Optics: measuring and testing – By shade or color – With color transmitting filter
Reexamination Certificate
1999-09-17
2002-04-23
Rosenberger, Richard A. (Department: 2877)
Optics: measuring and testing
By shade or color
With color transmitting filter
C250S458100
Reexamination Certificate
active
06377346
ABSTRACT:
OBJECT OF THE INVENTION
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.
PRIOR ART
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 &mgr;l. 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 &mgr;l. 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 &mgr;l of liquid or 1 &mgr;l 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 &mgr;l. 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 &mgr;l, and possibly even as little as 1 &mgr;l. 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. Excit
Harju Raimo
Korpi Jarmo
Nurmi Jarmo
Väisälä Mikko
Kubovcik & Kubovcik
Rosenberger Richard A.
Wallac Oy
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