Maintaining spectral quality over long measuring periods in imaging mass spectrometry

Abstract

The invention relates to imaging mass spectrometry on thin sample sections, in particular using MALDI, where a high lateral image resolution means that a plethora of mass spectra has to be acquired and the image acquisition runs over many hours. The quality of the mass spectra deteriorates considerably over time in such cases. The invention is based on the finding that the decrease in spectral quality of continuous measurement series over many hours is only partially caused by a decrease in detector gain, and that another significant cause is a decrease in the number of usable ions per ion generating pulse, which is attributable to several phenomena that are difficult to regulate. The invention now proposes to instead regulate only the detector gain, and such that not only the decrease in the detector gain is compensated, but also the decrease in the number of usable ions per ion generating pulse.

Claims

1. A method for imaging mass spectrometry on thin sample sections, from which a large number of mass spectra are continuously acquired in a mass spectrometer with ion detector via a pixel pattern in order to record a distribution of signals in the thin sample sections, where regulating an ion detector voltage enables ion currents, which are measured by the detector across a selected mass range in the mass spectra and summed, to be kept constant at a target value in the long term over the measurement of many mass spectra in order to maintain the quality of the mass spectra over a period of many hours until the end of the measurements.

2. The method according to claim 1, wherein the ionization of molecules of the thin sample section is realized by matrix-assisted laser desorption (MALDI).

3. The method according to claim 2, wherein the ion currents measured include at least a portion of the matrix ions.

4. The method according to claim 1, wherein the mass spectra are acquired by a time-of-flight mass spectrometer, ion cyclotron resonance mass spectrometer, or mass filter.

5. The method according to claim 1, wherein the ion currents measured represent an ion current over the complete mass spectrum (total ion count) or a part of it.

6. The method according to claim 1, wherein a clock-pulse rate of the ion detector voltage regulation has a predetermined value at the start of the measurement and changes to a lower predetermined value over the further course of the measurement.

7. The method according to claim 1, wherein the ion currents are determined by forming averages across several mass spectra.

8. The method according to claim 7, wherein a sliding average is formed across several hundred to several thousand pixels, or wherein a series of averages across several hundred or several thousand pixels (“section averages”) in each case is formed.

9. The method according to claim 7, wherein the ion detector voltage is virtually changed continuously with a predetermined temporal drift value during the acquisition of the mass spectra, and the drift value is corrected when the average of the ion currents no longer remains constant over time.

10. The method according to claim 9, wherein a temporal constancy of the ion currents is determined by straight lines, which are applied to curves of the averages and whose gradient is used to calculate drift values.

11. The method according to claim 9, wherein a derivative curve of the curves of the averages is formed by calculating differences between successive averages, wherein a distribution curve of the variances of this derivative curve is formed, and a deviation of the distribution centroid from zero is used to calculate an ion current drift correction.

12. The method according to claim 1, wherein the signals in the thin sample section originate from peptides, lipids, phosphorylated molecules, pharmaceutical agents and/or composite markers for unusual tissue states such as carcinogenic degenerations.

13. The method according to claim 1, wherein an acquisition rate for the mass spectra is in the kilohertz range, while around 10 to 1,000 mass spectra per pixel are added together to form a sum spectrum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention can be better understood by referring to the following figures. The elements in the figures are not necessarily to scale, but are primarily intended to illustrate the principles of the invention (largely schematically).

(2) FIG. 1 depicts an idealized ion current curve during a scan across five different types of tissue within a thin section with a good drift value setting for continuous adjustment of the detector voltage (x-axis: #pixel; y-axis: ion current in arbitrary units). The sections a1 to e1 represent the total ion currents as the five different types of tissue are being scanned. The scan duration for the 100,000 pixels of conventional size shown can be assumed to be around one hour.

(3) FIG. 2 shows the same initial situation, except that in this case the correction of the detector voltage is not optimal (or there is no correction at all), so the measured ion current decreases continuously, despite the gain being kept constant, for example.

(4) FIG. 3a depicts the derivative of the ion current curve from FIG. 1. The derivative was generated by simply forming the differences between successive measured values. Apart from the spikes, which originate from the transitions between the tissue types, the values are distributed precisely about zero.

(5) FIG. 3b depicts the spread about zero, enlarged by stretching the intensity axis.

(6) FIG. 4 shows the spread of the measured values from FIGS. 3a and 3b in a distribution curve. When the spikes are not taken into account, the distribution is Gaussian. The centroid of the Gaussian is precisely zero here, and therefore indicates that the correction of the detector gain is correct.

(7) FIG. 5 shows, by way of contrast, the Gaussian distribution as obtained from the measured curve in FIG. 2. The centroid of the distribution is no longer at zero. The deviation of the centroid from zero can be used to calculate a better drift value for the readjustment of the detector voltage.

DETAILED DESCRIPTION

(8) While the invention has been illustrated and explained with reference to a number of embodiments, those skilled in the art will recognize that various changes in form and detail may be made to it without departing from the scope of the technical teaching defined in the attached patent claims.

(9) As mentioned above, the invention is based on the finding that the decrease in spectral quality is only partially caused by a decrease in the detector gain and quite significantly by a decrease in the number of usable ions per generating pulse (e.g. laser shot) also. The term “usable ions” is here deemed to mean all ions which have been produced in the ion source, e.g. during a laser shot in laser-desorbing ionization methods, and arrive at the detector at the right time, i.e. they generate a signal which can be evaluated. The decrease in the number of usable ions (per laser shot) means that fewer and fewer ions per mass spectrum are measured over the course of the acquisition time. The signal-to-noise ratio steadily decreases: the quality of the spectra, and hence their evaluability, drops continuously. This effect is naturally aggravated by the simultaneous decline in detector gain.

(10) The introduction described measures for a MALDI time-of-flight mass spectrometer in particular which can bring about a situation whereby sufficient numbers of ions of each ionic species are still being generated for every mass spectrum of a pixel at the very end of a long acquisition series (sometimes after over 20 hours). However, without compensating measures, the signals can become so small that they cannot be separated sufficiently well from the omnipresent noise. Such measures consist in selecting a less volatile matrix material and cooling the mass spectrometer, the aim of both measures being to slow down the vaporization of the matrix material. Further measures concern the design of a pulsed laser with a long service life, the design of ion sources which are less susceptible to becoming electrically charged as a result of deposit formation, and stabilization of the high-voltage conditions in the ion optics. Furthermore, suitable designs can also largely reduce the effects of temperature on the focal length of the pulsed laser, and thus on the spot diameter and the energy density in the laser spot. Although measures of this kind are sufficient to provide enough ions per pixel until the end of the acquisition, they usually do not prevent the continuing decline in the evaluability of the associated mass spectra because the signals become smaller and smaller.

(11) The evaluability of the mass spectra can also deteriorate when the mass resolution in the mass spectrum diminishes; in other words, when the mass signals broaden or shift. This can be caused in particular by charging effects on surfaces which are close to the flight path. The mass resolution can be kept largely constant over a wide mass range by applying a method which has become known under the name of “pan” (cf. DE 196 38 577 C1, corresponding to U.S. Pat. No. 5,969,348 A and GB 2 317 495 B). This method uses a time-delayed acceleration of the ions (DE=delayed extraction) followed by a continuous change in the acceleration voltage in time.

(12) In order to keep the quantity of ions generated per laser shot constant, one might have the idea of regulating the energy density in the laser spot. But regulating the energy density of the laser beam in such a way has been found to be unsuitable, since the mass spectra change qualitatively with the energy density. In particular, the ratio of the molecular ions formed in the plasma of the vaporization cloud to the fragment ions formed spontaneously in the surface of the sample (by so-called in-source decay, ISD) changes, and thus also the character of the mass spectra. Furthermore, the strength of the ionization depends to an extremely critical degree on the energy density, so the balance for setting the energy density in the successive laser shots is very difficult to maintain. Any regulation can disturb the carefully created balance of the mass spectrum with regard to the distribution of the analyte ions in an unpredictable manner and thus destroy the homogeneity of a tissue image.

(13) The invention therefore proposes that the detector gain be regulated in such a way that not only the decrease in detector gain is compensated over continuous measurement series lasting for hours, which are required for imaging mass spectrometry of a two-dimensional thin sample section, but also the decrease in the number of usable ions. Unlike the case described in the cited patent U.S. Pat. No. 8,193,484 B2, it is thus not the detector gain which is kept constant, but the quality and evaluability of the measured mass spectra. This sounds very straightforward here, but has been found to be quite essential for the imaging mass spectrometry of larger thin tissue sections or for thin tissue sections for which a very high lateral image resolution is required. This means that a satisfactory signal-to-noise ratio can be maintained right to the end of acquisition series, which take many hours, and the evaluability of the mass signals in the mass spectrum can be kept constant.

(14) This aim of constant evaluability can be achieved if the sum of the amplified ion currents of a mass spectrum, which are measured by the detector and then digitized, is kept constant in the long term across a selected mass range of the mass spectrum, e.g. from around m/z 500 to 1,100 for lipids, as quite light analyte substances.

(15) Most preferable are embodiments in which the total ion count is kept constant over the whole mass spectrum. In a special embodiment, spectral acquisition with the MALDI method, which normally masks out large portions of the ion current of the light matrix ions at the start of the mass range, even includes larger mass ranges of the light matrix ions, usually in the range up to around m/z 1,000, including matrix cluster ions. It has been found that the sum of all the ion currents, including the ions from the matrix substance, remains constant over time, and is therefore more suitable as a controlled variable, than only the current over the ions from the substances of the tissue sample, probably because a greater quantity of matrix ions remains when tissue molecules are ionized to a lesser degree.

(16) The detector gain must be regulated very gently here. Changes caused by different types of tissue in the thin section should be averaged over as large an area as possible. It is therefore preferable to observe the change in the averages of the ion current over long measuring times and larger sections of the thin section. If jumps occur in the averages of the ion currents, these measurements must be rejected for the purpose of regulation. The regulation should be very robust. However, many experiments have shown that such regulation can be achieved with the measures described here.

(17) In a particularly preferred embodiment, an initial drift is specified for a continuous change in the detector voltage, which is dimensioned, for example 0.002 to 0.005 volts per second, such that no disadvantages for the spectral quality are to be expected even if the drift is over- or underestimated, until sufficient measurement data are available to allow the ion current signal regulation proposed here to take effect. The detector voltage is then changed continuously with this drift value. The change in the average of the (total) ion current over several million measured values (thousands of pixels) in each case is then used as the basis for continuously monitoring whether the drift value specified is sufficient for the compensation or needs to be changed. This embodiment has the advantage that the regulation of the detector gain is never abrupt, but that only occasional adjustments of the drift value are necessary.

(18) FIG. 1 depicts an idealized ion current curve during a scan across five different types of tissue within a thin section with a good drift value setting for continuous adjustment of the detector voltage (x-axis: #pixel; y-axis: ion current in arbitrary units). The sections a1 to e1 represent the total ion currents as the five different types of tissue are being scanned. The scan duration for the 100,000 pixels of conventional size shown can be assumed to be around one hour. Within the individual tissue types, the average of the ion currents remains very constant in each case, but exhibits small jumps from one type of tissue to another due to the difference in molecular content. The molecules of certain types of tissue, for example very lipid-rich tissue, are much easier to ionize and therefore supply larger ion currents (e.g. d1). The change in the ion current averages from one type of tissue to another is chosen to be exaggeratedly large here for illustrative purposes. As already noted above, the average of the ion currents from the different types of tissue can remain almost constant over a scan when large sections of the light matrix ions are also measured.

(19) FIG. 2, in contrast, shows a situation in which the correction of the detector voltage is not optimal because, for example, only the change in the gain of a secondary electron multiplier is corrected, or there is no correction at all, so the measured ion current decreases continuously despite this corrective measure. In this case, the detector voltage must be corrected for the ion current drift.

(20) A sliding average over a few hundred to a few thousand pixels can be formed as the basis for determining a change in the spectral quality, but it is also possible to more simply form a series of averages across a few hundred or a few thousand pixels in each case. The latter shall be called “section averages” here.

(21) There are many ways of determining the correction of the drift value for the continuous change in the detector voltage:

(22) For example, it is possible to simply adapt a straight line to the curves of the sliding average or the section averages across each of the sections of a homogeneous type of tissue, and to use its gradient for the regulation, i.e. the gradient along the sections a1, b1, c1, etc. from FIG. 1 or 2. In FIG. 1, the gradient is zero; in FIG. 2, the average values decrease over time. It is preferable to determine each of the sections between the jumps individually to avoid the singularities at the transitions between the different tissue types.

(23) A particularly successful step has been found to be the formation of a derivative of the averages as a function of time. A preferred method calculates the differences between successive averages in each case, as shown in FIGS. 3a and 3b. The variation of these differences forms a curve which (ideally) should correspond to a Gaussian distribution about a zero point. When the spectral quality is well regulated, the differences should vary precisely about the zero value; if the center of this Gaussian distribution deviates from zero, the drift value for the detector voltage must be changed accordingly. FIGS. 4 and 5 present such distribution curves of the spreads; FIG. 4 for a well-regulated change in detector voltage, FIG. 5 for a drift value requiring correction.

(24) It is also possible to form the sum of the averages of the ion currents over complete scanning lines in each case, and to use the change over the sequence of scanning lines to correct the ion current drift.

(25) The detector voltage is usually controlled via a digital-to-analog converter. Controllers with depths of 14 to 16 bits are used here to achieve a high control accuracy. Despite the fine control, it is found that changing the control by one unit takes many seconds, in contrast to the example above, i.e. it cannot be done continuously, but only incrementally. It can therefore be advantageous to specify after how many seconds the control is to be changed by one unit and to change this timespan, where necessary.

(26) It has been found that bigger changes in the evaluability occur at the start of the measurements than during the remaining measurement period. The causes for this can be manifold and have not been fully explained. It is therefore advantageous to track these changes separately. These changes can particularly be taken into account by observing the amplified ion currents or their average value(s) over shorter time intervals at the start of the measurement, for example over 10 seconds each instead of several minutes in the middle part and at the end of a measurement period.

(27) The invention has been described above with reference to different, specific example embodiments. It is to be understood, however, that various aspects or details of the embodiments described can be modified without deviating from the scope of the invention. In addition to the MALDI method, other types of pulsed ionization such as SIMS can also be used for the pixel scanning of a thin sample section. Furthermore, mass filters or ion cyclotron resonance mass spectrometers are conceivable as the mass analyzer instead of time-of-flight mass spectrometers. The invention should therefore not be restricted to the examples explained. Furthermore, features and measures disclosed in connection with different embodiments can be combined as desired if this appears feasible to a person skilled in the art. Moreover, the description above serves only as an illustration of the invention and not as a limitation of the scope of protection, which is exclusively defined by the appended Claims, taking into account any equivalents which may possibly exist.