DEVICES AND METHODS FOR DIRECT-SAMPLING ANALOG TIME-RESOLVED DETECTION

Abstract

Devices and methods for sampling an analog signal to perform data analysis are disclosed. The sampling devices and corresponding methods include a detector module that measures a response generated from a sample, an analog to digital converter that samples the analog signal, received from the detector module, and converting it into a digital signal, a sampling rate of the converter being faster than the response of the sample; and a logic circuit coupled to the converter that processes the digital signal to generate a reduced digital data signal, the logic circuit processing the digital signal acquired from the converter to generate a continuous data transfer to a processing system.

Claims

1. A sampling device for measuring an analog signal from a detector comprising: a light detector module for measuring a response generated from a biological sample in response to illuminating light from a light source and generating an analog signal; an analog to digital converter to receive the analog signal from the detector module and convert it into a digital signal; a logic circuit coupled to the analog to digital converter that processes the digital signal; and wherein the logic circuit that processes the digital signal continuously transfers the processed digital signal to a data storage device.

2. The sampling device of claim 1, further comprising a memory device coupled to the logic circuit for buffering processed digital signals from the logic circuit, the memory device further including a data processor.

3. The sampling device of claim 2, further comprising an interface coupled to the memory device for streaming the processed digital signals to an external display device.

4. The sampling device of claim 1, further comprising a reference clock coupled to the analog to digital converter for generating a sampling clock signal.

5. The sampling device of claim 1, further comprising a data clock coupled to the logic circuit for providing a reference clock pulse for processing of the digital signal.

6. The sampling device of claim 1, further comprising a reference clock coupled to the logic circuit for providing a reference clock pulse for processing of the digital signal.

7. The sampling device of claim 1, wherein the biological sample is selected from the group consisting of a tissue sample, a cell sample, and one or more biological molecules.

8. The sampling device of claim 1, wherein the detector module is selected from the group consisting of a photomultiplier tube, multianode photomultiplier tubes, a hybrid photomultiplier tube, hybrid photomultiplier tube arrays, a photomultiplier tube with multilevel discrimination, an avalanche photo diode, avalanche photo diode arrays, a photodiode, a CCD, and a CMOS.

9. The sampling device of claim 1, wherein the analog to digital converter has a sampling rate of at least 1 MS/s.

10. The sampling device of claim 1, wherein the logic circuit includes at least one of a field-programmable gate array, an application specific integrated circuit, a discrete integrated circuit, or a dedicated integrated circuit.

11. The sampling device of claim 1, wherein the logic circuit is configured to perform at least one of: a Fourier transform function, a Fast Fourier transform function, a X function, a Y function, a Z function, a threshold function, or an averaging function.

12. The sampling device of claim 11, wherein the logic circuit is configured to generate a plurality of different components to characterize the biological sample.

13. The sampling device of claim 12, wherein the components include at least one of coefficients of the Fourier transform, multiple harmonic components of the analog signal, or a folding average.

14. The sampling device of claim 1, wherein the processed digital signals are used for at least one of FRET, fluorescence anisotropy, photon migration, fluorescence lifetime, nonlinear spectroscopy, CARS, time-resolved CARS, cellular imaging, tissue imaging, diagnostic applications, flow cytometry, image cytometry, gel readers, FCS, time-resolved FCS, PCH-FIDA, ultrasound, impedance measurements, plate readers, capillary electrophoresis, or imaging treatment.

15. The sampling device of claim 1, wherein the light detector module comprises a parallel data channel which is configured to detect a plurality of analog signals.

16. The sampling device of claim 15, wherein the analog signals correspond to different wavelengths of a spectral response of the sample.

17. The sampling device of claim 1, wherein the processed digital signal discriminates between a plurality of photon events from the response and noise events from the detector.

18. The sampling device of claim 1, wherein the processing is further analyzed to segment the response into discrete components.

19. A sampling device for performing data analysis for photon migration comprising: a light source to emit illuminating light; a photomultiplier tube that detects a response from a tissue sample to the illuminating light and generates an analog signal; an analog to digital converter for sampling the analog signal received from the photomultiplier tube and converting the analog signal into a digital signal; a logic circuit coupled to the analog to digital converter for processing the digital signal received from the analog to digital converter; wherein the sampling rate of the converter is faster than the time of flight or phase delay of the response of the sample to the illuminating light, and wherein the logic circuit is configured to process the digital signal continuously acquired from the analog to digital converter such that data analysis is obtained.

20. The sampling device of claim 19, further comprising a computer and wherein the processing includes performing at least one of a Fourier transform function, a Fast Fourier transform function, an X function, a Y function, a Z function, a threshold function, or an averaging function.

21. A method for processing an analog signal comprising: positioning a biological sample relative to a light source; subjecting the biological sample to illuminating light to generate a response; detecting the response of the biological sample with a detector module to generate an analog signal of the response generated from the sample; sampling the analog signal received from the detector module, and converting the analog signal into a digital signal with an analog to digital converter, wherein the sampling rate is faster than the response of the sample to the illuminating light; processing the digital signal continuously acquired from the analog to digital converter using a logic circuit; and further processing the processed digital signal with a computer connected to the logic circuit.

22. The method of claim 21, further comprising the step of segmenting the digital signal into discrete components.

23. The method of claim 21, further comprising continuously streaming the discrete components to an external display device

24. The method of claim 21 wherein the analog to digital converter comprises a gigahertz digitizer and wherein the method further comprises transferring data from the gigahertz digitizer to a computer with a first in first out (FIFO) buffer and further processing the processed digital signals with a computer.

25. The method of claim 21 wherein the step of detecting the response of the biological sample with the detector module comprises detecting light with a photomultiplier tube.

26. The method of claim 21 further comprising clocking the rate of the analog to digital converter with a reference signal from the light source operated at a pulse rate.

27. The method of claim 21 further comprising detecting a fluorescence lifetime signal from the biological sample with the detector module.

28. The method of claim 21 wherein processing the digital signal with the logic circuit comprises processing the digital signals with a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

29. The method of claim 21 wherein detecting the response comprises detecting a first light signal with a first photomultiplier tube and a second light signal with a second photomultiplier tube.

30. The method of claim 21 wherein subjecting the biological sample to illuminating light includes illuminating the biological sample with a pulsed light source or a modulated light source.

31. The method of claim 21 wherein sampling the analog signal is performed at a rate of 1.6 GHz.

32. The method of claim 21 further comprising displaying fluorescence lifetime data for the biological sample on a display.

33. The device of claim 19 further comprising a computer connected to the logic circuit, the computer being connected to a display.

34. The device of claim 19 further comprising a reference signal to generate a clock signal to clock the analog to digital converter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] So that those skilled in the art to which the subject invention appertains will readily understand how to make and use the method and device of the subject invention without undue experimentation, preferred embodiments thereof will be described in detail herein below with reference to certain figures, wherein:

[0028] FIG. 1A illustrates the decay curve of fluorescence lifetime in TCSPC;

[0029] FIG. 1B illustrates the time of arrival of successive trials when a sample is excited with a pulsed source in TCSPC;

[0030] FIG. 1C illustrates a histogram of arrival times vs. the photon distribution constructed from successive trials in TCSPC;

[0031] FIG. 2 illustrates the phase of an emission signal relative to the excitation signal in frequency domain lifetime spectroscopy;

[0032] FIG. 3 illustrates the reduction in sensitivity of a duty cycle of a PMT detector;

[0033] FIG. 4 is a schematic of a conventional frequency domain fluorometer;

[0034] FIG. 5 is a chart showing examples of application areas that benefit from the frequency domain versus the time domain approach to time-resolved measurements;

[0035] FIG. 6 illustrates one embodiment of the direct sampling frequency domain apparatus and method of the subject invention in which a ADC08D1500 converter is used;

[0036] FIG. 7 illustrates a time trace according to the subject invention of the fluorescence of rhodamine 110 measured under low count rate conditions;

[0037] FIG. 8 illustrates a time trace according to the subject invention of the fluorescence of fluorescein under high emission intensity;

[0038] FIG. 9 illustrates the Fourier transform obtained of 2.7 microsecond time traces measured for the fluorescein and rhodamine 110 samples according to the subject invention;

[0039] FIG. 10 shows one embodiment of a sampling device employed on a time-resolved acquisition card according to the subject invention;

[0040] FIG. 11 illustrates a multichannel configuration of a sampling device employed using a time-resolved acquisition card within the context of photon migration application according to one embodiment of the subject invention; and

[0041] FIG. 12 illustrates the configuration of two time-resolved acquisition cards shown in FIG. 10 according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which like reference numerals represent the same or similar elements.

[0043] The subject invention provides an improved method for time-resolved detection that combines the dynamic range of frequency domain (FD), the sensitivity of time-correlated single photon counting (TCSPC), while being able to simultaneous acquire all harmonics in parallel in a single measurement. The subject invention achieves several advantages and benefits including, but not limited to: sensitivity improvement of a factor of two to four over traditional frequency domain approaches, simultaneous acquisition of all harmonics; high speed acquisition faster than the both the frequency domain and TCSPC; the ability to handle high signal levels; and low cost and scalability.

[0044] According to the subject invention, in a fluorescence application, for example, a signal is sampled faster than the decay of the fluorescence. In the case of photon migration, a signal is sampled faster than the illumination modulation frequency. Thus, a time-resolved signal can be directly measured from a detector, without the analog mixing used in traditional FD measurements, such as heterodyning or homodyning. This dramatically increases the sensitivity, as the detector duty cycle remains at 100%. In addition, the devices and methods of the subject invention does not incur the sensitivity penalty from the noise added in the mixing process.

[0045] The direct measurement of the subject invention samples all harmonics simultaneously in a single measurement, unlike mixing, in which a single frequency is selected in a measurement. However, unlike TCSPC, the direct approach is not limited to less than one photon per laser period, giving the subject invention much greater throughput than either TCSPC or traditional FD. In addition, the sampling rate of the subject invention is faster than the response time of the detector, e.g. PMT. This enables the ability to sample individual photoelectron pulses from the PMT. Thus, the devices and methods of the subject invention allow for the discrimination between photon counts and darks counts as in TCSPC, which further increases low-light sensitivity over traditional FD.

[0046] FIG. 6 illustrates a prototyping kit from National Semiconductor which can be used, for example, in achieving the sensitive, rapid time-resolved acquisition of the subject invention. In FIG. 6, a sample 2 is excited by a source. The photon response from the sample 2 is reflected through the objective lens 4. A dichroic 6 or other filter is provided between the lens 4 and the detector 10, to selectively pass light of a certain colors (while reflecting other colors). The filtered analog signal is couple to the anode of a PMT 10, for example, an R7400 Hamamatsu PMT, which is connected to one of the analog inputs of the ADC 18 at the other end. In one embodiment, a ADC08D1500 converter is used as the ADC 18 shown in FIG. 6. The 50 ohm termination of the ADC 18 input converts the output current of the PMT 10 into a voltage for digitization.

[0047] The configuration of the subject invention illustrated in FIG. 6 has several advantages. First, the output of the PMT 10 is directly compatible with the analog input of the ADC 18 chip without any intervening electronics. Second, the device enables single photon sensitivity. Ordinarily, signals from PMT's are not continuous because they result from the superposition of pulses created from the amplified cascade of an individual photo or dark electron. However, even though the measurement of the subject invention is analog, photons can be discriminated from the dark current in the subject invention, because the fast sampling rate of the subject invention allows individual pulses to be distinguished.

[0048] In one exemplary embodiment, an R7400 series PMT 10 is used. Given the specifications of this detector, the magnitude and duration of a pulse due to a single photoelectron can be estimated. The PMT gain is therefore nominally specified as 10.sup.6, and the nominal output rise-time is 0.78 ns (corresponding to a pulse of about 1.6 ns). Since input capacitance of the digitizer is less than a picofarad, significant pulse broadening is not expected. So, a single photon would generate a pulse of 10.sup.6 photoelectrons over 1.6 ns, which, dividing charge over time, would general a peak current of 0.1 mA. This corresponds to a signal of 5 mV across the 50 ohm input impedance of the digitizer.

[0049] FIG. 7 illustrates a time trace outputted according to the subject invention where the fluorescence of rhodamine 110 was determined under low count rate conditions. With the ADC 18 configured with a sampling interval of 0.667 ns and voltage resolution of 2.5 mV, individual photon events are made distinguishable. In the absence of an event, the dark current does not the threshold of 2.5 mV, so the measured dark level is zero, as in photon counting.

[0050] FIG. 8 shows data analysis according to the subject invention where the fluorescence of a fluorescein sample was measured under conditions of high emission intensity. In this case, many photons are detected per laser pulse and the individual photoelectron pulses are no longer isolated. Under these conditions, photon counting is no longer possible, but our analog detection continues to make valid measurements. In one instance, the fluorescence decay is obtained by averaging the recovering signal after each laser pulse shown in FIG. 8. The subject invention therefore allows analysis of the analog signal as a function of space (e.g. binning into spatial pixels in an image for instance) and time (as a time series which can be arbitrarily binned). Thus, decays can be successfully observed from both the high and low rate samples, under both extremes where individual photons were observed and where they were indistinguishable. From a single measurement, multiple harmonic components of the decay are simultaneously resolvable.

[0051] FIG. 9 illustrates the FT of fluorescein and rhodamine 110 data. In each fluorescence sample, the data was collected in a 2.7 microsecond measurement, and time traces measured for both samples are shown. In both cases, distinct peaks are observed at multiple laser harmonics. From a 2.7 microsecond measurement, the lifetime was determined using standard FD analysis. Using fluorescein as a standard reference with 4 ns, the lifetime of the rhodamine 110 sample was found to be 3.09 ns as compared to the known value of 2.99 ns.

Dynamic Range

[0052] Dynamic range can be described as the range between the maximum and minimum signals detectable by the instrument. As noted above, conventional FD measurements excel at measuring high signals, but have lower sensitivities, while complementary TCSPC measurements have high sensitivity, but cannot measure high intensity samples. The devices and methods of the subject invention combine the best advantages of both traditional FD measurements and TCSPC techniques, as well as exceed the dynamic range of steady photon counting or analog measurements.

[0053] In one embodiment of the subject invention, an ADC08D1500 converter 18 is employed as the analog-to-digital converter. The high sampling speed of the ADC08D1500 converter 18 makes its eight bit resolution sufficient to capture signals from detectors. For low signal levels, as shown in FIG. 7, a 2.5 mV resolution is sufficient to measure single photon events. The maximum photon rate measurable by the ADC08D1500 converter 18 can be calculated from the peak level of the single photon response. In the exemplary configuration shown in FIG. 6, the maximum measurable negative voltage is −325 mV. Approximating the maximum peak level of the single photoelectron response as −10 mV corresponds to a maximum of 32 photons arriving simultaneously, where simultaneously means within the 1.6 ns minimum pulse time of the PMT 10. The maximum measurable detected photon flux is on the order of 32/1.6 ns or 20 GHz. Thus, the subject invention has the benefit of increasing significantly the typical linear range of a PMT detector.

[0054] In the devices and methods of the subject invention, the upper end of dynamic range may be detector limited. Accordingly, since the high sampling rate means an instantaneous measurement will not integrate many photons, the eight bit resolution of the ADC08D1500 12 is sufficient to capture single photon events through intensities which will saturate the detector 10. Thus, time-resolved measurements are made as rapidly as the detector linearity will allow.

Exemplary Application: High-Sensitivity, Direct Sampling Analog Acquisition Card for Rapid Parallel-Acquisition Frequency-Domain Spectroscopy

[0055] As noted above, traditional FD measurements suffer from low sensitivity. However, lack of sensitivity is not an intrinsic feature of the frequency domain approach; rather it is a result of the instrumental methods which have been used to acquire FD data. In conventional devices, sensitivity is lost due to the need to reduce the modulation frequencies to match the slower sampling speeds of the digitization electronics. This reduction was performed by analog homodyning or heterodyning techniques, which both suffer from at least 50% signal loss from the duty cycle of the detector and lower sensitivity due to the noise introduced from the RF mixing. Because of the detector duty cycle problem, only one frequency can be mixed at a time, requiring a separate measurement of each modulation frequency, significantly increasing the acquisition time for multi-frequency measurements of conventional devices and methods. These disadvantages, however, are overcome by the subject invention.

[0056] Turning to FIGS. 10-11, the subject invention enables second channel analog-to-digital conversion for dual channel acquisition, employed using a time-resolved acquisition card 12. The time-resolved acquisition card 12 connected to detectors 10 includes the ADC 18, the logic circuit 22, and associated clocks (laser reference clock 16 and pixel clock 20), an optional first-in-first-out memory and an optional interface 26.

[0057] In one embodiment, a single analog-to-digital converter, for example, an ADC08D1500 converter 18 contains two independent analog-to-digital converters. Each of these converters are capable of making simultaneous measurements from two distinct inputs. The ADCs are independent in the sense that each input is separately measured in parallel (not multiplexed). However, the ADCs remain simultaneous because they both share the same sampling clock 16. Therefore to feed the converter 18, an analog front end for two 50 ohm terminated is created, analog single-ended inputs on the board for connection to two detector channels 10.

[0058] In one embodiment of the subject invention, it is possible to employ modulated excitation sources to excite a sample (for example, light emitting diodes (LEDs) or laser diodes), rather than pulsed light sources. This alternative increases the flexibility and portability of an application employing the devices and methods of the subject invention, thus reducing the costs of measurements produced.

[0059] In one embodiment, the device of the subject invention adds reference and scanning sync inputs to enable imaging (not shown). A laser reference 16 is created by using an external sampling clock, for example, the National Semiconductor LMX2346 single chip clock synthesizer to generate the ADC sampling clock from a pulsed or modulated laser reference signal. For an 80 MHz laser, a 1.6 GHz sampling clock will be synthesized to drive the ADC to synchronously sample the detectors 10 twenty times per laser period.

FPGA Implementation of FD Time-Resolved Measurements

[0060] As shown in FIGS. 10 and 11, the digitized signal will be transferred from the ADC08D150018 to a logic circuit 22, here, a Xilinx Virtex4 Field Programmable Gate Array (FPGA) on the card 12. Parallel data transfers will be made at full-speed through dedicated high-speed I/O lines on the card 12.

[0061] In this embodiment, the FPGA 22 is as critical component of the direct sampling method as the gigahertz digitizer itself. The bandwidth of the raw data from the two channels is 3.2 gigabytes/second, which is vastly greater than the capacity of even the fastest computer workstations and storage devices which typically operate with maximum sustained transfer rates of about 50 MB/s. Clearly for direct sampling method to prove practical, on-board data preprocessing is essential to reduce the data before it is transferred to a host computer. The FPGA 22 runs fast enough to process the raw data as it is being acquired, as well as controlling the ADC.

[0062] In our method, the information in a photon migration, fluorescence lifetime, or other time-resolved spectroscopy experiment, is contained in the Fourier transform coefficients of the raw data. However, only the coefficients at the laser harmonic frequencies contain information.

[0063] For 1.6 GHz sampling of an 80 MHz laser, the maximum number of harmonics measured is 10 by the Nyquist theorem. Although, depending on the signal-to-noise of an experiment, only the first several harmonics may contain information.

[0064] Since the information is contained in the harmonic coefficients, a dedicated algorithm in the FPGA 22 can be written to calculate the coefficients in real-time as the data is continuously acquired. The coefficients can be calculated on a per laser period basis, but only transfer the total for each coefficient at the end of a pixel integration period.

[0065] For example, for 5 microseconds of pixel integration using an 80 MHz pulsed laser, 400 laser pulses will occur and 8000 samples per channel will be acquired during the pixel. However, if desired, it is possible to retain only the coefficients from the total intensity and the first seven harmonics. Four bytes per harmonic, corresponding to 16 bits per coefficient can be used since both sine and cosine coefficients are present per harmonic. The zero harmonic is the total intensity, so the full 32 bits can be used for it. Overflows are avoided since the maximum total intensity per pixel is 8000*256 which fits in 21 bits. Similarly, overflows are avoided in the other coefficients, since there are only 400 total samples integrated per point in the laser period. In this case, 36 bytes per pixel per channel are sufficient. For two-channels, this corresponds to 14 MB/s which is about half the real, sustainable bandwidth of USB 2.0.

[0066] The hardware is flexible enough that choices of the pixel integration time and the number of harmonics to transfer can be changed on the fly during a desired experiment. Thus, data collection can be adjusted for the intensity and signal-to-noise of a particular application. This becomes more important when the acquisition is scaled to a large number of channels. Long integration times can be accumulated on the main computer from 5 microsecond integrations, since at that rate the main computer can efficiently accumulate the coefficients into 32, 64 bits, or more.

[0067] Xilinx freely provides an FFT core for the FPGA 22, so it would be possible to calculate a full FFT over the chunk of raw data acquired during the pixel integration time. However, in the instance where only the first several laser harmonics are desired and the laser period and pixel integration time of the measurement are known in advance, it is possible to optimize the computation by calculating the coefficients as sine and cosine weighted moments at only the harmonics of interest. This approach has already been used for video rate FLIM. The FPGA 22 can perform this calculation as a multiply and accumulate operation from preloaded sine and cosine tables. The tables needs only as many entries as there are samples per laser period, which are usually 20. Both the tables and the results can be processed in circular buffers, with a pointer incremented by the sample clock 16. Each coefficient from a laser period is summed the corresponding coefficient from the previous laser periods and only the total is transferred at the end of the end of the pixel integration. Performing the integration over the laser period instead of the pixel integration time also minimizes the effect of artifacts such as instrument drift photobleaching in the sample.

[0068] FIG. 12 illustrates how two time-resolved acquisition cards 12, shown in FIG. 10, can be configured according to one embodiment of the subject invention. The cards 12 are connected to an end computer 14, which may be used for further analysis, storage and display. Additional channels can be implemented through the use of additional acquisition cards, detectors, filters, gratings, or prisms.

USB Transfer Module and FIFO Memory

[0069] The FPGA 22 gives the card 12 the ability to continuously acquire time-resolved and stream it directly to the host computer for real-time display. This is essential for applications, such as video-rate imaging, which require the rapid acquisition of data and is not possible with RF signal analyzers. This is also important when the number of channels is scaled up. To accommodate this capability, a small amount of fast First In First Out (FIFO) memory 24 can be added to augment the transfer FIFOs in the CY768013A USB controller 26. This memory is not used to store the measurements, but is used for streaming to buffer the data so it is not lost if the host computer delays a transfer from the card 12. This will ensure robust performance during continuous acquisition. In another embodiment, a universal serial bus, for example, a Labview® USB driver, can be used for data transfer and display. The USB driver 26 captures the data sent to the external storage device, for example, a PC, through an interface (for example, USB port), from the sampling device in order to enable display and streaming of the data to a disk.

Representative Applications

[0070] There are several products and areas where this invention will be of benefit. The following are a few examples.

[0071] Photon Migration. Photon migration can analyze the composition of a tissue based on the scattering and absorption coefficients of scattered near infrared light. The tissue is illuminated with light that is modulated at frequency in the range of 10 MHz to 1 GHz and the modulation and phase shift of the scattered light is measured. Information about the tissue composition and physiology can be then calculated by employing diffusion theory models. Photon migration offers promising therapeutic strategies for neonatal care, stroke diagnosis, tissue oxygenation, breast cancer identification, and sleep apnea diagnosis. There is a strong move towards instrumentation with tens to hundreds of channels. TCSPC is too expensive to scale with multiple channels. However, the method of the subject invention allows multiple harmonics to be captured simultaneously and with higher sensitivity than standard FD.

[0072] FLI Microscopy. Current fluorescent lifetime imaging microscopy instrumentation suffers from a number of limitations with the dynamic range and speed. When combined with multifoci imaging, fluorescent lifetime imaging microscopy systems have the potential to offer real time in vivo lifetime imaging. Current lifetime imaging systems based on TCSPC are limited to image acquisition rates on the order of minutes to tens of minutes, far too slow for most biological applications. The fast lifetime acquisition capabilities of the present invention, however allow much higher image frame rates without saturation problems with the electronics.

[0073] FRET. The Zeiss LSM510 has proven to be an enormously popular product, enabling biologists to conduct FRET studies far more easily than previously. The addition of spectral resolved lifetime microscopy will greatly aid FRET studies in cell culture, and more importantly, tissue based FRET studies where FRET based on purely spectral approaches suffer from uneven absorption and scattering of different wavelengths. Additionally, lifetime provides an additional contrast modality, further increasing the number of components that can be visualized in image, and aiding spectral unmixing of multi-wavelength images.

[0074] Cytometric Studies. Both Flow and image cytometry require small integration and the ability to handle high photon fluxes. TCSCP is uncompetitive in this area as shown in FIG. 5, and the DSAL card will have a significant competitive advantage. Furthermore, FD lacks the sensitivity for dimmer, but biologically important, aspects of the cellular physiology.

[0075] Multi Wavelength Frequency encoded Excitation and Detection. Multiple excitation wavelengths modulated at different frequencies can be employed to further increase the discrimination of multiple components.

[0076] Pharmaceutical Assays. The high throughput rate of DSAL lends itself well to the industrial pharmaceutical setting for high throughput assays based on lifetime analysis of compounds, cells and tissues. Again, TCSPC is not well suited for this regime, and the DSAL data acquisition approach has the opportunity to capture a significant portion of the market for lifetime assays.

[0077] Although the subject invention has been described with respect to preferred embodiments, those skilled in the art will readily appreciated that changes or modifications thereto may be made without departing from the spirit or scope of the subject invention as defined by the appended claims.