Patent Application
20080008479
(1) Field of the Invention
The invention relates to a method for detecting light signals, in which a light signal impinges on an optoelectronic converter, where the light is converted into an electric signal, and in which, after the conversion, the electric signal is distributed to several evaluation channels. Here, in each evaluation channel (i) a signal evaluation is performed, which is different from the signal evaluations for the other evaluation channels and (ii) a result signal is produced. The invention relates to the problems found in optic examination methods, especially in microscopy, where many different types of detection methods can be used. Each of the methods has advantages and disadvantages and some of the methods may beneficially be used only in special examination methods. For this reason, detection methods are frequently used alternately or in combinations. The invention also relates to a detector module for detecting and evaluating light signals as well as the use of such a detector module in a laser-scanning method.
(2) Description of the Related Art
In microscopy, in general, and in laser-scanning microscopy, in particular, there are a multitude of different examination methods, each requiring a detection method adjusted to the examination method. Each of these detection methods has its own characteristic and is generally particularly well suited for one or several of the examination methods, but poorly adapted for other methods.
A widely used method is the integration of signals within a defined measuring period with a subsequent analogue-digital conversion, for example. Usually condensers are used for the integration and collect a charge in a predetermined measurement time. Light signals are converted into electric signals by an optoelectronic converter, so that the charge collected in the measuring time corresponds to the light intensity. Between two integration processes, the condenser must be discharged and/or deleted, so that a certain downtime develops, in which no integration can occur. Up to 30% of the overall time is required to discharge the integration condenser, in which time no signal can be detected; as a consequence loss of sensitivity develops. In this case the so-called odd/even variant is often used as a solution. Here, while one condenser is prepared for the next integration process, i.e. its charge being deleted, another condenser is used for integration. This integration method has a very wide dynamic range when the integration time constant is adjusted appropriately. The loss in sensitivity can be avoided in the odd/even variant; however, in this method lines develop in the image, which result from the tolerances of the two integrators and their parts during switching.
Another method is to count individual photons. This method is very sensitive; however it only has a narrow dynamic range and is therefore only operational to a limited extent. The same applies to the so-called 2D-photon counting, such as for example described in US 2003/0183754 A1. When a photon multiplier, for example a photomultiplier-tube (PMT), is used as a detector, the highest possible voltage must be connected for detecting individual photons in order to receive a signal. When several photons simultaneously impinge the detector, they have no influence on the intensity of the signal, because the signal is already at maximum level with a single photon. There is therefore no difference between the impingement of few or many photons; the dynamics are therefore very low.
Furthermore, a method that can be easily implemented is the so-called oversampling method. This method is particularly well suited to scan a changing signal because the signal is scanned at a higher scanning frequency than actually necessary to represent the band of the signal. This way, during the measuring time the signal-to-noise ratio can be influenced.
In addition to these standard methods in laser scanning microscopy, additional detection methods are used specially adjusted to the examination methods. In so-called fluorescence-life-measurements (FLIM), a pulsed illumination system is necessary and very fast with digital signal processing in the pico second range. The pulse time, i.e. the time at which the molecules are excited to fluorescence, plays an important role. For this reason, processing of the signals usually occurs in different steps: for example the preprocessing is frequently performed in the detection module, while the final processing may occur in a computer for example. In order to determine the life of a fluorescence excitation, the time between the excitation and the detection of the signal must be determined.
A similar, special detection method is used for the fluorescence-correlation spectroscopic measurements (FCS-measurements). Here, bonding features of molecules are determined within the cofocal volume detected by a laser-scanning microscope. For this purpose, the emission signals of fluorescent molecules must be detected; here too the temporal progression and interval the signals are detected are of decisive importance.
For simultaneous or alternating use of several detection methods, the prior art suggests different solutions. In US 2006/0203241 A1, a device for the spectral selection and detection of spectral ranges of a light beam are disclosed, in which the light beam is split into several, even spectrally different, partial beams. Each of the partial beams impinges a different detector, with the detectors each having different detection features and/or different detection methods. This construction is very expensive because for each partial beam a separate detector with a separate optoelectronic converter is used.
From an article by W. Becker et al., “Proceedings of SPIE,” vol. 4431, pages 94-98, a method for detecting an object with the help of a laser scanning microscope is known. The detection device includes two detection channels, with prior to the detection the signal first being split optically into two channels. Each of the channels has therefore a separate optoelectronic converter. A separate evaluation device is connected to each converter.
In contrast thereto, the solution disclosed in DE 102 53 108 B4 shows an improvement. Here, the light is detected by a single detector, the detection signal is then distributed to two channels via highpass filters and/or lowpass filters. According to the disclosure of DE 102 53 108 B4, these filters are necessary to achieve a clean channel separation. In one of the two channels pulses are created by a pulse former, which serves for further processing. The detection signal originally connected to the input of the second channel no longer requires any attention, so that the second channel essentially acts as a pulse counter, which can also register the temporal intervals between the pulses. The system presented in DE 102 53 108 B4 is a very special detector, which can only be used for certain problem conditions, such as for example FCS-measurements.
The object of the invention is therefore to develop a method and an arrangement which has a range of use as wide as possible and can flexibly be adjusted to different examination conditions, in particular in microscopy and laser scanning microscopy.
This object is attained in a method of the type mentioned at the outset in that one or more result signals being selected or displayed for further processing according to a predetermined, variably adjustable selection criterion. When, for example, a sample is examined microscopically, the light originating in the sample is detected, with independent on the examination method after the distribution to several channels an evaluation occurs in each channel. Only thereafter a selection of one or more signals is made. This way a user has various, flexibly selectable result signals available; however in principle a single detector is sufficient. Even during the measurement and/or life, i.e. online, a change of the selection of one or more other evaluation principles may occur.
Here, the electric signals are advantageously individually amplified and/or filtered after their distribution to several channels. In particular in a distribution of the signal over a multitude of parallel channels, the amplitude of the signal drops, which can be compensated for by amplification. Furthermore, each signal can individually be filtered more or less or not at all in reference to the subsequent evaluation as well as filtered by a highpass or a lowpass filter.
In a particularly preferred embodiment of the method, the result signals are selected using logical switching, preferably an FPGA (Field Programmable Gate Array). The logical switching is switched according to the adjusted selection criteria, so that one or more result signals are selected. A programmable switching, such as FPGA, allows the utmost flexibility as well as a quick adjustment to a modified selection criterion.
Advantageously, the result signals are automatically selected using the selection criterion. For example, a user enters a selection criterion, if one has not already been selected automatically, which is transmitted to the logical switching, which then switches the signal channels such that the predetermined result signals are selected and forwarded. Of course, a selection of the signals can also be selected manually by the user.
Preferably, the result signals are displayed for further processing via an LVDS-interface (low voltage differential signaling interface). Using this interface the selection criterion is also transmitted to the logical switching and the logical switching is switched accordingly. In an LVDS interface, instead of common base voltages amounting in digital systems to approximately 5 Volts, lower voltages of approx. 1.2 Volts are used. Additionally two wires are used for signal transmission, with the difference of the voltages, approximately 0.3 Volts, being decisive for the logical state. The disadvantages of classical interfaces, such as the development of high-frequented electromagnetic alternating fields in voltage and current fluctuations, can be avoided here.
An application for this method is preferably the microscopic or laser-scan microscopic examination of samples, with light emitted by the sample being detected. As already mentioned at the outset, in particular, there are many different examination methods, some of which need special signal evaluation methods. Here beneficially the determination of selection criteria and the selection of the result signals occur depending on the adjusted examination method or methods. In other words, the adjusted examination method also determines a selection criterion or at least a part of a selection criterion. When, for example, in a method seven selection channels with the appropriate signal evaluation methods are provided and a user selects a special selection method, which preferably needs the signal processing of channels 3, 5, and 6, the selection criterion may comprise a sequence of seven binary numbers and/or a seven-digit binary number, with the third, fifth, and sixth numbers being 1 and the other numbers being 0. This sequence/number may be transmitted via the interface to the logical switch, which then adjusts such that it selects the signals of the channels 3, 5, and 6 for forwarding. The other signals are evaluated, though, but nor further processed.
A user may now determine which examination methods should be used, for example by checking the approximate fields in a selection menu at a connected PC or a control unit of the microscope. However, the selection of the evaluation method and thus the determination of selection criteria can also occur automatically, when for example at the PC or the control unit, an appropriate examination method is selected for a sample. Here, too, an appropriate manual selection by a menu control is possible.
For microscopic and particularly laser-scan-microscopic applications there preferably occurs at least an oversampling evaluation, an integration evaluation, an FCS-evaluation, a photon counting evaluation, and/or an FLIM evaluation. The number of different signal evaluations provided is generally unlimited, more or less signal evaluations may be provided depending on the purpose of the application of the method according to the invention.
Preferably a PMT is used as the optoelectronic converter, based on its wide spectrum of use. Its sensitivity can be variably adjusted depending on the voltage provided so that for example at highest sensitivity individual photons can be registered, but also such that at low sensitivity exclusively high intensities can be registered, distinctly separating themselves form the background noise. In addition to PMT photon diodes or APD's (Avalanche-photo diodes), photon converters on a semiconductor base may also be used. The latter may also be provided as single-photon avalange photodiodes (SPAPD) and are then exclusively suitable for counting photons, which are predestined for use in FCS measurements and life term measurements.
When the detection signal is optically distributed to several channels, several optoelectronic converters may be used, with specific signal evaluations being connected to each of them. The converters with their evaluations switchings may be integrated in a single detector module, in which at the input of the light initially an optic beam splitting occurs. The ultimate selection of the result signal for forwarding may also be performed by a single FPGA, though.
The invention also comprises a detector module for detecting and evaluating light signals, which comprise an optoelectronic converter for converting a light signal into an electric signal, connected to a distribution switching for distributing the electric signal to several evaluation channels with an evaluation module in each evaluation channel, which (i) performs a signal evaluation, which are different from the signal evaluations for the other selection channels, and (ii) creates a result signal. In such a detector module the object is attained in that a selection unit is provided in which one or more result signals are selected or displayed for further processing according to a predetermined selection criterion.
Initially, an optic signal is detected by an optoelectronic converter and converted into an electric signal. The electric signal is then distributed to different evaluation channels, for which a distribution switching is provided. In the simplest case, this represents a junction with an input and several outputs. Each output corresponds to an evaluation channel. The signals of the various evaluation channels are then each fed to an evaluation module. Here, the signal is evaluated and a result signal is created accordingly. In the last step the selection unit selects one or more result signals depending on a predetermined but variably adjustable selection criterion and displays them.
Such a detector module can be designed so that it can be used in different optic examination devices, such as telescopes, microscopes, material examinations, and other analysis arrangements. The selection criterion may for example be determined by a user when determining the examination method for a certain sample. A selection may occur via an interface or directly via one or more switches at the detector module itself. When the detector module is used, for example in a laser scanning microscope, the examination methods can be selected by a connected control unit. Depending on the selected examination method the selection criterion can then be automatically or manually be determined, for example via a menu control, and be transmitted to the detector module.
In contrast to the prior art, several selection principles are simultaneously implemented in one detector module, one or more of which can be selected in a flexible manner. The selection and switching may also occur during the measuring. Due to the fact that the preparation of the signals occurs directly in the detector module, signal processing times and transmission times are of minor importance. Since several measurements can occur simultaneously, stress on the sample is reduced.
Preferably, signal amplifiers and/or frequency filters may be provided, individually adjustable in the detector module for one or more evaluations channels. In particular in case of a high number of selection channels, such signal amplification is useful after the distribution of the signal, because the signal has been weakened by the distribution. Using a filter, further special parts of the signal can be filtered out. These filters of course may also be integrated in the selection module.
Preferably, a selection switching is provided in the selection unit of the detector module, which can be controlled. By control of the selection switch, depending on the selection criterion, resulting signals can be flexibly selected. It is particularly preferred for the selection switch to be implemented as an FPGA. This represents a freely programmable logic circuit that can be appropriately programmed by predetermining the selection criteria. When the selection criteria are changed, an adjustment and/or reprogramming of the switch occurs in the FPGA. Instead of an FPGA, a CPLG (complex programmable logic device) or another programmable logic switching may be used.
Preferably, an LVDS interface can be provided in the selection unit for displaying the result signals as well as for transmitting the selection criterion to the selection unit. Other bi-directionally operating interfaces may also be used. When the detector module is used, for example with a laser scanning microscope (LSM), one or more selection criteria and/or result signals for determining the selection criterion can be selected, for example via a control unit provided at the LSM. Accordingly, this may of course also be performed as early as the selection of the examination method(s) without interference from the user. Then, via the LVDS-interface, a respective signal with the selection criterion is transmitted to the selection unit and the selection switching is programmed accordingly, so that only signals from the selection channels whose evaluation modules implement the selected evaluation methods are selected and forwarded to a PC for a graphic display, for example.
As an optoelectronic converter, a PMT (photomultiplier tube) is preferably provided at the detector module. In such tubes the voltage serving to multiply the secondary electrons can be adjusted over a wide range, thus making a PMT suitable both for high intensity as well as for individual photon measurements. Alternatively, other photon converters, such as photodiodes, APD, or SPAPD can be used as well.
Several different photon converters may be used simultaneously in the detector module; however, the detection beam must be appropriately optically distributed. For each converter, a separate switch for distributing selection modules must be provided but also a connection may be provided so that, for example, first the signals of one converter are processed and then the signals of the other converter are processed using the same selection module.
The use of line detectors or detectors in the form of arrays for the above-mentioned photon converters is possible as well, when for example the detection beam is spectrally split prior to the detection. The individual spectral channels are then consecutively evaluated with the same evaluation module. Therefore for each spectral channel a separate evaluation switching may be provided having different evaluation modules.
A common selection unit is provided for all selection channels. However, there is only a single interface by which the data is forwarded from the detector module.
For the use in microscopy, in particular in laser scanning microscopy, an oversampling, an integration, an FCS, a photon-counting, and/or an FLIM-module are provided as selection modules in the detector module. Further selection modules may also be and/or become integrated. Of course, however, fewer evaluation modules may also be sufficient. The above-mentioned list includes the most common evaluation methods, though. The detection module may also be embodied such that individual selection modules can be supplemented or exchanged. This is particularly advantageous when the space available is limited. When the selection modules are configured as plug-in modules, for example, an exchange is possible without any elaborate redesign or readjustment because the entire detector module is not exchanged.
The detector module according to the invention is particularly suitable for the use in a laser scanning microscope, because different examination methods can be realized, each of which requires different signal evaluation methods. Such a laser scanning microscope, in which the detector module is used, usually has an adjustment or control unit, by which one or more examination methods can be selected to examine a sample. The selection criterion is then preferably determined automatically using the predetermined examination methods and transmitted to the selection unit of the detector. The selection of the evaluation method can alternatively also occur manually, for example by an appropriate menu control, via the control unit of the microscope.
In the following, the invention shall be explained in greater detail using an exemplary embodiment making reference to the following drawings in which:
In describing preferred embodiments of the present invention illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.
In
In a radiation-forming unit 8, the radiation of different wavelengths is first collimated and then changed with regard to the radiation profile so that the beam, when emitted, illuminates an essentially rectangular field in a profile level, for example, with the distribution of the intensity along the longitudinal axis of the rectangular field not being equivalent to the distribution of a formal curve but to a square wave. This linear beam is suitable to create a square wave illuminated field in the cross-section. The beam emitted by the radiation forming unit 8 serves as an illumination beam 9 for illuminating a sample 10. For this purpose, the illumination beam 9 is deflected via a primary color splitter 11 to the scan module 2. Here, the illumination beam 9 is deflected according to an instruction before it is deflected via the microscope module 3 to the sample 10 in the focus of a lens (not shown) of the microscope module 3 so that the sample 10 can be scanned. Emitted radiation focused by the sample 10, for example a reflecting radiation or excited fluorescence radiation, returns into the scan module 2 via the microscope module 3. Here, the time-modified beam is reconverted into a resting beam, the radiation emitted by the sample being “de-scanned.” After leaving the scanning module 2 the light emitted by the sample passes through the primary beam splitter 11 and impinges the detector module 4. Here the light emitted by the sample is detected and analyzed. Here, it may be provided in the detector module 4 that light of different wavelengths is selectively examined spectrally.
The design of the detector module 4 is shown in
In the example of the
In
The detector module shown in
Modifications and variations of the above-described embodiments of the present invention are possible, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2006 030 530.2 | Jul 2006 | DE | national |
