Patent Application
20120156714
This invention relates generally to an integrated cytometric sensor system and method. In particular the invention relates to a highly integrated and low cost electronic optical sensor device, methods and algorithms for enumeration of microscopic cells, particles or molecules distributed in a sample. The invention also relates to a wide dynamic range optical sensor for use in a flow cytometer.
Flow cytometry is a powerful method of analysis to determine the cellular/biological content of various types of samples, and in particular samples that contain living cells. In clinical applications, flow cytometers are useful for myriad of applications including lymphocyte counting and classification, for immunological characterization of leukaemias and lymphomas, and for cross-matching tissues for transplants.
In most flow cytometry techniques, cells in a fluid solution are caused to flow individually through a light beam, usually produced by a laser light source or a monochromatically filtered LED source. As light strikes each cell, the light is scattered and the resulting scattered light is analyzed to determine the type of cell. The cell may also optionally be labelled with a marker linked to a fluorescent molecule, which fluoresces when light strikes it and thereby reveals the presence of the marker on the cell. In this fashion, information about the surface components of the cell can be obtained. Examples of such fluorescent molecules include FITC (fluorescein isothiocyanate), TRITC (tetramethyl rhodamine isothiocyanate), Texas Red (sulforhodamine 101), and PE (phycoerythrin). Intracellular components of the cell, such as nucleic acids, may be stained, and subsequently detected by fluorescence. Examples of such compounds include ethidium bromide, propidium iodide, YOYO-1, YOYO-3, TOTO-1, TOTO-3, BO-PRO-1, YO-PRO-1, and TO-PRO-1. In addition, smaller molecules or proteins in the plasma may be detected using stained microbeads or quantum dots.
Light scattering measurements are widely used in flow cytometry to measure cell sizes and to distinguish among several different types of cells. It is known that incident light is scattered by cells at small angles (approximately 0.5-20 degrees) from the axis of the incident light that interrogates the cells, and that the intensity of the scattered light is proportional to the cell volume. The light scattered at small angles is referred to as forward scattered light. Forward scattered light is useful in determining cell size, thus aiding in distinguishing which cell type is being detected.
The ability to measure cell sizes is influenced by the wavelength employed and the precise range of angles over which light is collected. For example, material within the cells having a strong absorption at the illuminating wavelength may interfere with the size determination because cells containing this material produce smaller forward scatter signals than would otherwise be expected, leading to underestimates of cell size. In addition, differences in refractive index between the cells and the surrounding medium may also influence the small-angle scatter measurements.
Different cell types may be further distinguished on the basis of the amount of orthogonal light scatter (or right angle side scatter) they produce. Cells having a high degree of granularity, such as blood granulocytes, scatter incident light at high angles much more than cells with low granularity, such as lymphocytes. As a result, forward and side scatter measurements are commonly used to distinguish among different types of blood cells, such as red blood cells, lymphocytes, monocytes, and granulocytes.
Much prior art exists for cytometer instruments including improvements to the light source, see for example WO2006104699, assigned to Beckman Coulter Inc., and WO0129538, assigned to Beckton Dickinson. Methods to reduce instrument cost, as disclosed in US 2007117158, Coumans et al. Methods to provide increases in multi-parametric detection EP 0737855, Beckton Dickinson, and methods to enable a portable cytometer instrument are disclosed by US2003/0142291, assigned to Honeywell International Inc., and WO2007103969 and US2007127863, assigned to Accuri Instruments Inc.
To obtain meaningful information about the numbers and types of cells in the sample or of the concentration of markers on cell surfaces, the samples must be calibrated with respect to the amount of light scatter or fluorescence associated with standardized populations of the cells. Calibration of the instrument is typically accomplished by passing standard particles through the instrument, and measuring the resulting scatter or fluorescence typically using synthetic standard materials (e.g., polystyrene microbeads). These microbeads are made to be extremely uniform in size, and to contain precise amounts of fluorescent molecules to serve in calibrating the photodetectors used in detection of fluorescent probes. U.S. Pat. No. 5,380,663 (A), assigned to Caribbean Microparticles Inc., discloses one such method for calibrating a flow cytometer, which employs combined populations of fluorescent microbeads and a software program matched to each combined population of microbeads.
A significant amount of training is required to learn how to use these instruments properly, and these instrument systems can require service several times a year. As a result, the high costs associated with purchasing, using and maintaining these high-end instruments make them financially inaccessible to small clinics and doctor's offices. Physicians can get access to these instruments by utilizing commercial laboratories that will collect the blood samples from the clinic or physician's office and transport the samples back to the laboratory for analysis on their high end haematology systems, which are typically based on flow cytometer techniques. The results are then sent to the doctor. This process is very time-consuming, and more often than not the doctor will not have the results until the next day, at the least, or up to a week, typically.
Many haematology analyzers now incorporate flow cytometric techniques and other advances, which make the instrument relatively compact and more efficient through incorporation of semiconductor microelectronics where applicable; however, in the end these systems are functionally based on the Coulter instruments used by clinical laboratories in the late 1970's and early 1980's. While the low-end cytometer based haematology analyzers have reduced in price to $30,000-$50,000, they are still beyond the budget of many small-to-medium sized hospital laboratories and physician offices, lack the flexibility and wide application support that the higher-end cytometers possess and still come with high maintenance and support costs.
Photomultiplier Tubes (PMT's) have been the incumbent photodetector of choice, within flow cytometers, for reasons of high dynamic range and sensitivity. However, a number of problems exist in that these PMT's are expensive ($1,000 per channel), require very high voltage operation (>1,000 V), which leads to large and expensive power management electronics, and require high maintenance.
Recent advances in solid-state photodiode detectors have started to replace the forward scatter PMT. PCT patent publication number WO 01 94938, assigned to Idexx Lab Inc., describes a lensless cytometer whereby a photodiode is used to collect forward scatter. No optical filters are in the light path between the flow system and photodiode. Hence, light at all wavelengths is detected by the photodetector, even fluorescence. US2007097364 and US2007207536 describe a cytometer and fluorescent biosensor, respectively, using photodiode arrays.
In addition, recent advances in avalanche- and Geiger-mode photodiode detectors (referred to as Silicon Photomultipliers, or SiPM detectors for short) are showing much promise in replacing the side scatter and fluorescence PMT's which require much more sensitivity and dynamic range, and therefore specificity, than the forward scatter PMT's. The main advantages are their much lower cost (<$50) and size, lower voltage operation (<50 V) and higher photon detection efficiency. However, they still suffer from low dynamic range, leading to lower sensitivity and specificity compared with the PMT, and large dichroic filters and beam shaping optics are still needed. US 2006250604 (A1) describes one such cytometer based haematology analyzer, which uses avalanche photodiodes.
The use of dichroic filtering as a method to discriminate light across the UV, Visible and IR spectra is well known. JP 58044406 and U.S. Pat. No. 5,341,238 describe dichroic filtering techniques on glass and oxide semiconductor surfaces, respectively. SiPM's, as with PMT's, cannot discriminate different wavelengths of light. So the large and costly dichroic filters ($300-500 each) remain.
US patent publication number US 2007145236, Kiesel et al., discloses an integrated circuit (IC) that includes a photosensor array, some cells of which are reference cells that photosense throughout an application's energy range, while other cells of which are subrange cells that photosense within respective subranges. The subrange cells can receive photons in their respective subranges from a transmission structure that has laterally varying properties, such as due to varying optical thickness. The reference cells may be uncoated or may also receive photons through a transmission structure such as a gray filter. Subrange cells and reference cells may be paired in adjacent lines across the array, such as rows. Where photon emanation can vary along a path, quantities of incident photons photosensed by subrange cells along the path can be adjusted based on quantities photosensed by their paired reference cells, such as with normalization. However a problem with this sensor is that it is not suitable for cytometric systems as the sensing of the radiation emitted is performed along a path whereas cytometers detects the radiation emitted from a target point.
US Patent publication number US2002/154315, Myrick, discloses an optical filter systems and optical transmission systems, an optical filter compresses data into and/or derives data from a light signal. The filter way weight an incident light signal by wavelength over a predetermined wavelength range according to a predetermined function so that the filter performs the dot product of the light signal and the function. The approach outline by Myrick is akin to a CCD camera, with many pixels. However this filter is not suitable for use in a cytometric system.
US Patent publication number US2007/084990, Coates, discloses an integrated spectral sensing engine featuring energy sources and detectors within a single package that includes sample interfacing optics and acquisition and processing electronics. The miniaturized sensor is optimized for specific laboratory and field-based measurements by integration into a handheld format. Design and fabrication components support high volume manufacturing. Spectral selectivity is provided by either continuous variable optical filters or filter matrix devices. Coates relates to a Spectrophotometer or Spectrometer, which are specific to measuring fluids or solutions which is not suitable for cytometer applications.
In summary, an objective of the present invention is to provide a highly sensitive, low-cost optical sensor with integrated optical filtering and high dynamic range, to enable low-cost, portable and easily maintainable flow cytometer to overcome the above mentioned problems.
According to the invention there is provided, as set out in the appended claims, a flow cytometric system comprising a first sensor positioned axially to a light source; a channel comprising means for receiving a sample target and interposed between said first sensor and light source; and a second sensor placed at an angle to said first sensor adapted to sense side scattering and/or fluorescent components and said first sensor is adapted to sense a forward scattering component in response to light illuminating the sample target in said channel.
The integrated cytometric system of the present invention provides integrated optical filtering and high dynamic range, to enable low-cost, optically efficient, miniaturised, portable and easily maintainable flow cytometer. Incumbent PMT arrangements require numerous dichroic mirrors and filters to split the wanted wavelengths for detection by separate PMT's, resulting in long and inefficient optical signal paths. The numerous optical components of such systems, each generate optical noise, reduce optical transmission, and any misalignment due to mechanical movement all contribute to sub-optimal optical capture. The integrated cytometric sensor arrangement allows for its placement adjacent the detection zone, and its parallel optical detection paths, each requiring a single filter, provides for a more efficient optical capture arrangement.
Any misalignment of the incumbent PMT optics, due to mechanical movement from transportation or operation, further reduces its optical capture efficiency. The integrated cytometric sensor offers alignment-free optics, as both the filter and detector arrays are integrated into a single package, and each parallel optical path requires only one filter, thus maximising optical transmission, minimising optical noise and removing any mis-alignment errors due to mechanical movement.
With its small size, additional integrated cytometric sensors can be placed about the detection zone to either increase optical gain by capturing more scattered and fluorescent light. Since the integrated cytometric sensor requires low-voltage operation (−30 V) compared with high-voltage PMT based systems (−1,000 V), the electronic design and power consumption of the resulting diagnostic instrument is much advanced.
In one embodiment the first or second sensor comprises a plurality of photodiode pixels;
In one embodiment the second sensor is positioned orthogonal to said first sensor.
In one embodiment a third sensor can be placed at an angle to said first or second sensor. The third sensor can be placed orthogonal to said first sensor and directly opposite said second sensor to sense side scattering and fluorescent components at 180 degrees to scattering and fluorescent components incident on said second sensor to provide further information.
In one embodiment at least one or more of said photodiode pixels are voltage biased in one or more of the following modes: normal, avalanche or Geiger modes, for wide dynamic sensor range operation. By altering the reverse bias voltage, thus putting each photodiode into one of normal, avalanche or Geiger mode, the dynamic range of incident scattering and fluorescent power to which the filter cell array is sensitive to is greatly increased, thus significantly increasing the resultant instrument sensitivity and specificity.
The invention significantly advances the art to develop such a highly integrated cytometric sensor, by using arrays of SiPM's, with each SiPM element having monolithically integrated optical filters that are separately configured in normal, avalanche and Geiger modes to overcome issues of low dynamic range, sensitivity and specificity.
In one embodiment at least one filter cell comprises a metal-dielectric based arrangement. The metal-dielectric filter comprises integrated metal wires, separated by insulating dielectric layers, arranged in metal grids to form Fabry-Perot cavities.
In one embodiment at least one filter cell comprises a thin-film based arrangement. The thin film layer may comprise areas of different dielectric constants allowing filter cells with different defined filter characteristics across the sensor.
In one embodiment the defined filter characteristics are provided by an array of different dichroic filter materials, with a distribution of different dielectric constants.
The invention embeds a plurality of either patterned thin film filters, or metal-dielectric filters, or both, deposited on a photodiode pixel array, each pixel biased in either, photon counting, normal, linear avalanche or Geiger mode, in an embodiment that integrates multiple dichroic filters and photodetectors (photodiodes) into one monolithic solid state sensor with high photon sensitivity and dynamic range, thus advancing the miniaturisation, operation and cost reduction of analytical instruments.
In one embodiment the invention provides a transparent window cap comprising additional thin film filters to compliment the filter characteristics monolithically deposited on the sensor.
In one embodiment the filter cells are adapted to detect different wavelengths of light when light is incident on said sensor, said different wavelengths are dependent on the scattering and fluorescent signal components representative of specific biological targets.
In one embodiment the set filter characteristics comprises one or more of the following filters: band-pass, high-pass (long-pass), low-pass (short-pass), out-of-band and/or band-stop filters.
In one embodiment a first sensor is positioned axially to an incident light source generated from either a laser beam or monochromatically filtered LED, to sense forward scattering components. A second sensor can be placed orthogonal to said first sensor to sense side scattering and fluorescent components to provide further information on the biological target.
In one embodiment a second sensor is placed at an angle to said first sensor to sense side scattering and fluorescent components to provide further information.
In one embodiment a beam stop is positioned between the forward scatter sensor and light source, to minimise the saturating effects within the sensor from the optically high-power laser incident directly on to the sensor.
This sensor of the present invention incorporates multiple optical filters, with different pass/stop-band characteristics across visible and near-infrared spectra, monolithically integrated on an array of photodiodes.
The invention relates to the integration of multiple optical filters and photodiodes (currently separate components in analytic instruments) into a highly integrated solid-state cytometric sensor; and embodiments into analytical instruments such as flow cytometers, scanning cytometers or fluorimeters which significantly simplifies, miniaturises and lowers their operation and cost.
The invention also provides for the use of these cytometric sensors in a typical cytometer instrument, and the data signal path from sensor output through to instrument display.
In one embodiment at least one filter cell output is post-processed with fluorescent biomarker compensation algorithms by conditioning the filter output by predetermined fractions to compensate for fluorescent interference from other biomarker profiles.
In one embodiment, the filter cell outputs are amplified and over-sampled into the digital domain for post-processing and later interpretation for user-readable display. Such post-processing algorithms may include calibration algorithms, gain optimising algorithms or signal conditioning algorithms. These post-processing algorithms may be implemented in a digital signal processor, hardwired into an ASIC, or integrated into the filter cell ASIC, using CMOS integration techniques.
In a further embodiment there is provided an integrated sensor, comprising:
In another embodiment there is provided a method of detecting different wavelengths representative of specific biological targets in a flow cytometric system comprising the steps of:
In another embodiment there is provided a cytometric system comprising a first sensor positioned axially to a light source; a channel comprising means for receiving a sample target and interposed between said first sensor and light source; and a second sensor placed at an angle to said first sensor adapted to sense side scattering and/or fluorescent components and said first sensor is adapted to sense a forward scattering component in response to light illuminating the sample target in said channel.
In a further embodiment there is provided a wide dynamic range optical sensor, for use in a flow cytometric system, comprising:
In one embodiment the wide dynamic range sensor comprises a plurality of optical filters positioned on top of said photodiode pixels.
In one embodiment each optical filter comprises a set filter characteristic and cooperates with one or more of said plurality of photodiode pixels to define a filter cell.
In one embodiment the wide dynamic range sensor comprises a transparent window cap comprising additional thin film filters.
In another embodiment of the present invention there is provided a method of analysing a sample target in a flow cytometric system comprising the steps of:
In one embodiment a plurality of axially and orthogonally placed sensors may be placed along the sample path for each incident light source generated by one laser beam split into a plurality of light sources.
In one embodiment a plurality of axially and orthogonally placed sensors may be placed along the sample path for each incident light source generated by a plurality of lasers.
There is also provided a computer program comprising program instructions for causing a computer program to carry out the above method and control the sensor according to the invention which may be embodied on a recording medium, carrier signal or read-only memory.
The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:
When a photodiode is reverse biased, a photon incident onto its pn (or PiN) junction region creates an electron-hole pair upon impact ionisation. The reverse bias drives this electron to the anode and, as long as the bias voltage is relatively low, does not further amplify within the photodiode, thus creating a current collected at the anode that is proportional to the number of incident photons. This operation is termed normal reverse bias mode. Once the photodiode reverse bias is increased towards, but remains lower than, the reverse breakdown voltage, electron-hole pairs created by incident photons create a cascade of further electron-hole pairs, thus amplifying the effect of the first electron-hole pair generated by the incident photon. This describes the avalanche mode of operation. This cascade effect is not self sustaining but still results in moderate amplification with current gains typically in the 101-103 range. This gain is higher the closer the reverse bias voltage approaches the reverse breakdown voltage. In Geiger mode, the photodiode is biased beyond its reverse breakdown voltage so that the incident photons create a rapid cascade of electron-hole pairs by impact ionisation. The high reverse-bias voltage sustains and amplifies the cascade. In this mode, the photodiode exhibits very high gain (typically >105) and is highly sensitive to single photons but can only quantify low incident photon numbers since the high gain quickly saturates to the voltage rails. When in Geiger mode the SPM can be also be configured as a photon counter (photon counting mode). For very low levels of light, when the frequency of photons hitting the sensor is so low that the current pulse created by an incident photon is extinguished before the next incident photon arrives, the SPM is seen to be able to detect individual photons. These pulses are counted using the signal acquisition electronics (such as a comparator connected to a digital counting circuit) to create a photon counter.
Once the output current pulse is generated, quenching circuits may be used to quickly bring the photodiode out of these modes, especially avalanche and Geiger modes, and back to the reversed biased state prior to the onset of the cascading effects from incident photons. The quicker the cascade can be ‘quenched’ the more incident photon events can be sensed or counted. Passive quenching circuits typically use capacitors and resistors to limit the current flowing through the photodiode in avalanche and Geiger modes thus terminating the cascading effects. Active quenching circuits typically sense the increase in current and reduce the bias voltage across the photodiode bringing it out of the cascade.
By altering the reverse bias voltage, thus putting each filter cell into one of normal, avalanche or Geiger mode, the dynamic range of incident scattering and fluorescent power, to which the filter cell array is sensitive to, is greatly increased. For example, if two filter cells have the same filter characteristic, but one is in avalanche mode and the other is in Geiger mode, then the former can quantify high numbers of incident fluorescence/scattering photons without saturating, whereas the latter is sensitive to low numbers of incident fluorescence/scattering photons, even down to single photon detection.
It will be appreciated that the following sensor description shows the filter cells all integrated onto one semiconductor die. Equally, the cytometer apparatus later described may use a cytometric sensor with separate die, having one or more filter cells, mounted into one package.
The second independent filtering component comprises a plurality of thin film layers 15 monolithically built atop the die and metal interconnect layers, with each thin film layer having different dielectric constants. Each single thin film layer may comprise areas of different dielectric constants 16, allowing filter cells with different filter characteristics across the sensor die. This thin-film based filter component is described in greater detail in
The sensor design interfaces through IO pads 17 connected to the substrate carrier via wirebonds 18. Other methods of mounting the die that do not obscure the photodiodes may be used. The sensor die can be protected with a package 19 that has a transparent window cap 20. This window cap may either be transparent or may have deposited thin film filtering layers 21 to compliment the filter characteristics monolithically deposited on the sensor die. In this way the light 22 incident on the package can be filtered by a combination of the window cap and monolithic filters (thin films and/or metal-dielectric) before detection by the photodiode pixels.
When the photodiode detectors of the filter cells are biased in high-gain avalanche and Geiger modes, thermal or shot noise creates false output pulses known as dark current pulses. These dark current interferer pulses may be filtered using decision-driven gating methods. For example, no filter cell outputs due to scattering or fluorescent light are generated unless the biological targets intersect the laser beam. Typically, no fluorescence signals are produced unless light scattering is produced. Therefore, any dark current pulses may be filtered out when no light scattering is detected.
The need for fluorescent biomarker compensation is illustrated in
The fluorescent light components are emitted at a plurality of wavelengths defined by the properties of the fluorescent markers which may have been mixed with the biological targets. The emitted light component axial to the incident laser beam 57, denoted as the forward scattering component, is focused by the forward lens 58 on to the forward cytometric sensor 59. Other embodiments may not use this forward lens. A beam stop 60 may be placed axially between the detection zone and the cytometric sensor, in front of the forward lens, if used, to avoid the laser beam damaging the forward scatter sensor when the biological targets are not interposed. Extra diagnostic information is collected by interrogating side scattering and fluorescent components 61 at an angle, Φ, 62 to the axial and an angle, Ψ, 63 to the fluidic channel. Φ and Ψ are normally 90° each.
This emission component is focused by the side lens 64 on to the side cytometric sensor 65. Other embodiments may not use this side lens. Both forward and side cytometric sensors interface to an ASIC 66 and DSP 67, whose operation is previously described with respect to
ASIC or microelectronic circuit assembly 66 and DSP 67 take the output signals from each SiPMs and the bias information from every SiPMs for each measurement and combine them to create a single wide dynamic range measurement. The dynamic range of the combined measurement is significantly larger than the individual dynamic ranges of the SiPMs operating at a single bias condition.
It will be appreciated that the invention anticipates that any combination of SiPM bias modes is suitable, photon counting, normal, avalanche or Geiger. Additionally, one or more sensors which are biased in any one of these modes, but with different bias voltages within that mode of operation may also be utilised.
It is envisaged that the embodiment described with respect to
It will be appreciated that the invention can be incorporated in other similar analytical instruments. This invention enables the various embodiments of such instruments presented herein. Such instruments include, for example, and without limitation, immunoassay analyzers, clinical haematology analyzers, flow and scanning cytometers, fluorimeters, and chemistry analyzers. The specific biological targets can be in a fluid sample or a non-fluidic sample, so long as the sample can be subjected to a light source and detection of different wavelengths can be achieved.
It will be appreciated that the invention illustrates a single laser source in
The embodiments in the invention described with reference to the drawings comprise a computer apparatus and/or processes performed in a computer apparatus. However, the invention also extends to computer programs, particularly computer programs stored on or in a carrier adapted to bring the invention into practice and control operation of the cytometric sensor. The program may be in the form of source code, object code, or a code intermediate source and object code, such as in partially compiled form or in any other form suitable for use in the implementation of the method according to the invention. The carrier may comprise a storage medium such as ROM, e.g. CD ROM, or magnetic recording medium, e.g. a floppy disk or hard disk. The carrier may be an electrical or optical signal which may be transmitted via an electrical or an optical cable or by radio or other means.
The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09169552.8 | Sep 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/062963 | 9/3/2010 | WO | 00 | 3/2/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61240009 | Sep 2009 | US |
