text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 23, 2002
|Filing date
|Sep 17, 1999
|Priority date
|Sep 17, 1998
|Fee status
|Also published as
|Publication number
|Mikko Väisälä, Jarmo Nurmi, Jarmo Korpi, Raimo Harju
|Original Assignee
|Wallac Oy
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Referenced by (18), Classifications (15), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
The object of the present invention is a system for measuring biochemical and medical samples, the said measuring being carried out by imaging. The objects of imaging are mainly regular macro-size sample matrices, gels, Petri dishes or completely free-form samples, such as, for example, biological sections. The signal to be imaged is ultraviolet light, visible region light, or infrared light.
Light-producing mechanisms are:
1) luminescence, such as chemiluminescence and bioluminescence, in which case the light p produced by each sample at different points of the sample is measured,
2) fluorescence, in which case the amount of emission light produced by special excitation light at different points of the sample is measured ed, and in addition
3) the amount of reflection, scattering, or absorption at different points of the sample, resulting from the illumination of the sample.
In known measuring devices many different types of sample plates are used, in which the number of wells may vary considerably. In a conventional sample plate there are, for example, 96 sample wells, in which case the amount of solution required for each well is 200 μl. Another typical number of wells is 384 wells in a sample plate, in which case the amount of the solution required for each well is, for example, 200 μl. Although these amounts are small as such, in cases where, for example, 100 000 samples are measured during one sample run, the overall costs are considerable. It obviously makes a marked difference whether 50 μl of liquid or 1 μl of the same liquid is used for one sample, which may, for example, be a single patient sample. The costs relating to the consumption of liquid are directly proportional to the volume used. In the course of measurements, during one set of sample measurements, that is, a sample run, considerable amounts, i.e. several litres of used solution is produced, the said solution often being hazardous waste. The residues of solution often contain radioactive and/or toxic chemical compounds. When numerous sample runs are performed daily (on parallel equipment and in different laboratories), the amount of toxic solution waste produced is considerable. Thus there are obvious reasons to reduce the amounts of solution significantly, that is, in practice to reduce the sample well volume.
To reduce the amount of liquid and to speed up measurements, sample plates with 864 wells are now being used in several measuring devices, in which case the amount of solution required is, for example, about 10 μl. The aim has, however, been to reduce the size of the wells even further. There now already exist sample plates with 1536 wells in which the amount of solution required is now only 5-10 μl, and possibly even as little as 1 μl. In the near future the number of wells will increase further—sample plates with e.g. 9600 wells are being tested in laboratories.
Reducing the size of the sample wells has, however, caused problems, because a small-volume sample requires much better and more efficient measuring properties of the measuring device. Known devices do not usually meet these requirements without extremely complex constructions, or else their measuring times are unacceptably long, which affects the reliability of the measuring results and which also makes the use of parallel equipment compulsory in order to obtain a reasonable overall measuring time for the set of samples.
In luminescence and fluorescence measurements, the aim of reducing the volumes of the wells results in the amount of light from the sample well decreasing in proportion to the volume of the well. In luminescence measurements this means that either the measuring time must be extended correspondingly, or more sensitive measuring devices than the conventional ones have to be used. In fluorescence measurements the situation is different; the amount of fluorescent light produced is proportional to the efficiency, that is, intensity of the light of the excitation light. Especially when operating within the linear range of fluorescence, where the yield of emission light is directly proportional to the amount of excitation light, by doubling the intensity of the excitation light, for example, the amount of the emission signal obtained from the sample, that is, the amount of fluorescent light from the sample will also be double. In a measuring situation such as this it is obvious that the aim will be to increase the intensity of the excitation light considerably, so that shorter measuring times can be used.
In traditional fluorometry, one sample well is measured at a time. In such a case, the excitation light originating, for example, from a xenon flash lamp, is directed by means of focusing optics directly at the sample solution contained in one sample well. Each sample well is measured separately and in the same manner as the previous one.
In imaging, however, the situation is completely different. In this case, where the aim is to image all sample wells at the same time, the excitation light is directed at all the wells simultaneously. The easiest way to do this is by illuminating the entire sample plate with excitation light at one time. However, as the size of the sample plate may, for example, be 80 mm×120 mm, and the surface area of one well in a single plate comprising 1536 wells may be 1.5 mm×1.5 mm, it is obvious that in order for the imaging to be successful, considerably more excitation light is required for imaging than for fluorometry, if the measuring times are to be of the same magnitude.
From this it follows that in fluorescence measurement it is difficult to obtain sufficiently powerful excitation light in the sample plate area, that is, in each sample well. It is also desirable that the uniformity of the excitation light field, that is, its intensity distribution over different parts of the sample plate should be as uniform as possible. The overall sensitivity of the measurement, that is, how small a specific part of a sample will be detected, is determined by that point in the excitation field which has the lowest intensity.
A continuous light source, for example, an arc lamp or a halogen lamp or any other device generating light continuously, is sufficient for prompt fluorescence. However, for time-resolved fluorescence a pulse mode light source is required, for example, a flash lamp or a pulsed laser. The length of the light pulse is of decisive importance for the sensitivity of the device, a property which in turn depends on the decay time of the fluorescence in the sample.
A pulse mode light source may be one of the following:
a) a flash lamp
b) a pulsed laser, such as the combination of an XeCl excimer laser and a dye laser or, for example, a nitrogen laser
c) a combination of a continuous light source and a light chopper; continuous lamps include an arc lamp, a halogen lamp, a continuous laser, and other lamps that produce light continuously.
For example, the light of an arc lamp is interrupted by means of a light chopper in the excitation/illumination path. In practice, the operation of this type of combination is rather ineffective, depending, however, on the application.
Time-resolved fluorescence is achieved by using a combination in which the light source is a pulse mode lamp and the camera acting as detector can be gated. The gating of the camera is a rapid shutter function. This is required because the illumination path leading to the camera must be shut at the moment when the light source flashes. It is only after this that the illumination path of the camera is opened. In practice, the gating of the camera can be done mainly by means of the following combinations of devices:
a) a sensitive camera, in front of which is a mechanical light chopper,
b) a sensitive camera, in front of which is a liquid crystal shutter device, which is triple if necessary.
c) an intensified charge coupled device camera
d) a gatable camera
In prompt fluorescence, it is possible to use a powerful lamp, because it applies spectral filtering. Excitation light and fluorescent light, which are at different wavelengths, can be separated from each other by means of a spectral filter. A disadvantage of prompt fluorescence is, however, that prompt fluorescence also easily comes from other fluorescent parts of the sample than from the fluorescent tracer being measured. This type of fluorescence at another point typically emits light over a wide wavelength region, which means that it is also emitted in the emission wavelength of the fluorescent tracer being measured. Since the optical filter in front of the camera has been selected according to the wavelength of the tracer, prompt fluorescent light originating from another single point than the tracer being measured can also enter the camera. In the image, these points may overlap, which sometimes makes it impossible to say, when analysing and observing the image, whether the signal comes from the fluorescent tracer being measured or from other interfering fluorescent light in the sample, which causes a background signal. Another disadvantage of prompt fluorescent imaging is that excitation light may also enter the camera, which causes more interfering background signals, which further reduces the measuring sensitivity of the measurement, that is, imaging.
In the imaging device relating to the invention, which does not apply time-resolved imaging as in prompt fluorescence and luminescence imaging (another embodiment), there is a dimmer in place of the chopper 13. In this case the optics also incorporate a mechanical shutter controlled electrically, pneumatically or otherwise, or a liquid crystal shutter device to shut the illumination path leading to the camera before the imaging signal is read electrically on the ccd matrix of the camera, which signal is formed into a digital image.
For imaging time-resolved fluorescence, a light chopper is needed in front of the detector, that is, in this case a sensitive, cooled ccd camera, by means of which the emission light path is interrupted for the short interval during which a short fluorescent excitation light pulse originating from a pulse mode light source is directed at the sample. After this the light chopper opens the illumination path leading to the camera to allow the passage of the long-living fluorescent light emitted from the sample to the camera. The chopper may, for example, be a mechanical rotating light chopper or a liquid crystal shutter device (LCD), which is most preferably located on the aperture plane of the imaging optics, which means that light chopping takes place in a controlled manner, without disturbing shadows or dark areas or other irregularities attributable lighting, being formed in the image.
If a separate light chopper is not used, time-resolved imaging can be performed by using an Intensified Charge Coupled Device (ICCD) camera.
It has been shown in practice that time-resolved (TR) fluorescence measurement is advantageous in many respects. If sufficiently short and powerful excitation light intensity is obtained for the sample in this measurement, there will also be sufficient time for measuring fluorescence after the excitation light has been switched off. However, for the above reasons, the reduced size of the sample wells and the increased number of wells have caused problems.
It is difficult to obtain uniform and sufficiently powerful excitation lighting over the entire sample plate area. At the same time, the exposure time should nevertheless be short enough to enable efficient measurement immediately after the excitation light has switched off. Long measuring times are unacceptable because in such a case the measuring device is too ineffective in practice.
Increasing the power of the excitation lamp has not provided a solution to this problem, because the pulse length of a powerful lamp is relatively long, for example, about 300 ps. The excitation fluorescence of many fluorescence measuring agents is halved already about 200 μs after excitation, and thus the fluorescent light is covered under the excitation light.
Differences between cameras
1. Cooled CCD camera, that is, c-CCD (=cooled Charge Coupled Device): good resolution, wide dynamic range, good sensitivity, difficult to gate
2. Intensified Charge Coupled Device or ICCD camera: limited resolution, limited dynamics, good sensitivity, easy to gate.
The lens systems used in connection with excitation light sources also cause problems when the size of sample wells is reduced. Normally, the light emitted from the lens disperses so that the beams of light enter the sample well at an oblique angle, which means that the sample wells in the centre of the sample plate are obviously illuminated in a different manner than the sample wells at the edges of the sample plate.
The efficiency of measurement is also impaired by the fact that as the number of sample wells increases, the intermediate walls of the sample wells make up an increased relative proportion of the sample plate surface area. From this follows that the sample fluid in the sample well obtains too small a proportion of the excitation light. To achieve uniform and sufficiently intensive excitation lighting, the light beams must enter the sample well at a sufficiently small angle. According to one embodiment, a small entry angle of the excitation light beams is obtained by positioning the light source sufficiently far away from the sample.
Imaging lens systems
The question here is, therefore, of imaging at a fairly considerable downscaling-ratio, that is, about 1:5, because the size of the sample plate is about 80×120 mm and the size of the ccd matrix is, for example, 25×17 mm with current technology. It is easy to calculate the above-mentioned downscaling ratio on the basis of these figures. It would be more advantageous if the downscaling required for imaging were not so great, but closer, for example, to the ratio 1:2. However, ccd matrices are not widely available commercially in other than the above magnitude. It is, however, likely that the situation will change in the near future, which means that imaging will become more efficient from the point of view of measuring technique.
When imaging a sample, an image of the sample is formed on the camera on the basis of the emission light (luminescence, fluorescence, absorbancy) emitted by the sample. When using conventional optics such as a conventional objective camera lens, the parallax error of imaging is considerable due to the downscaling ratio used. In such a case the edges of the sample plate are imaged much less effectively than the sample wells in the centre of the plate. In order to be able to eliminate this drawback and to maximize the collection of light, a telecentric lens system should be used in imaging. In this case the sample wells in the centre and edges of the sample plate are imaged with equal efficiency.
According to a dictionary of optics, a telecentric lens is: "a lens in which the aperture stop is located at the front focus, resulting in the chief rays being parallel to the optical axis in the image space, i.e. the exit pupil is at infinity" (The Photonics Dictionary, 1993: Telecentric Lens).
For reasons relating to the basics of optical design, the lens system which collects light from the sample plate area must be designed so that at least two types of glass with different refractive indices are used. Moreover, good resolution is required of the optics, since the sample wells are imaged in small size on the camera. This means that the telecentric lens system has, for example, about 20 separate lens elements or even more.
From this it follows in turn that the total transmittance of the lens system is limited mainly to the visible area, that is, the wavelength 400-800 nm. In order to be able to utilize the total transmittance of the said lens system, the lens elements must in practice be coated with Anti Reflex coating film (AR-coating). If the said AR-coating films were not used, total transmittance would remain rather low.
In the HTS method, the use of fluorescent label molecules is restricted by the large number of samples; measurement results remain unreliable when using fluorometers because with large numbers of samples the output of the excitation lamps decreases, resulting in the need to service instruments even during operation. If, however, a combination consisting of several flash lamps is used, the lamp unit will be sufficiently powerful and the intervals for changing the lamp bulbs will be sufficiently long.
The aim of the invention is to achieve a versatile and efficient imaging device for luminescence, prompt fluorescence, time-resolved fluorescence, and absorbancy, that is, as for transmittance and photometric measurements.
The aim is to carry out mainly the following types of measurements with the device relating to the invention:
1. Prompt fluorescence
2. Time-resolved fluorescence (TR fluorescence)
3. Fluorescence polarization
4. Luminescence, such as chemiluminescence and electroluminescence
5. Absorbancy
6. SPA (=Scintillation Proximity Assay)
The present imaging device provides a solution to the problem of how to realize a sufficiently powerful lamp unit which produces a sufficiently short light pulse. This type of unit comprising several flash lamps is the only possible lamp for many time-resolved fluorescence measurements. One example of these is homogeneous fluorescence assay LANCE, which is a trademark of Wallac Oy. No such powerful lamp is known with which measurement can take place within a sufficiently short measuring time. The measuring time should be short, e.g. 2 minutes, in order for the total measuring time in High Throughput Screening to be acceptable. The total number of samples in one HTS run may be, for example, 100 000 samples, that is, sample wells. Measuring such a large number of sample wells by traditional fluorometric means would take an extremely long time. The amount of hazardous waste produced would also be considerably higher. A combination of a powerful pulsed laser and a dye laser could in principle be used, but it is an unnecessarily complex and uneconomical device which is also difficult to service.
The intervals for changing the lamp bulbs of the light source become extremely long when a lamp combination consisting of flash lamps is used. This is of considerable importance in HTS runs where the aim is to minimize the number of stoppages. When using conventional continuous, that is, continuous wave lamp units, for example, mercury or xenon arc lamps or halogen lamps, the lamp changing intervals are without exception too short considering the demands of HTS screening. One of the few possible solutions when using continuous lamps would be that the lamp could be changed automatically, but it is rather difficult to arrange this to function reliably.
The device relating to the invention combines the absorption measurements of both the near ultraviolet region and of visible light, or, vice versa, transmittance measurements, that is, in practice photometric measurements: the absorption of near UV is required especially in the applicant's LANCE measurements. Absorption measurements in the visible region are in the nature of standards in terms of measuring technique.
The difficulties in using both absorption modes, such as near UV and visible region light mainly concern optics. It is almost impossible, or extremely uneconomical, to construct a telecentric lens with such a high light-collecting efficiency that would transmit effectively the wavelengths of both near UV and visible region light. Typically, a lens system which collects light efficiently and has good resolution can be made either in the near UV region, or alternatively in the wavelengths of visible light. This means that typically only absorption imaging in the visible region or absorption in the near UV light region are possible. In the application relating to the invention, on the other hand, a special scattering plate is used by means of which both near UV and visible light absorption measurements can be imaged.
The light source solution relating to the invention is advantageous for measuring numerous samples done by imaging, for example, especially in demanding applications such as HTS screening and both time-resolved and prompt fluorescence measurements. The device is able to image absorption measurements efficiently both in the near UV light region, that is, in 300-400 nm, and in the visible region, that is, 400-800 nm.
The size of a commonly used sample plate is 120×80 mm, which is imaged at one time, and a digital image is produced from it. In the example device, a sample area as large as 240×160 mm can be imaged in four parts one after another. The four images obtained in this way are combined into one by means of software. The sample may otherwise be of any shape, so long as it meets the boundary dimensions of the example situation. The amount of light produced by the sample and the desired resolution of details are decisive as regards the imaging efficiency of the device. The thickness chosen for the sample in this case equals plate thicknesses within the range of 0 . . . 30 mm.
In the device relating to the invention there are the following possible ways of illuminating or exciting the sample:
1. The sample can be illuminated and/or excited from above and/or below.
2. The sample can be imaged from above or below.
For measuring (imaging) the sample plate from below, the device is turned upside down.
Properties of the illumination system relating to the invention:
1. High average total pulse power
2. Narrow pulse width, which is required in the embodiment used as an example
3. The use of several lamps makes it possible to select the pulse energy of a single lamp so that the fluorescent sample will not become saturated. If a single lamp with high pulse power were used, the sample might become saturated, which would mean a sharp drop in the sensitivity of detection.
4. The use of several lamps also means that even if there should occur a fault in a single lamp, the overall efficiency remains almost unchanged in proportion to the number of lamps.
5. Using several pulse mode lamps also means that the intervals for changing the lamps are extremely long, for example, about six months. This is due to the fact that a pulse mode lamp is switched on only when it is really used. There is no wastage.
If, for example, a continuous arc lamp is used instead, the lamp will have to be changed at least at one month intervals. This is due to the fact that a continuous lamp is switched on all the time, that is, also when there is no sample in the sample space. It is not worthwhile, nor possible, to switch a continuous lamp off for example for a 15 minute waiting period, because the lamp reignites slowly due to the increase in the internal gas pressure inside the lamp, which is due to the warming up of the lamp following its use, this making it difficult, or even impossible, to reignite the lamp . In addition, stabilizing the arc lamp, that is, stabilizing its power within a desired wavelength region cannot be performed successfully if the lamp is switched on and off, for example, at 15 minute intervals.
A powerful light source is required in order for fluorescence measurement to be efficient. According to one embodiment, sufficiently strong excitation light is produced by using several lamps for the illumination, which can be switched on either simultaneously or successively by phasing.
If the lamps are switched on simultaneously, the simplest way is to position all the lamps in such a way that they can be directed towards the centre of the sample plate, and arrange the travel of the light beams by means of lenses so that the illuminating beams emitted from the lamps are distributed evenly across the entire sample plate from each lamp separately, but at the same time.
According to one embodiment, the lamps are situated on the circumference of a circle and their light is directed towards the centre of the circle, where there is a polygon mirror. In the polygon, there is an individual reflection plane for each lamp, the said plane reflecting the light beams of the lamp towards the centre of the sample plate. By means of the lenses, the light is distributed evenly over the entire sample plate area.
If the lamps are switched on successively by phasing, according to one embodiment the lamps are also situated on the circumference of a circle and their light is directed towards a rotating, for example, ellipse-shaped mirror in the centre of the circle. The rotating mirror is in an inclined position, for example, at a 45 degree angle, so that when the mirror rotates, it reflects the light from each flash lamp in turn onto the sample plate, preferably along the same optical path. This is implemented by triggering each flash lamp at the precise moment when the rotating mirror is in such a position during its movement that the illuminating beam emitted from the flash lamp at the moment of triggering strikes the reflecting surface of the rotating mirror so that the light beams are directed further towards the sample plate. The speed of rotation of the rotating mirror should not be too high, so that the random time lag resulting from the minor natural inaccuracy of the triggering moment between the triggering event and the flash of light from the lamp will not hinder the travel of the light beams in the desired optical path towards the sample plate. While the mirror is still rotating towards the next flash lamp, this lamp is being triggered in the same manner as the previous lamp and the light beams from this next lamp are directed in the same manner as above towards the sample plate. This function takes place at each individual flash lamp while the mirror is still rotating, with the result that during one full rotation of the rotating mirror, each flash lamp is triggered once, that is, each of the lamps has flashed once at precisely the appropriate moment, which corresponds to that point on the rotational angle of the mirror at each individual lamp, where the light beams are directed towards the sample plate.
The function described above only succeeds when pulse mode light sources are used which can be triggered. In such a case each flash lamp is controlled at full power which means for each individual lamp that the electric pulse fed to each individual lamp is exactly as high as the frequency complying with the rotation of the rotating mirror, that is, as high as the frequency, or cycle time, allows for each individual lamp. For example, if the speed of rotation of the rotating mirror is 6000 rpm (roots per minute), that is, one rotation lasts for 10 milliseconds, and if a lamp unit using, for example, eight flash lamps is used as a light source, in that case each of the eight lamps is triggered at 10 millisecond intervals, each at the precise moment when the rotating mirror is at the individual lamp in question. Since each individual lamp is triggered at 10 ms intervals, this means that each lamp is triggered 100 times per second, that is, at a frequency of 100 Hz. In the device application relating to the example a flash lamp with a maximum permitted average power of 50 watts (W) is used. In such a case, when the lamp is used at a frequency of 100 Hz, this means that 0.5 joules of energy is supplied to the lamp at each triggering. The said amount of energy is thus supplied to each lamp separately at the precise moment which corresponds to that point on the rotational angle of the rotating mirror at which it is at the individual lamp. Thus, in this example, each individual lamp is supplied with 100 Hz, that is, at intervals of 10 milliseconds, 0.5 joules energy is supplied to each lamp, which corresponds to an average power of 50 watts per each lamp separately. From this follows that the overall output of the lamp system as a whole is on average eight times 50 watts, that is, 400 watts. The overall output is, therefore, directly dependent on the total number of lamps.
In practice this means that each lamp is controlled, or triggered, with the highest average power permitted, that is, with 50 W. Due to the movement of the rotating mirror, each of the eight lamps in the lamp unit can be used at the full power permitted. In this way an average power output of 400 watts is produced by the system of eight lamps relating to the example.
The length of the pulse of a flash lamp is mainly only about 1 microsecond. However, the light pulse has a "tail" extending up to about 50 microseconds, the said tail limiting the sensitivity of measurements when the aim is to achieve extreme sensitivity. The operation of the flash lamp can be made more efficient by incorporating a so-called "tail light chopper" in the lamp unit. In that case each excitation light pulse should be shortened by positioning a device which functions like a fast shutter in front of the lamp, in order to cut off the excitation illumination path after each excitation light pulse. Thus, if the operation of this type of chopper were rapid enough, the light tail of the lamp could be cut, for example, 3 μs after the moment of triggering the lamp, and in this way the flash lamp would produce an excitation light pulse lasting at longest 3 ps in total, which would extend the scope of application of the said lamp.
When measuring the fluorescent homogeneous sample referred to in the invention, it is sufficient if, for example, the light tail of the lamp is cut off about 30 μs from the moment of triggering the lamp.
The tail light chopper can be implemented in several ways. Its operation can be combined with the operation of the rotating mirror, in such a way that if the rotating mirror rotates fast enough, due to this movement of the mirror, the tail of the light pulse is cut off timewise as the rotating mirror rotates further towards the location of the next lamp. In time-resolved measurements it is also important that enough time remains after excitation for measuring the emission signal before the next excitation pulse is directed at the sample. The rotating mirror can be rotated faster, in which case the cutting of the light tail becomes more accurate. In such a case it may, however, be more advantageous if the next lamp being triggered is the one after the next (in other words, every other lamp in the lamp system is triggered), in order to utilize the emission light of the fluorescence in an efficient manner. In this case there should preferably be an odd number of lamps in the lamp system, so as to make use of all the lamps. If there is an even number of lamps in the unit and the type of lamp triggering method described is to be used, care should be taken that the even number of lamps to be triggered is alternated at regular intervals. The selection can be automated by means of an electric coupling. This means that in a system comprising eight lamps, four lamps (such as, for example, lamps 1, 3, 5 and 7) are triggered first, and then after a regular interval, the remaining four lamps (that is, lamps 2, 4, 6 and 8 would be due to be triggered). When the group of lamps to be triggered is changed at regular intervals, all the lamps are used up evenly.
The structure of the lamp unit is not limited by the requirement of having an even or odd number of lamps.
The lamps in the lamp unit can also be triggered in other ways. The order of triggering can in itself be selected freely, because the selection is done by means of an electric circuit coupling or the program command of a computer processor, or a combination of these, and because this may give the advantage that the method of use, or the point of operation of an individual lamp can be made more appropriate considering, for example, the lamp's service life or its UV light production, than by triggering the said lamps in strict order as described above.
Structurally, the tail light chopper may also be a separate mechanical light chopper which is located after the lamp so that the length of the aperture or the lengths and shapes of the apertures in it determine the true length of the light pulse. This type of tail light chopper is timed, that is, synchronized with an emission light chopper and the rotating mirror situated in the lamp unit, so that these operate together in a synchronous manner.
It is not worth using the rotating mirror system if continuous light sources are used as lamps. This is simply due to the fact that when the mirror rotates, and when each lamp is switched on all the time (because in such a case it would be a question of using continuous wave lamps, that is, continuous lamps), every time that the mirror is not exactly at the individual lamp, the optical power will be directed past the mirror most of the time, and thus not at the sample. On the other hand, if a fixed mirror polyhedron was to be used instead of the rotating mirror, in such a case both pulse mode and continuous light sources could be used. In that case the overall service life of the continuous light sources would naturally also depend on the number of lamps in the system, because whenever the service life of one continuous lamp came to an end, a new lamp would be switched on and operation could continue uninterrupted while the lamp was being changed.
The mechanical light chopper in front of the camera and on the emission light path must be synchronized with the movement of the rotating mirror in the lamp unit. The rotating mirror and the emission light chopper will then always at a certain phase rotate with respect to each other. Phase locking is performed electronically by means of the synchronizing pulses obtained from the emission light chopper and the rotating mirror. The synchronizing pulses are obtained from opto-electronic readers, or so-called N-coders, which rotate with the emission light chopper and the rotating mirror. If a tail light chopper is in addition used in front of the lamp, this also gives synchronizing pulses according to which the electronic unit guides the operation of the rotating means in a controlled manner.
In the solution described, the sensitivity of luminescence has been maximized, taking into account the requirements of structural simplicity. The result is affected by the structures of the large and small telecentric lenses, their coatings and the properties of the camera. For the duration of luminescence measurement, the mirrors in the mirror unit 22 are moved away so as not to obstruct the illumination path leading to the detector 11, and the filter wheel containing filters which is in the filter unit 12 is moved into such a position that there are either no filters in it or else there is a special luminescence filter. The rotating discs of the light chopper 13 are moved by means of the motors rotating the discs and the electric circuit coupling controlling the motors, into such a position that the illumination path leading to the detector 11 is unobstructed.
The invention is described in the following by means of examples, with reference to the appended drawings in which
FIG. 1 shows the device relating to the invention diagrammatically, as seen from the side.
FIG. 2 corresponds to FIG. 1 and shows a second embodiment of the device.
FIG. 3 shows diagrammatically the shutter of the device shown in FIG. 1.
FIG. 4 corresponds to FIG. 3 and shows the shutter in another position.
FIG. 5 shows diagrammatically the light source of the device shown in FIG. 1, as seen from above.
FIG. 6 shows the light source shown in FIG. 5 as seen from another direction.
FIG. 7 shows a diagrammatic section of the sample well.
FIG. 8 corresponds to FIG. 7 and shows the sample well in another situation.
FIG. 9 shows a diagrammatic side view of the excitation illumination of the sample plate so that the travel of the light beams emitted from one individual lamp is shown schematically.
FIG. 10 shows a diagrammatic side view of a second embodiment of the excitation light system where there is a fixed, stationary mirror polyhedron in place of the rotating mirror.
FIG. 11 shows a detail of FIG. 10.
FIG. 12 shows the mirror unit of the excitation light source shown in FIGS. 10 and 11.
FIG. 13 shows a third embodiment of the excitation light system, which is simple, but not as compact as a system which uses a solid dividing mirror.
FIG. 14 shows diagrammatically the principle of measuring fluorescence.
FIG. 15 shows diagrammatically an excitation and measuring principle.
FIG. 16 shows diagrammatically an excitation illumination principle.
FIG. 17 shows diagrammatically another measuring principle.
FIG. 1 shows a measuring device according to one embodiment for measuring, or in this context for imaging, different types of samples, such as, for example, fluorescent homogeneous assays. The measuring device 10 includes a detector 11, an emission filter wheel 12 in conjunction with the detector, and a set of objective lenses for emission 14. The emission objective 14 is comprised of sets of telecentric lenses 14 ab and 14 c. The set of lenses 14 ab comprises two separate sets of lenses 14 a and 14 b.
The lens groups 14 a and 14 b of the set of lenses 14 ab are situated on either side of the light chopper 13. Between the lens groups 14 a and 14 b is located the optical aperture plane on which the light chopper 13 is situated. The imaging unit 36 includes a detector 11, filters 12, a light chopper 13 and sets of telecentric lenses 14 ab and 14 c. This imaging unit can be moved in the vertical direction to make focusing on samples of different heights possible. The camera 11 also incorporates a vertical fine adjustment movement 42 for fine adjustment of focusing. This is required when the emission filter is changed, because the light wavelength region to be imaged usually also changes, which means that the image of the object forms at a slightly different distance from the object in each wavelength region.
The filter wheel 12 can also be replaced by individual filters which are located in a slide or a separate filter storage unit. This unit may, for example, have shelves for different filters and a lift or other type of filter changing apparatus, which fetches and puts the desired filter into place in the measuring device, if required.
The measuring device 10 comprises a light source 15 with several flash lamps 27 and condensing lenses 30 in front of them. The light from the flash lamps 27 is taken via the excitation light filter wheel 18 to the excitation light objective 19 by means of a rotating mirror 28. The wavelength region of the light emitted by the flash lamps 27 is so wide that the desired light wavelength band, which may be either a UV band or a visible region band, can be supplied to the sample by means of the excitation light filter wheel 18. The desired band width is selected by means of a filter. Should polarized light be required in the measurement for exciting or illuminating the sample, the filter wheel also incorporates a polarization filter, either as a separate filter or combined (simultaneously) with a band filter.
In the excitation illumination path there is also a partly reflecting mirror (e.g. 0.2% reflecting mirror) 33, which reflects a small proportion of the light through a lens 35 to a feedback photodiode 34. Also, should it be desirable to increase the efficiency of lamps' operation, a tail light chopper 38 can be used.
The measuring device 10 also includes mirror units 21 and 22, fixed mirrors 23 and 24 and, above the sample 17, a double-acting transparent scattering plate 20 for absorption measurement of both visible light and UV light.
The mirror unit 21 is an optical path switch, the mirror 21 a of which is hinged and can thus be turned in front of the optical path of the excitation light, to an angle of 45 degrees. This causes the excitation light to be directed to the sample plate from above via the mirror unit 22.
The mirror unit 22 comprises a dichroic mirror 22 a and a half-transmitting mirror 22 b, which are located in slides so that they can optionally be moved to the illumination path or away from it. The scattering plate is also located in the slide, and can thus be brought into use when necessary. The scattering plate 20 can also be moved vertically in order to press it against the sample plate 17.
In the visible light region, absorption takes place in the sample when the sample is illuminated from below. The scattering plate 20 acts as a scattering plate, which means that an image of the light that passes through the sample is formed on the plate.
Since the lens system of a typical imaging device does not transmit UV light beams well due to the type of glass and the coatings, in the device relating to the invention the scattering plate 20 is double-acting. The plate acts as a fluorescent light wavelength converter plate in absorption measurements in the UV region, which means that visible light is obtained on the detector. Absorption of UV light takes place in the sample, which means that the UV light which is able to pass through the sample to the converter plate is indirectly proportional to the absorption taking place at different points of the sample. The intensity of fluorescent light is directly proportional to the intensity of the UV light that has passed through.
Alternatively, the scattering plate 20 used in the visible light region is replaced by a separate fluorescent converter plate when UV absorption is to be measured.
The sample table 16 of the measuring device 10 can be moved in both the x and y directions in the plane of the sample, so that any sample 17 can be moved to the detector 11 for assay. For movement in the direction of the z axis, the detector 11 is equipped with a height adjustment means. The structure of the adjustment means is not shown in FIG. 1. The sample plates are located in the storage unit 31, from where the desired sample plate 17 a can be transferred to the measuring device for measurement. The bar code reader 32 identifies the sample plates 17 to be transferred to the sample table of the measuring device 10. When the sample plate has been moved in the xy direction to the detector 11, the light shutter 37 closes, which means that the measuring device is light-tight.
In FIG. 2, the device in FIG. 1 is shown upside down. All the measuring methods described above are possible with this device. This means that the pipetting of the sample can advantageously be carried out from above, for example, in flash luminescence measurement (which requires almost real-time pipetting) and/or the excitation of the luminescence of the sample by means of electrodes in electroluminescence measurement.
FIG. 3 shows diagrammatically the structure of the chopper 13 of the measuring device 10 shown in FIG. 1. In this embodiment the chopper 13 comprises two rotating discs 13 a and 13 b, which are mainly circular. On the edge of each disc 13 a and 13 b are formed two notches 25 on the opposite edges of the discs. Correspondingly, the notches 25 could also be apertures formed on the edges of the discs 13 a and 13 b. In FIG. 3 the discs rotate in the same direction and they are positioned so as to partly overlap. The rotational movements of the discs 13 a and 13 b are synchronized with respect to each other so that the notches 25 in the different discs will always meet between the discs. This means that the discs 13 a and 13 b form a light chopper which is used for cutting the emission light path leading to the measuring detector 11, for the purpose of time-resolved imaging.
To increase the speed of rotation of the discs further, the shutter may be underpressurized. If necessary, the underpressurized space is filled with a small-molecule gas, such as helium, in which case the friction on the discs is low, that is, the friction of the gas molecules against the shutter discs is considerably smaller than it would be if the filler gas used in the underpressurized state was air. With lower friction , the rotational resistance also decreases and the rotating motor is able to rotate the light chopper discs faster and/or more easily, and a fast, silent device is obtained.
In FIG. 3 the chopper 13 discs 13 a and 13 b are shown in such a position that the discs rotate clockwise and the shutter aperture 26 formed by the notches 25 is about to open.
In FIG. 4, on the other hand, the chopper 13 discs 13 a and 13 b are in such a position that the aperture 26 is already completely open.
In the embodiment shown in FIGS. 3 and 4 the chopper has two discs 13 a and 13 b and each of them has two notches 25. By means of this structure, the speed of opening of the aperture 26 formed by the notches 25 can be made twice as fast as that of a structure provided with one shutter disc. If necessary, the number of notches 25 in the discs 13 a and 13 b may be even greater, for example, three or four. With the chopper structure shown, the time differences required in time-resolved fluorescence measurement can be controlled accurately between the flashes of the excitation light and the time of measurement. The chopper 13 in a measuring device 10 is most preferably located between the lens parts 14 a and 14 b of the emission lens 14. The set of lenses 14 ab is comprised of two separate lens groups 14 a and 14 b, one of which is located on one side of the light chopper and the other on the other side. Between the lens groups 14 a and 14 b is the optical aperture plane of the set of lenses 14 ab. The light chopper is positioned between these sets of lenses 14 a and 14 b, on the aperture plane.
FIG. 5 shows diagrammatically an example of an embodiment of a light source 15 which comprises four flash lamps 27, each of which is also equipped with a condensing lens 30. In the centre of the flash lamps 27 there is a mirror 28 which is inclined at an angle of 45° with respect to the vertical plane, as shown in FIG. 1. FIG. 1 also shows that the mirror 28 is provided with a rotating motor 29. When the mirror 28 is rotated, it is alternately in each light path leading from the flash lamp 27 while it rotates. In such a case the said flash lamp is triggered electrically, which means that the light pulse, that is, the illuminating beam of short duration which is emitted from the lamp, travels via the mirror surface of the rotating mirror, through the filter 18 and the excitation light objective 19 to the mirrors 21 and 22 or the mirrors 23 and 24, finally reaching the sample. The triggering of a single flash lamp is such a rapid occurrence that the rotating mirror has time to rotate only very little (by a fraction of the angular degree) during the duration of the light pulse emitted from the lamp, and thus this illuminating beam of short duration but high intensity has ample time to be reflected forward from the mirror surface of the rotating mirror.
FIG. 6 shows axonometrically the positioning of the lamps 27, condensing lenses 30 and the mirror 28 with respect to each other when using four lamps.
FIG. 7 shows a diagrammatic section of a sample well in a known device. The figure shows that the light beams enter the sample well 39 of the sample plate 17 at a relatively wide angle α1. This means that the sample wells at different points of the sample plate receive different amounts of excitation light/illumination light. In FIG. 7, the illuminated area is shown diagrammatically by shading with oblique lines.
In FIG. 8, the beams of light enter the sample well 39 of the sample plate 17 according to the excitation illumination method relating to the invention at the angle α2, which is much smaller than that shown in FIG. 7. This means that the sample wells at different points of the sample plate receive substantially the same amount of excitation light illumination light.
FIG. 9 shows a sample plate excitation illumination system which applies a known manner of illumination. Due to this, the illumination of the sample plate 17 is uniform. The beams of light arrive at the sample plate at a small angle α2
FIG. 10 shows an excitation illumination system which consists of a fixed mirror polyhedron 40 in the centre of the circle. The light from the lamps 27 is directed through the lenses 30 to the mirror polyhedron 40, and from there on to the excitation objectives 19 specific to each lamp, which image the aperture planes of the excitation optics on the sample plate 17.
FIG. 11 shows a detail of FIG. 10. The angle of one mirror surface 40 a of the mirror polyhedron 40 is slightly over 45 degrees.
FIG. 12 shows the mirror polyhedron 40 axonometrically.
FIG. 13 shows one embodiment for illuminating the sample plate 17. Above the plate 17 are several lamps 27 and their optics positioned adjacent to one another, the lamps being directed towards the sample plate 17.
FIG. 14 shows a general fluorescence measuring diagram. With a sufficiently low intensity level of the sample's excitation light is achieved the fact that measurement takes place in the linear measurement area 41.
FIG. 15 shows the difference in principle between prompt and TR fluorescence measurements. In the figure it can be seen that the excitation light pulse should be short in order for TR measurement to be successful.
FIG. 16 shows a comparison of the durations of light pulses emitted from different types of flash lamps as a function of time. The lifetime curve 43 of a single powerful lamp is of tong duration. The average pulse 44 obtained with several small lamps is of short duration.
FIG. 17 shows the duration of the fluorescence 45 of a sample. FIG. 17 shows that the fluorescence has already been reduced to a half by the time the light from the powerful lamp has dimmed in FIG. 16. This means that TR fluorescence measurement cannot be carried out efficiently by means of a single powerful flash lamp. FIG. 16 also shows the effect of the tail light chopper 46 placed in front of the lamp for shortening the length of the light pulse. It has, however, been shown in practice that this light chopper does not improve the situation. Despite the chopper, a powerful lamp causes so much background interference that efficient TR measurement cannot be carried out.
A light source according to a preferred embodiment of the invention comprises two or more flash lamps, and the measuring device comprises a timer for switching the lights on simultaneously or timed in phases as desired. The following advantages are achieved by means of the device relating to the invention:
a high average pulse power of the light source
narrow light source pulse width, which is required in certain time-resolved measurements
optical power guarantees sufficiently short measuring times in fluorescence measurement
the intervals for changing the light source bulbs are notably long in comparison to a continuous light source
the structure of the light source is economical in comparison to other light sources with corresponding pulse power
the same device can be used to carry out absorption measurements of both the UV region and the visible region by imaging on extremely high-density sample plates.
According to a preferred embodiment of the invention, the light source of the measuring device comprises a rotating mirror on the different sides of which the flash lamps are located so that when the mirror rotates, the light beams from the lamps are directed one at a time, and the light beams from all lamps thus once during one rotation of the mirror, via the rotating mirror at the sample, either directly or through other optical means such as mirrors.
The light source of one preferred measuring device comprises eight flash lamps, which are at an angle of 45° with respect to each other, between which lamps a mirror provided with a rotating motor and a vertical rotation axis is placed, the said mirror being inclined at an angle of 45° with respect to the vertical plane.
The frequency of each flash lamp in the light source is, for example, 200 Hz, and they are controlled to flash at an interval of 0.625 ms with respect to each other, so that during a 5 ms period, a total of eight flashes of light are obtained.
In the light source of the measuring device the lamps may also flash simultaneously, in which case the directions of the illuminating beams are controlled by fixed mirror surfaces instead of a rotating mirror. If, therefore, the flash lamps are arranged to flash simultaneously, a rotating mirror is not used, but a fixed mirror system instead.
The measuring device preferably comprises a light chopper, which is synchronized with the timer of the light source. The chopper comprises, for example, at least two rotating discs or the like, on the circumference of which, or its vicinity, there is at least one notch or opening, and which discs are positioned so as to overlap at least partly, so that as the discs rotate, the openings in the adjacent discs form the chopper aperture when they meet at the same point.
In one embodiment the chopper comprises at least two discs rotating in the same direction, the said discs being mainly circular and at least two notches or openings being formed on the edges of both discs, at opposite edges of the discs.
Equipped with the components shown in FIG. 1, the measuring device 10 is a multifunction device, with which, for example, the following assays can be carried out:
In a fluorescence assay, the light emitted from the light source 15, which light is continuous wave light or pulse light, is directed towards the sample 17 through an objective 19 and mirrors 21 and 22. Both a dichroic mirror 22 a and a half-transmitting mirror 22 b may be used. Fluorescence measurement is carried out by means of a detector 11 and an emission objective 14. If the light source is a flash lamp, the light chopper 18 is in that case permanently in the open position. If, on the other hand, the light source is a continuous wave lamp, an electrically controlled shutter is used in place of the light chopper.
In the example shown in FIG. 1 the light source is comprised of four flash lamps 27, which means that the intensity of the light of the excitation light source 15 is four times greater than with a single lamp. In the example case, the flash lamps 27 are arranged to flash in phases one after the other, in which case the rotating mirror 28 is used to direct the light emitted by the lamps 27 at the sample 17. Directed via the mirror 28, the light beams of all lamps 27 can be directed in turn along the same optical path.
In time-resolved fluorescence measurement the light source 15 is always a pulse mode light source (e.g. a flash lamp), the light emitted from which is directed towards the sample 17 through an excitation objective 19 and mirrors 21 and 22. Depending on the measurement, either a dichroic mirror 22 a or a half-transmitting mirror 22 b is used. In time-resolved fluorescence measurement, a light chopper 13 and a detector 11 are needed in the emission light path when the detector used is a cooled CCD camera. If, on the other hand, the detector 11 is an Intensified Charge Coupled Device camera, or ICCD camera, a gatable intensified charge coupled device camera is used as a light chopper and no mechanical light chopper is required.
In the example shown in FIG. 1, the optical power of the excitation light source 15 comprising four flash lamps 27, which flash at different phases, can be made four times greater than with a single flash lamp.
By using separate small flash lamps, the excitation illumination period of the measuring device can be determined much more accurately than by using one large lamp, because the illumination time of a small lamp is considerably shorter than that of a large one. The background interference can also be reduced because the "tail" of a single powerful flash lamp is relatively of much longer duration than that of a less powerful flash lamp.
When measuring, that is, imaging luminescence, the mirror unit 22 is not in use, and thus the dichroic mirror 22 a or the half-transmitting mirror 22 b are not in use. The scattering plate 20 is not in use either. However, filters, a shutter and a dimmer may be in use if necessary.
If, for example, luminescence is measured and there is no light chopper in the device, a solid black plate is used as a camera shutter in place of the filter in the filter wheel, by moving which plate away from the front of the camera and correspondingly to the front of the camera, a shutter-like on-off action can be obtained in the camera.
In a photometric assay the light emitted by the light source 15, which is continuous wave light or pulse light, is directed by means of mirrors 23 and 24 below the sample 17. According to the example shown in FIG. 1, the light source 15 comprises four flash lamps 27 which flash at different phases in order to increase optical power. Photometric measurement is carried out by means of a detector 11 and an emission objective 14.
Absorption measurement is carried out by using visible light from below the sample and by means of a scattering plate 20.
In shadow imaging of UV absorption, the light source 15 is a UV light source, the light emitted by which is directed by means of mirrors 23 and 24 onto the underside of the sample 17. According to the example of an embodiment shown in FIG. 1, the light source 15 comprises four flash lamps 27 which flash at different phases. Since the wavelength range of the light emitted by the flash lamps 27 is wide, UV light can be directed at the sample 17 by means of the filter 18. Above the sample 17 is placed a light wavelength converter plate 20, which converts the ultraviolet light into visible light, the intensity of which is measured by means of a camera and which is proportional to the amount of UV light directed at the converter plate, that is, it is proportional to the absorption of UV light. If necessary, an opal glass may also be positioned below the sample 17. In such a case the shadow imaging of UV absorption is carried out by means of a detector 11 and an emission objective 14.
The light source 15 of the example of an embodiment shown in FIG. 5 comprises four flash lamps, where the rotational frequency of the rotating mirror is 200 Hz. This means that the interval between two successive pulses of one lamp is 5 ms. According to the invention, the lamps are controlled so that they flash at a 1.25 ms time difference with respect to each other, which means that during one 5 ms period, a total of four flashes of light are obtained. The combined pulse frequency of the lamps achieved with the construction described in the example is in this case 800 Hz. This is four times greater compared with that obtained with a single lamp. By means of this solution the optical power of the light source 15 is quadrupled compared with the optical power obtained with one lamp. When even greater optical power is required, the number of flash lamps can be further increased, for example, to eight flash lamps.
In the example of an embodiment shown in FIGS. 3 and 4, the speed of rotation of the chopper 13 discs 13 a and 13 b is 24000 rpm, that is, the rotational frequency is 400 Hz, in other words the wheel rotates at 400 rpm. If the light chopper disc has two apertures 26, it will open 48000 times per minute, that is, 800 times per second. The opening frequency of the apertures is in that case 800 Hz.
Since one flash of light emitted by the flash lamp should correspond to each aperture of the light chopper, the total frequency of the pulse light source should also be 800 Hz. This means that in the example of the embodiment shown in FIGS. 4 and 5, it is advantageous to modify the light source so that it incorporates eight flash lamps flashing at 1.25 ms intervals. The frequency of each individual lamp is 100 Hz, that is, an individual lamp flashes 100 times per second. This means that the rotational frequency of the rotating mirror should be 100 Hz, or 6000 rpm. During one rotation of the mirror, each individual flash lamp is triggered always at 1.25 ms intervals, which means that when the rotation is completed, 8 times 1.25 ms, that is, 10 ms have elapsed, which corresponds to a frequency of 100 Hz. During one rotation in this case eight flashes of light are obtained and the combined pulse frequency of the lamps is 800 Hz. The frequency of a single lamp is still 100 Hz.
One flash of light from the flash lamp occurs during each interval in the opening of the apertures of the light chopper, and during the next interval the next lamp flashes etc. What is essential is the synchronization between the lamps and the camera. In the device relating to the example, one flash occurs between the openings of the apertures. The flash of excitation light takes place, for example, about 10 μs before the aperture begins to open.
Four apertures in the chopper discs, speed of rotation of chopper discs 24000 rpm, flashing interval 1.25 ms/2=0.625 ms, eight lamps in the light source, speed of rotation of rotating mirror 12000 rpm.
If the sample plate has 384 wells, the excitation time is 1 s and the time spent for reading the light intensity data on the camera's ccd matrix electronically, that is, the reading time, is 30 s for a full resolution image, then the sample is subjected to 800 excitation light pulses when the overall frequency of the excitation light source is 800 Hz. If the sample plate has 1536 wells and volume of a single well is 5 μl, 8000 excitation light pulses will be required. The measuring time is in this case 10 s and the reading time 30 s. If the volume of the well is 1 μl and there are 1536 wells in the sample plate, 40000 pulses are required, that is, a measuring time of 50 s. A short (reading) time reading, that is, a 2 s fast reading, is also possible in all measurements, but in this case the noise increases, markedly impairing the end result of imaging.
Instead of the mechanical chopper shown in FIGS. 3 and 4, a liquid crystal shutter device, or LCD, can also be used. It is, however, slower and its light dimming capacity is not sufficient for all applications, but it may allow light to pass through, which means that the proportion of the disturbing background signals of the number of detectable signals increases. Another disadvantage is its sensitivity to temperature, since the wavelength band of the light passing through is dependent on the temperature of the component.
The cooled CCD camera (c-CCD) and the light chopper can be replaced by an Intensified Charge Coupled Device, or ICCD camera. Its resolution is, however, poorer and its dynamics inferior to those of a c-CCD camera.
On the aperture plane, that is at the location of the chopper, a dimmer and shutter may also be substituted for it.
The chopper 13 discs 13 a and 13 b may also be stopped at exactly the desired point, which means that the chopper is suitable for luminescence measurement.
If the excitation light power is a 0.2 J/pulse, then at a flash frequency of 100 Hz of one lamp, an average power of 20 W is obtained for one lamp. If there are eight lamps in the device, the average power is 160 W.
The chopper disc has four apertures and there are eight flash lamps in the lamp unit. When the lamps are triggered, 0.2 joules of energy are supplied to each one in turn. This means that the frequency of each individual lamp is 200 Hz, in other words the overall frequency of the entire lamp system is 1600 Hz, which means that an average output of 320 W is obtained with the lamp unit.
Depending on type, the service life of an arc lamp is typically about 300-1000 h, which means a maximum of about 40 days' use, after which the lamp has to be changed. After the said period of use, the intensity of the lamp has decreased to about a half, and at the same time the inner surface of the globe of the lamp has typically become covered in metal detached from the electrodes and other impurities. A lamp which has become cloudy on its inner surface will heat up more than its normal use would require and may even cause a risk of explosion of the lamp if the lamp is not changed in time. This is due to the fact that while arc lamps are switched on, there is considerable overpressure inside them, which rises the hotter the temperature at which the lamp has to operate as it ages, and this causes the risk of explosion. With flash lamps, on the other hand, there is no such direct risk, or it is at least substantially less, because when a flash lamp is switched on, its pressure is substantially lower than that of an equally powerful arc lamp and may even be close to normal air pressure.
In the device relating to the invention, the highest power permitted for the flash lamps used is 0.5 J. The lamp may, however, be used at a lower voltage, which lengthens its service life considerably. This gives it a service life which is many times longer than that of an arc lamp. The same output is obtained from the lamps, but the changing interval is prolonged. This means longer service intervals. Another advantage of the device is that even if one or more lamps blow, this does not mean that measurement is discontinued. The blown lamp is detected by power measurement and compensated for by the programme, which means that the measuring device will still function well.
The device relating to the invention makes it possible to obtain an excitation light distribution that is uniform in intensity and applicable to the fluorescence measurement of macro-size sample plates, which distribution it has previously been difficult to achieve while striving to keep the duration of measurement within reasonable limits.
It is obvious to a person skilled in the art that the different embodiments of the invention may vary within the limits of the claims presented below.
|Cited Patent
|Filing date
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|Kozponti Elelmiszeripari Kutato Intezet
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|Spectroscopic analysis
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|Chen-Yu Tai
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|Trey Enterprises Corp.
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|Olympus Corporation
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|Jurgen Muller
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|Biospace Mesures
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|U.S. Classification
|356/417, 250/458.1
|International Classification
|Cooperative Classification
|European Classification
|Dec 7, 1999
|Sep 30, 2005
|Fee payment
Year of fee payment: 4
|Oct 23, 2009
|Fee payment
Year of fee payment: 8
|Oct 23, 2013
|Fee payment
Year of fee payment: 12


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*You can also use the HD-E4100 with the Rhin-O-Tuff HD4101 EZ Book Former.
*The E4100 can attach to the top of your binding punch to save space in a crowded office or production area.
*The HD-E4100 can be used as a stand alone coil inserter or can be mounted as a module to any vertical Rhin-O-Tuff Punches.


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|Community News - Community News
|Written by Muslim Link Staff
|Wednesday, 08 December 2010 13:09
Area Leaders See Social Disease CausingFrustration, Depression, Broken Marriages
Over the last decade, the Muslim Link has reported on social diseases which have taken root in the Muslim community. Widespread in the United States, the presence of ills like domestic violence and homelessness among Muslims is now acknowledged by community leaders, and – albeit at a slow pace – some masajid and Islamic organizations are devoting resources to address these ills.
Comments (8)
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My Guitar
Borce Trajkovski
- January 7, 2015
( 37,419)
♩♪♬ My Guitar ♬♪♩
The best virtual guitar simulation for Android™
Play guitar on your device like on a real guitar!
The best virtual guitar simulation for Android™
Play guitar on your device like on a real guitar!
■ 4 guitar types
- steel acoustic
- nylon acoustic
- clean electric
- muted electric
■ 4 play modes:
- solo mode
- tapping mode
- chord mode
- combined mode
■ all 325 chords
■ studio quality sound
■ multi-touch support
■ integrated recorder
■ 6 sound effects
■ play velocity
■ string muting
■ string damping
■ string bending
■ string animation
■ right and left handed
■ 5 neck colors
■ 4, 5, 6 or 7 frets
■ an capo option
■ virtual vibrato arm
■ virtual volume knob
■ chords load & save
■ tracks load & save
■ MIDI export
■ MIDI over WiFi
■ MIDI note velocity
■ MIDI note damping
■ MIDI volume control
■ MIDI pitch bend
■ tablet support
■ advanced vibrations
■ fullscreen on 4.4+
■ free
NOTE: Unlike the other available guitar apps this app can actually be used to play like on a real guitar (in solo mode), holding the strings with one hand and strumming with the other. Also, for those that don't know how to play a real guitar there is a default "tap mode" that plays the guitar by tapping on the frets. In "chord mode" you can program a chord to be played when you strum on a particular fret. In the innovative "combined mode" you can program a chord to be played when you strum on a particular fret but only when you start strumming from the first string. After you play the chord you can tap on the frets to play like on a "tap mode" enabling to play songs's chords and solo parts simultaneously.
THIS IS FIRST IN MY BOOK! Best guitar app ive seen. i love how even the neck width changes so now i can play the songs that i play on my real guitar easily. This is awesome. it is also sooo much more portable than my guitar.
Does what I want but not right size I wanted a fretboard I could practice chords to help memorize finger positioning. I have a "Phablet" (Note2) and the phone setting was too wide. The Tablet settings made it just slightly too small. Maybe you could allow for more size options or make it so we can adjust on screen by pinching fingers to zoom in and out until it's just right and then be able to save the size in preferences. If you can do that, I'll give it 5 stars.
AWESOME!!! I love it! Even though I have an actual guitar, I am definitely having fun on this one. To help me sing, I can use this instead to find the right notes. And yes, I can read staff not tabs.
Awful The guitar makes an out of tuned sound and it doesn't even play the notes I try to play correctly. I thought I would be able to use this app while my real guitar was in repair... Now I know its probably better to just be patient.
Lol love this game So whant to play guitar well look for your self to see a fun game to play😜
Simply Awesome !!! This is the first time m rating an app on google.. Hats off... amazing speechless.. a big thanks dev team..
What's New
V2.1 changes: Bugfixes.


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This year's Congress has 27 speakers coming from around the world. There are English Barristers coming from London, a Venezuelan lawyer also from London, a Ph. D. professor coming from Dubai, UAE, a Belgium attorney/mediator form Paris, as well as attorneys and professors coming form New York, California, Chicago and Des Moines. It is an array of speakers never before assembled in Des Moines, Iowa. Please plan to attend and support this important event.
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text:
- Genre:
- Action Games
- Release Date:
- 1/11/2005
- Publisher:
- LucasArts
- Developer:
- Pandemic
- Gamesite:
- Click Here
By: mechgod
a good game
Views: 200 | Posted: 05/25/2011
By: metzman
This is a cut together ingame-material montage from Video Blog #19 of Mercenaries 2. I edited all the footage together in a nice order and sticked a matching Mercenaries OST track to it. Hope you like it.
Views: 11,730 | Posted: 05/25/2011
By: wouterdeweer
a video of the E3 2008: Mercenaries 2; world in flames
Views: 405 | Posted: 05/25/2011
By: Scarrier
EDIT: I know the quality sucks. I used a low-resolution digital video camera and a TV when I made it, but at least it's possible to see what goes on. This is a short video clip from the game "Mercenaries: Playground of Destruction"(Copyrighted by LucasArts and Pandemic Studios). It includes music by Sons of Angels (a.k.a Crush 40), if anyone knows who they are.("Rush into the Crazy World" on their album "Sons of Angels - Thrill of the Feel") Video was made using my AIPTEK DV4500 DVC and Ulead Video Studio 7, which came with my camera.
Views: 1,119 | Posted: 05/25/2011
By: johanks86
Mercenaries 2: World In Flames Gameplay Vid. Enjoy Mushroom Lovers!
Views: 933 | Posted: 05/25/2011
By: johanks86
A new video from Mercenaries 2: World In Flames for Xbox 360 & PlayStation 3.
Views: 557 | Posted: 05/25/2011
By: MACHCLASSIFIED
Trailer for Mercenaries 2, a game I am very excited about.
Views: 860 | Posted: 05/25/2011
All That and a Bag of Chips- Take me to your leader Tell me what you think. Thanks.
Views: 892 | Posted: 05/25/2011
By: tigersnake86
New Mercenaries 2 Trailer original link here: http://www.youtube.com/watch?v=A_KZwIg10iU [url=http://tigersnake86.gametrailers.com/gamepad/index.php][img]http://users.gametrailers.com/showimage.php?id=93705[/img][/url]
Views: 2,294 | Posted: 05/25/2011
By: Tryzon
If you havent seen the first, just look for my other vids. Anyway, this shows what happens once I get bored of the dangling chopper. Brief hilarity ensues.
Views: 933 | Posted: 05/25/2011


text:
Border Battle
Families Of The Missing In Baja California Protest Students' Disappearance
- Nov. 10, 2014
- By Jill Replogle
The disappearance of 43 students from a rural teaching college in central Mexico has galvanized family members of missing persons in other states, like Baja California.
Dozens Arrested In Imperial County Drug Bust
- Oct. 21, 2014
- By Jill Replogle
Authorities arrested 43 people in predawn raids Tuesday that prosecutors said are linked to a large-scale methamphetamine and heroin trafficking organization in Imperial County.
Second Leader Of 'Los Palillos' Gang Gets Consecutive Life Sentences
- Oct. 15, 2014
- By City News Service
The "Los Palillos" gang was involved in multiple murders between 2004 and 2007, including two victims whose bodies were dissolved in acid.
'Los Palillos' Boss Gets Nine Life Terms For Killings, Kidnappings In San Diego
- Oct. 14, 2014
- By Kelly Wheeler / City News Service
Los Palillos splintered off from Tijuana's Arellano Felix drug cartel in 2002. It set up operations in San Diego, murdering nine people.
What Does 28K Pounds Of Cocaine Look Like? Coast Guard Shows Off Seized Drugs (Video)
- Oct. 7, 2014
- By Beth Ford Roth
The Coast Guard Cutter Boutwell arrived at Naval Base San Diego on Monday morning with a very unique cargo — 28,000 pounds of cocaine seized in 18 different busts. The wholesale value of the haul? An estimated $423 million.
$1M Grant Awarded To Decrease Meth Getting Into U.S.
- Oct. 6, 2014
- By Associated Press
An estimated 70 percent of smuggled methamphetamine enters the country through the San Diego Port of Entry.
Hope Dwindles For DNA Evidence At Tijuana Body Dump
- Sept. 3, 2014
- By Jill Replogle
More than 22,000 people have gone missing in Mexico since the country declared war on drug traffickers in 2006. Some family members of the missing hoped to find answers at a body disposal site in Tijuana. But so far, they have only frustrated hopes.
Why Nicaraguan Kids Aren't Fleeing To U.S.
- July 29, 2014
- By Jill Replogle
Nicaragua is Central America's poorest country, yet its children aren't fleeing to the border along with their Salvadoran, Guatemalan and Honduran neighbors. Experts say that's because of the country's low crime rate, effective police force and unique migration history.
Tijuana Blocks Three Narco-Corrido Stars From Annual Fair
- July 14, 2014
- By Jill Replogle
The city of Tijuana is trying to keep three singers of so-called "narco ballads" from performing at the city's annual fair.
Arellano Felix Cartel Leader Captured In Tijuana
- June 24, 2014
- By KPBS
Fernando "El Ingeniero" Sanchez Arellano was arrested Monday afternoon by the Mexican Army, according to reports.
Border Patrol Seizes Nearly $1M Of Hard Narcotics In San Diego County
- June 3, 2014
- By Quinn Owen
Border patrol agents arrest four men and seize nearly $1 million worth of heroin, meth and cocaine last week.
Border Patrol Seizes Nearly $500,000 Worth Of Narcotics In San Diego
- May 23, 2014
- By Quinn Owen
The U.S. Border Patrol intercepted two shipments of hard narcotics this week in San Diego.
Mexico Forbids Drug Lord's Extradition Even As Negotiations With U.S. Continue
- April 25, 2014
- By Lorne Matalon
Sinaloa cartel leader Joaquín Archivaldo Guzmán Loera, known as El Chapo, has been indicted in San Diego and three other U.S. cities. After his arrest in February in Mexico, U.S. authorities began negotiating for his extradition.
Mexico's Drug-Related Homicides Are Down But Not In Tijuana
- April 16, 2014
- By Megan Burke, Maureen Cavanaugh, Peggy Pico, Jill Replogle
A University of San Diego report on drug violence in Mexico finds that homicides are down overall in the country, but the number of killings in Tijuana is up from 2012.
Border Patrol Seizes Drug-Laced Lollipops At San Clemente Checkpoint
- March 6, 2014
- By Quinn Owen
U.S. Border Patrol agents seized approximately 23 pounds of drugs with a value of $160,000 at the San Clemente checkpoint Wednesday.


text:
"We have found a great location that is easily accessible by trucks and rail, allowing us to locally serve the Arizona market and fulfill local demand. We believe the facility will provide us with a wonderful opportunity for an even greater number of farmers on the west coast to use our EM® Effective Microorganisms® products to improve their soil and grow stronger, healthier crops and livestock," stated Eric Lancaster, Executive VP of TeraGanix, Inc.
By being able to supply the demand in that region locally, TeraGanix has reduced the impact of drought and brought local revenue and employment into that area. The local distribution will furthermore lower the cost of shipping for its west coast consumers, saving farmers money on their overall irrigation and fertilizer costs.
To learn more about TeraGanix, Inc. and its farming and gardening supplies, please visit: http://www.teraganix.com/.
About TeraGanix
TeraGanix, Inc is the exclusive distributor of the original, authentic EM•1® Effective Microorganisms® in the US & Canada. Introduced in 1982, EM Technology® is widely used in over 100 countries to promote healthy soil for healthier plants and crops, clean water, odor control, waste management, and healthier people and animals. EM•1
|Source: PRWeb
Copyright©2014 Vocus, Inc.
All rights reserved


text:
Does anyone know when or why they added this change?
Does anyone know when or why they added this change?
what do you mean? do archavon and emalon give honorable kills now?
it might be just because, since you get PvP gear from them, you also get HK's from them.
Because of the high frequency of negativity and overall "my class/race/faction sucks" (both for IRL and ingame issues) across all boards, I have reverted to primarily lurking and only posting when I need help. I'm hoping this can be fixed, but if I don't do this, I will likely end up depressed and disgusted with humanity as a whole.
Yeah, go kill Archy or Emma and look at your combat log directly after.
Looks like 3.2 did, never got honor from them before.
Obvious question inc: how much honor did they give ?
All i remember is Archavon giving something like 113 honor
They give hk's i can confirm that


text:
The Sidney & Lois Eskenazi Hospital
A new day in the health of our community is here. The Sidney & Lois Eskenazi Hospital opened Dec. 7, 2013. Learn more about the new Eskenazi Health campus.
Music as Medicine
Supporters of the Marianne Tobias Music Program enable tremendous musicians like Sullivan Fortner, the 2015 Cole Porter Fellow in Jazz of the American Pianist Association, to summon the joyful and soothing strains of music to Eskenazi Health.
An Extraordinary Gift from Sidney and Lois Eskenazi
Sidney and Lois Eskenazi contributed one of the greatest philanthropic gifts ever to the people of Central Indiana, and one of the largest ever made to a public hospital in the United States. Learn more about Sidney and Lois Eskenazi.
Donor Stories: Why we give to Eskenazi Health
Contributors to Eskenazi Health Foundation include leaders in Central Indiana's philanthropic community, Eskenazi Health employees and physicians, many businesses and foundations, and men and women from across our community. Learn more.


text:
|The Lounge | Champions | The Wire | Schedule | Audio | Arcade | The Top Ten | Historical | Email | Video
The Basics Of Dumbells And Dumbbell Workouts
Dumbbells are integral to a long term fitness and personalstrengthening plan. Dumbbells are individual weights thatare best used as part of your overall fitness workout eachday.
A standard but effective dumbbell exercise is generallyconducted as follows. Place one dumbbell in each hand anduse you arms to lift the dumbbells up to shoulder height.Now, lower your arms down so you arms are by your side.Repeat as many times as necessary.
You can also perform exercises for strengthening both arms,by working with one dumbbell and one arm at a time.
Dumbbells and dumbbell weights can be purchased as sets ofpairs or as a complete dumbbell set that offers manydifferent weight increments depending on your currentfitness level and what you fitness goals are in the future.
Adjustable dumbbells can be purchased for use in the home -if you want to do some quick dumbell arm workouts each day- or borrowed at fitness clubs - if you are want to use thedumbbells as part of a great fitness program that involvestreadmills, bikes, bench presses and skipping ropes.
You can usually buy dumbbells weights in 5 poundincrements. This is ideal for people on a long termtraining program because it means that they can purchaseweights that match their strength level as they improvetheir fitness.
Dumbbells and dumbbell weights come in many differentcolors, designs, textures, and materials so you canpurchase dumbbells to suit your own personal style and tomatch the decor of your fitness area at home.
If you want to set up a home display then you shouldprobably opt for chrome or cast iron dumbbells which aresimple, easy to use, and attractive. You can also chooserubber encasing because it will minimize noise and thepotential for floor damage if you accidentally drop adumbbell during your dumbell workouts.
Chrome dumbbells are the most popular type of dumbbells forhome use.
If you are serious about your ongoing fitness program thenyou can also purchase a dumbbell rack for storing yourdumbbell set.
Without a rack, your dumbbells will clutter up your fitnessroom by taking up unnecessary floor space because you canstore your dumbbells vertically or horizontally away fromthe walk through areas.
The type of rack you purchase ultimately depends on thetype of dumbbell set you purchase. Similarly the type ofdumbbell set your purchase depends on your height, weight,body type, and fitness level and your level of strength.
Paul Johnson works tech support for New York based InternetPublishing company, DigiLectual Inc. He uses Dumbellworkouts as part of his overall, balanced fitness workouts.
Find out why dumbbells are the best type of free weightexercises. Get reviews and useful information. Click ==> http://www.dumbells-info.com/
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