment signal(s).

In certain embodiments, where the alignment light source and the sample illumination light source have different wavelengths, the overlap of the two signals may be used to align the illumination region of the sample and the collection device. For example, the overlap of the two signals can be monitored during measurement to ensure that the components are aligned. Because the sample emission wavelength is typically different than the wavelength used to illuminate the sample and the wavelength used to provide the alignment signal, these two light sources should not substantially interfere with light emission/scattering measurements. Where the emission wavelengths are close to or the same as the illumination source or the alignment source, the wavelengths of either of the light sources can be altered to avoid any interference.

In certain embodiments, the optical fibers and optical fiber bundles described herein can be obtained commercially from numerous sources. The exact number of optical fibers in a particular bundle can be selected by the user depending on the desired measurements to be performed, the desired shape of the alignment signal and the like. Fiber pigtails can be produced, obtained or used to optically couple the optical fibers to a receiver, detector or other selected device.

In accordance with certain examples, the instrument configurations described herein may be controlled or used with, at least in part, a computer system. The computer systems may be, for example, general-purpose computers such as those based on Unix, Intel PENTIUM-type processor, Motorola PowerPC, Sun U1traSPARC, Hewlett-Packard PA-RISC processors, or any other type of processor. It should be appreciated that one or more of any type computer system may be used according to various embodiments of the technology. Further, the system may be located on a single computer or may be distributed among a plurality of computers attached by a communications network. A general-purpose computer system according to one embodiment may be configured to perform any of the described functions including but not limited to: data acquisition, -31 -movement of the optical elements or other components for alignment, data analysis and the like. It should be appreciated that the system may perform other functions, including network communication, and the technology is not limited to having any particular function or set of functions.

For example, various aspects may be implemented as specialized software executing in a general-purpose computer system. The computer system may include a processor connected to one or more memory devices, such as a disk drive, memory, or other device for storing data. The memory is typically used for storing programs and data during operation of the computer system. Components of computer system may be coupled by an interconnection mechanism, which may include one or more busses (e.g., between components that are integrated within a same machine) and/or a network (e.g., between components that reside on separate discrete machines). The interconnection mechanism enables communications (e.g., data, instructions) to be exchanged between system components. The computer system typically is electrically coupled to an interface on the system such that electrical signals may be provided from the system to the computer system for storage and/or processing.

The computer system may also include one or more input devices, for example, a keyboard, mouse, trackball, microphone, touch screen, and one or more output devices, for example, a printing device, status or other LEDs, display screen, speaker. In addition, computer system may contain one or more interfaces that connect computer system to a communication network (in addition or as an alternative to the interconnection mechanism). The storage system of the computer typically includes a computer readable and writeable nonvolatile recording medium in which signals are stored that define a program to be executed by the processor or information stored on or in the medium to be processed by the program. For example, the sampling rates and times may be stored on the medium. The medium may, for example, be a disk or flash memory. Typically, in operation, the processor causes data to be read from the nonvolatile recording medium into another memory that allows for faster access to the information by the processor than does the medium. This memory is typically a volatile, random access memory such as a dynamic random access memory (DRAM) or static memory (SRAM). It may be located in storage system, as shown, or in memory system. The processor generally manipulates the data within the integrated circuit memory and then copies the data to the medium after processing is completed. A variety of mechanisms are known for managing data movement between the medium and the integrated circuit memory element, and the technology is not limited thereto. The technology is not limited to a particular memory system or storage system.

The computer system may also include specially-programmed, special-purpose hardware, for example, an application-specific integrated circuit (ASIC). Aspects of the technology may be implemented in software, hardware or firmware, or any combination thereof.

Further, such methods, acts, systems, system elements and components thereof may be implemented as part of the computer system described above or as an independent component.

In some examples, the computer system may be a general-purpose computer system that is programmable using a high-level computer programming language. The computer system may be also implemented using specially programmed, special purpose hardware.

In the computer system, the processor is typically a commercially available processor such as the well-known Pentium class processor available from the Intel Corporation.

Many other processors are available. Such a processor usually executes an operating system which may be, for example, the Windows 95, Windows 98, Windows NT, Windows 2000 (Windows ME), Windows XP or Windows Vista operating systems available from the Microsoft Corporation, MAC OS System X operating system available from Apple Computer, the Solaris operating system available from Sun Microsystems, or UNIX or Linux operating systems available from various sources. Many other operating systems may be used. In addition or alternative to a processor, the computer system may include a controller such as for example and 8-bit or 16-bit controller. Other controllers such as 32-bit or higher controller may also be used in place of a processor or in addition to the processor of the computer system.

The processor and operating system together define a computer platform for which application programs in high-level programming languages can be written. It should be understood that the technology is not limited to a particular computer system platform, processor, operating system, or network. Also, it should be apparent to those skilled in the art that the present technology is not limited to a specific programming language or computer system. Further, it should be appreciated that other appropriate programming languages and other appropriate computer systems could also be used.

In certain examples, the hardware or software is configured to implement cognitive architecture, neural networks or other suitable implementations. For example, desired emission or scattering wavelengths may be stored in the system and used where a desired assay or measurement is to be performed. Such a configuration permits recall of known parameters for use in successive measurements, which can simplify the functionality and increase the overall ease of use by an end user.

One or more portions of the computer system may be distributed across one or more computer systems coupled to a communications network. These computer systems also may be general-purpose computer systems. For example, various aspects may be distributed among one or more computer systems configured to provide a service (e.g., servers) to one or more client computers, or to perform an overall task as part of a distributed system. For example, various aspects may be performed on a client-server or multi-tier system that includes components distributed among one or more server systems that perform various functions according to various embodiments. These components may be executable, intermediate (e.g., IL) or interpreted (e.g., Java) code which communicate over a communication network (e.g., the Internet) using a communication protocol (e.g., TCP/IP). It should also be appreciated that the technology is not limited to executing on any particular system or group of systems. Also, it should be appreciated that the technology is not limited to any particular distributed architecture, network, or communication protocol.

-34 -Various embodiments may be programmed using an object-oriented programming language, such as SmaliTalk, Basic, Java, C++, Ada, or C# (C-Sharp). Other object-oriented programming languages may also be used. Alternatively, functional, scripting, and/or logical programming languages may be used. Various aspects may be implemented in a non-programmed environment (e.g., documents created in HTML, XML or other format that, when viewed in a window of a browser program, render aspects of a graphical-user interface (GUI) or perform other functions). Various aspects may be implemented as programmed or non-programmed elements, or any combination thereof.

In certain examples, a user interface may be provided such that a user may enter desired parameters such as, for example, the sampling rate, excitation light wavelength, the emission or scattering wavelength, the number of sampling points, slit widths, the arrangement of alignment/collection fibers, and the like. Other features for inclusion in a user interface will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure. In some examples, the user interface may permit dragging and dropping to facilitate rapid alignment of the collection fiber optic. For example, one of the spots shown in FIGS hA and llB can be selected and dragged over the other spot.

The software can send a suitable signal to move the collection optical fiber, the alignment optical path, the illumination optical path, or other suitable components, until a desired spot overlap is achieved. In this manner, alignment of the collection device with the illumination region of the sample can be greatly simplified for the end user. In some examples, the alignment process may be blind, and the user need only press an alignment button or click an alignment box in the user interface to align the collection device and the illumination region of the sample.

In accordance with certain examples, the devices and systems disclosed herein may be used to detect light emission and/or scattering from many different types of assays.

Illustrative assays include, but are not limited to, fluorescent assays, solid phase assays, chemical reactions, binding assays, hybridization assays, enzymatic assays, clinical diagnostic assays, immunoassays including, but not limited to, ELISA assays, or may be used in analytical measurements and/or the study of electronic structures of molecules.

In some examples, the systems disclosed herein may include additional components such as, for example, an autoloader. The autoloader may be configured to load samples sequentially into and out of the system such that the system may perform measurements without user intervention or monitoring. The autoloader may comprise, for example, a robotic arm and/or motor that can securely grip the samples and load them into a desired position in the system. The system may include other electrical components such as operational amplifiers, gain control devices and the like. The system may include a bar code reader so that each sample may be encoded with a bar code and the measurements of each sample can be associated with its respective bar code. Additional components and features for including in the devices and systems disclosed herein will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure.

In certain embodiments, the configurations disclosed herein may be packaged in the form of a kit which includes the optical module or accessory and optionally includes instructions for using the module or accessory. In certain examples, the module of the kit may include a plurality of optical elements including, but not limited to, those described in reference to the alignment optical fiber.

In certain examples, a method comprising aligning a light emission signal received by a collection optical fiber with an alignment signal provided by an alignment optical fiber to align the collection optical fiber with a region of illumination of a sample is provided. In some examples, the method further comprises positioning the light emission signal within the alignment signal, as described herein.

In certain examples, the exact component or components of the optical device that is moved can be one or more of the collection optical fiber, an optical element in an optical path between the collection optical fiber and the sample, the light source, the sample position or other components of the system can be moved. Where one or more components is moved, a motor coupled to that component may be actuated to move the component a desired distance or to change the angle of that component with respect to the -36 -sample or other component in the system. In some examples, two or more components may be independently moved to facilitate alignment of the illumination region and the sampling region.

In certain embodiments, a method of aligning the sampling region of a collection optical fiber with an illumination region of a sample in a Raman spectroscopic device is described. In certain examples, the method comprises detecting an alignment signal from an alignment optical fiber, detecting an emission signal from the sample using a collection optical fiber, and aligning the sampling region and the illumination region by positioning the detected emission signal within the detected alignment signal.

In some examples, a sampling region can be selected to be the center position of the detected emission signal after the position of the detected emission signal is moved within the detected alignment signal. Such centering or centroiding increases the overall accuracy of obtaining data only from a sample region of interest. As described herein, the exact component or components of the optical device that is moved can be one or more of the collection optical fiber, an optical element in an optical path between the collection optical fiber and the sample, the light source, the sample position or other components of the system can be moved. Where one or more components is moved, a motor coupled to that component may be actuated to move the component a desired distance or to change the angle of that component with respect to the sample or other component in the system. In some examples, two or more components may be independently moved to facilitate alignment of the illumination region and the sampling region In certain examples, alignment can be facilitated by providing the devices and methods described herein. For example, to facilitate alignment an illumination region and a sampling region in an optical system configured to detect an emitted light signal, an alignment optical fiber can be provided that is configured to be optically coupled to the optical system to provide an alignment signal. Instructions to position the detected emitted light signal within the alignment signal to align the sampling region and the -37 -illumination region can also be provided. In certain instances, software that included a suitable program to implement the alignment process can also be provided.

In the arrangements described above an illumination signal is provided by an illumination source, and an alignment signal is projected onto a sample by an alignment fiber.

Components of the optical arrangement can then be controlled in order to align the collection fiber with the illumination signal, as guided by the position of the alignment signal(s) on the sample.

In an alternative arrangement it is possible to provide a system that is substantially self- aligned. This arrangement uses substantially the same components as shown in FIGS 1- 12, but the illumination signal is provided via the alignment fibers. Thus, referring to FIG. 5A, the illumination signal may be provided via the outer fibers 505, 510, 515 from where it can then be projected onto the sample. The illumination signals would be substantially aligned with the collection fiber 520 because the fibers already point at substantially the same region of the sample space. In fact, there would be a slight mis-alignment between the illumination signals and the collection fiber 520 because the collection fiber 520 would point at a central position, equally offset from the three illumination signals. Such an arrangement would be particularly useful for a spatially offset Raman collection system.

Equivalently, it would be possible to achieve the same effect by reversing the illumination fibers and the collection fiber from the above example. Continuing to refer to FIG. 5A, the illumination signal may be provided via the central fiber 520, and the collection fiber may be provided as a bundle with the outer fibers 505, 510, 515. Thus, the illumination signal would be projected onto the sample and each collection fiber 505, 510, 515 would be offset from the central illumination signal by an equal amount. This arrangement would provide a substantially "self-aligned" system because no alignment procedure would need to be followed. In fact, the system actually has a built-in mis-alignment which would be suitable for spatially offset Raman collection applications.

-38 -In one aspect, an optical device comprising an alignment optical fiber configured to provide an alignment signal, a receiver comprising a collection optical fiber and optically coupled to the alignment optical fiber, the collection optical fiber configured to receive an emitted light signal from a sample, and a detector optically coupled to the receiver and configured to detect the received emitted light signal and the alignment signal, in which the received emitted light signal is moved to position the received emitted light signal within the alignment signal to align the collection optical fiber with an illumination region of the sample is provided.

In certain embodiments, the alignment optical fiber can be configured as an optical fiber bundle. In some examples, the collection optical fiber can be a central optical fiber positioned within the optical fiber bundle. In certain examples, the receiver can be coupled to a motor configured to move the receiver to position the received emitted light signal within alignment signal. In other examples, the optical device can include an optical element in an optical path between the sample and the receiver, the optical element coupled to a motor configured to move the optical element to position the received emitted light signal within alignment signal. In some embodiments, the optical device can include a light source configured to provide light to illuminate the sample and optically coupled to the alignment optical fiber to provide light for the alignment signal.

In other examples, the optical device can include a first light source to provide light to illuminate the sample and a second light source optically coupled to the alignment optical fiber to provide light to the alignment optical fiber for the alignment signal. In certain examples, the first light source and the second light source can have the same wavelength.

In additional examples, the alignment signal comprises a ring of light and the emitted light signal is centrally positioned with the ring of light. In other examples, the optical device can include an optical steering unit configured to direct illumination light from a light source to the sample and to direct emitted light from the sample to the collection optical fiber. In some examples, the device can include a motor coupled to an optical element in the optical steering unit, the motor configured to move the optical element to direct the emitted light from the sample to the collection optical fiber and to position the received emitted light signal within the alignment signal.

-39 -In certain embodiments, the optical device can be configured as a Raman microscope. In other embodiments, the optical device can be configured as a Raman spectrometer. In certain examples, the detector can be a photomultiplier tube, a photodiode, a charged coupled device, lnGaAs array, a camera, or a CMOS camera. En other examples, the position of the received emitted light signal can be spatially offset from the alignment signal.

In another aspect, an optical system configured to measure emitted light from a sample, the system comprising a light source, an optical steering unit optically coupled to the light source and configured to direct light from the light source to a sample, a sample space configured to retain the sample and to receive light from the optical steering unit, a receiver comprising a collection optical fiber configured to receive an emitted light signal from the sample in the sample space, an alignment optical fiber optically coupled to the receiver and configured to provide an alignment signal, and a detector optically coupled to the collection optical fiber and the alignment optical fiber and configured to detect the received emitted light signal and the alignment signal, in which a position of the received emitted light signal is moved to position the received emitted light signal within the alignment signal to align the collection optical fiber with a region of the sample illuminated by the light source is described.

In certain embodiments, the light source further provides light to the alignment optical fiber to provide the alignment signal. In certain examples, the optical system further comprises an additional light source optically coupled to the alignment optical fiber, the additional light source configured to provide light for the alignment signal. In some examples, the alignment optical fiber is configured as an optical fiber bundle. In other examples, the collection optical fiber is a central optical fiber positioned within the alignment optical fiber bundle. In additional examples, the optical system can include a motor coupled to the receiver, the motor configured to move the receiver to position the received emitted light signal within the alignment signal. In other examples, the optical system can include an optical element in an optical path between the sample space and the receiver, the optical element coupled to a motor configured to move the optical element to position the received emitted light signal within the alignment signal. In certain embodiments, the optical system can include an optical steering unit in an optical -40 -path between the sample space and the receiver, the optical steering unit comprising a motor coupled to an optical element in the optical steering unit, the motor configured to move the optical element to position the received emitted light signal within the alignment signal. In other examples, the alignment signal comprises a ring of light and the emitted light signal is positioned centrally within the ring of light. In some examples, the centrally positioned emitted light signal is spatially offset a distance from its central position after the emitted light signal is centrally positioned.

In certain embodiments, the optical system is configured as a Raman microscope. In other embodiments, the optical system is configured as a Raman spectrometer. In additional embodiments, the detector can be a photomultiplier tube, a photodiode, and a charged coupled device, a camera or a CMOS camera.

In certain examples, the alignment optical fiber can be configured as an accessory module that is optically coupled to the optical system through one or more optical ports. In some examples, the alignment optical fiber is positioned within the housing of the optical system.

In an additional aspect, a method comprising aligning a light emission signal received by a collection optical fiber with an alignment signal provided by an alignment optical fiber by positioning the light emission signal within the alignment signal to align the collection optical fiber with a region of a sample illuminated by a light source is provided.

In certain embodiments, the method can include moving the collection optical fiber to position the light emission signal within the alignment signal. In some embodiments, the method can include moving the collection optical fiber by actuating a motor coupled to the collection optical fiber. In additional examples, the method can include moving an optical element in an optical path between the collection optical fiber and the sample to position the light emission signal within the alignment signal. In certain examples, the method can include moving the optical element by actuating a motor coupled to the optical element. In additional examples, the method can include moving a light source configured to provide light to the sample to position the light emission signal within the alignment signal. In other examples, the method can include moving the light source by actuating a motor coupled to the light source. In some examples, the method can include moving the sample that provides the emitted light signal. In additional examples, the -41 -method can include moving the sample by actuating a motor coupled to the light source.

In other examples, the method can include moving the collection optical fiber and a light source configured to provide light to the sample to position the light emission signal within the alignment signal.

In another aspect, a method of aligning a collection optical fiber with an illumination region of a sample in a Raman spectroscopic device, the method comprising detecting an alignment signal from an alignment optical fiber, detecting an emission signal from the sample using a collection optical fiber, and aligning a sampling region of the collection optical fiber with the illumination region by positioning the detected emission signal within the detected alignment signal is provided.

In certain embodiments, the method can include selecting a center position of the detected emission signal to be the sampling region after the position of the detected emission signal is moved within the detected alignment signal. In certain examples, the method can include moving a light source that provides light to the sample to align the sampling region and the illumination region. In other examples, the method can include actuating a motor coupled to the light source to move the light source. In additional examples, the method can include moving the collection optical fiber to align the sampling region and the illumination region. In some examples, the method can include actuating a motor coupled to the collection optical fiber to move the collection optical fiber. In certain examples, the method can include moving an optical element in an optical pathway between the collection optical fiber and the sample to align the sampling region and the illumination region. In additional examples, the method can include actuating a motor coupled to the optical element to move the optical element. In other examples, the method can include moving a position of the sample that provides the emitted light. In certain embodiments, the method can include actuating a motor coupled to the sample to move the sample.

In another aspect, a method of facilitating alignment of an illumination region and a sampling region in an optical system configured to detect an emitted light signal, the method comprising providing an alignment optical fiber configured to be optically coupled to the optical system and to provide an alignment signal, and providing -42 -instructions to position the detected emitted light signal within the alignment signal to align the sampling region and the illumination region is described.

When introducing elements of the examples disclosed herein, the articles "a," "an," the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including" and "having" are intended to be open-ended and mean that there may be additional elements other than the listed elements. It will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that various components of the examples may be interchanged or substituted with various components

in other examples.

Although certain aspects, examples and embodiments have been described above, it will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that additions, substitutions, modifications, and alterations of the disclosed illustrative aspects, examples and embodiments are possible.

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Claims

CLAIMS1. An optical system comprising: an illumination source capable of projecting an illumination signal onto a sample space; a collection fiber having an end that is configured to receive a signal emitted from the sample space; an alignment fiber having an end that is configured to project an alignment signal onto the sample space, wherein the end of the collection fiber and the end of the alignment fiber are positioned in a known configuration; a camera capable of detecting the position of a projected alignment signal and a projected illumination signal in the sample space; and an adjuster, capable of adjusting the signal position of at least one of the projected illumination signal and the projected alignment signal in the sample space, as detected by the camera, so that the projected illumination signal and the projected alignment signal can be positioned with a selected configuration in the sample space.
2. The optical system of claim 1 wherein there is a plurality of fibers in a group consisting of one of alignment fibers and collection fibers, and wherein the ends of the fibers are arranged in a known configuration.
3. The optical system of claim 1 or claim 2 wherein there is a plurality of alignment fibers, the ends of which are configured in a known geometric configuration with respect to the end of the collection fiber.
4. The optical system of any of the preceding claims, in which the alignment optical fiber is configured as an optical fiber bundle.
5. The optical system of claim 4, in which the collection optical fiber is a central optical fiber positioned within the optical fiber bundle.
6. The optical system of any of the preceding claims wherein there is a plurality of collection fibers, the ends of which are configured in a known configuration with respect to the ends of one or more alignment fibers.
7. The optical system of any of the preceding claims wherein the adjuster comprises an optical element positioned in the path of one of the illumination signal and the alignment signal.
8. The optical system of any of the preceding claims wherein the adjuster is capable of adjusting the position of the end of the alignment fiber.
9. The optical system of any of the preceding claims wherein the illumination source is coupled to the alignment fiber so that the illumination source provides the illumination signal and the alignment signal.
10. The optical system of any of claims 1 to 8 further comprising an alignment source coupled to the alignment fiber for providing the alignment signal.
11. The optical system of any of the preceding claims further comprising a processor configured to: receive the detected position of the projected alignment signal and the projected illumination signal from the camera and calculate an adjustment to the position of at least one of the signals in order to position the signals in a selected configuration, wherein the selected configuration of signals in the sample space is related to the known configuration of the ends of the alignment fiber and the collection fiber.
12. The optical system of any of the preceding claims further comprising a detector coupled to the collection fiber capable of receiving and analyzing a signal emitted from the sample space. -45 -
13. The optical system of claim 12 in which the detector is an imaging spectrometer comprising at least one of a photomultiplier tube, a photodiode, a charged coupled device, a camera, and a CMOS camera.
14. A method for configuring an optical system comprising the steps of: projecting an illumination signal onto a sample space; configuring an end of a collection fiber to receive a signal emitted from the sample space; projecting an alignment signal onto the sample space from an end of an alignment fiber that is positioned in a known configuration with the end of the collection fiber; detecting the position of a projected alignment signal and a projected illumination signal in the sample space; and adjusting the position of at least one of the projected illumination signal and the projected alignment signal in the sample space, as detected, so that the projected illumination signal and the projected alignment signal can be positioned with a selected configuration in the sample space.
15. An optical system comprising: an illumination fiber having an end that is configured to project an illumination signal onto a sample space; and a collection fiber having an end that is configured to receive a signal emitted from the sample space in the vicinity of the projected illumination signal, wherein the end of the collection fiber and the end of the illumination fiber are positioned adjacent one another and with a known offset from one another so that the collection fiber is aligned with a point in the sample space that is substantially adjacent the projected illumination signal.
16. The optical system of claim 15, in which one of the illumination optical fiber and the collection optical fiber is configured as an optical fiber bundle, and the other is a central optical fiber positioned within the bundle.

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17. A method for configuring an optical system comprising the steps of: projecting an illumination signal onto a sample space from an end of an illumination optical fiber; and receiving a signal emitted from the sample space in the vicinity of the projected illumination signal with an end of a collection optical fiber, wherein the end of the collection fiber and the end of the illumination fiber are positioned adjacent one another and with a known offset from one another so that the collection fiber is aligned with a point in the sample space that is substantially adjacent the projected illumination signal.
18. An apparatus substantially as herein described with reference to and/or as illustrated in the accompanying drawings.
19. A method substantially as herein described with reference to the accompanying drawings. -47 -