CA2351419A1 - Method and device for automated chromophore-assisted laser/light inactivation (cali) - Google Patents

Abstract

The present invention relates to a device (1) for an automated chromophore- assisted laser inactivation (CALI), wherein said device includes a laser device (2) for radiating samples using a laser beam (3) in order to identify the function of biological molecules. A control organ for the laser device ( 2) and for the sample support (5) is used for carrying out a relative displacement in at least two directions between the laser beam (3) and said sample support (5) so that the samples can be radiated using the laser beam (3) or scanned by the same. The shape and/or the intensity of the laser beam (3) can be adapted to the size and/or to the type and/or to the number of th e samples. The control device (4) is designed so that the laser device (2) and its control organ and/or the control organ of the sample support (5) as well as the beam modification unit (6) can controlled in a centralised manner usi ng a system software. This invention further relates to a device and a method f or carrying out an automated CALI using a lighting unit in the form of a white- light source for radiating the samples with a light beam.

Description

Process and apparatus for automated chromophore assisted laser/light inactivation (CALI) The invention relates to an apparatus for automated chromophore-assisted laser/light inactivation (CALI) as defined in the preamble to claim l, and to a process for identifying the function of biological molecules by means of chromophore-assisted light inactivation.
The prior art discloses chromophore-assisted light inactivation based on a laser as light source.
Chromophore-assisted laser inactivation was described for the first time in Jay, D.G., Selective Destruction of Protein Function By Chromophore-Assisted Laser Inactivation, Proceedings of National Academy of Science, USA 85 (15):5454-8. This known technique can be employed selectively for time- and space-resolved inactivation of proteins. With the aid of this technique, a dye, which is normally the chromophore malachite green (MG), is chemically coupled to an antibody which has specificity for a target molecule.
After binding of the MG antibody has taken place, the complex is bombarded with pulsed, high-energy laser light. This results in the local formation, via the MG, of reactive, short-lived (lolls) hydroxyl free radicals, which modify the target molecule and can thus result in its inactivation. This is described in Liao, J.C., et al., Chromophore-Assisted Laser Inactivation of Proteins Is Mediated by the Photogeneration of Free Radicals, in Proceedings of the National Academy of Science, USA 91 (7):2659-63. Recently, besides MG, the fluorescent fluorescein (FITC) and the continuous-wave laser, i.e. a continuous laser, have been employed successfully for chromophore-assisted laser inactivation. This is described in Surrey, T. et al., Chromophore-Assisted Light Inactivation and Self-Organization of Microtubules and Motors, Proceedings of the National Academy of Science, USA 95:4293-8. It was found here that FITC is more potent than MG and results in faster target molecule inactivation. It was also found that use of FITC also required significantly less powerful continuous lasers to give a comparable effect than in the case of significantly more powerful pulsed or continuous lasers when using MG. It is evident from this that CALI still offers considerable scope for development and is thus capable of improvement and worthy of improvement. For the purposes of this application, the term chromophores is also taken to mean fluorophores.
For example, a whole series of other dyes are CALI-compatible or may even prove to be superior to the dyes used hitherto.
Furthermore, the entire CALI procedure has hitherto been carried out virtually exclusively manually, resulting in low throughput, extremely limited potential for standardization, the inclusion of human sources of error and risks for the experimenter.
Furthermore, CALI was predominantly used in its microvariant for the inactivation of target molecules at a single-cell level by means of a microscopically focused laser beam. However, in order to be able to realize the full potential of the macrovariant of CAL L
for example for protein inactivation in massive screening sets, it is desirable to automate and standardize the entire procedure and to render it inexpensive.
In detail, the cited article by Jay et al., and Lindan, K.G. et al., Spatial Specificity of Chromophore-Assisted Laser Inactivation of Protein Function, in Biophys. J. 61 (4):956-62 describe the use of a dye laser in the form of a Quanta Ray DCR3 (for example from Spectra Physics Inc., Mountain View, CA) Nd:YAG (neodymium-yttrium-aluminum garnet), a 10 Hz pulsed infrared laser with a pulse length of 8.5 nm and a wavelength of 1064 nanometers, in order, with its .second harmonic wavelength (wavelength 532 nm), to operate a downstream dye laser (Quanta Ray PDL2, from Spectra Physics) which contains the circulating, fluorescent laser dye DCM (Exciton chemical Co., Dayton, OH). The 620 nm pulsed laser light generated is in the vicinity of the absorption maximum of malachite green. This beam was deflected into the vertical by total reflection in a right-angled prism and focused on the desired diameter of about 2 mm by a convex lens.
The output energy of the 620 nm laser beam was measured at from 10 to 15 mJ/pulse at a pulse frequency of 10 Hz in CALI experiments. This corresponded to a mean peak power density per pulse of from 37 to 57 MW/cm2 and a mean laser output power of from about 80 to 100 mW. A
molar ratio of about 20:1 for MG-coupled antibodies (100 ug/ml) to ~-galactosidase target molecule (5.5 ug/ml) was employed. The coupling ratio for the antibody was about 6.5 MG molecules per antibody. The half-maximum inactivation time of ~-galactosidase under these conditions was about 150 s.
Furthermore, the above-described article by Surrey et al. describes that an argon-pumped adjustable continuous-wave dye laser (coherent 599 standing wave dye laser with rhodamine 6G, Coherent Radiation, Palo Alta, CA) was used for CALI malachite green. The wavelengths generated were 620 nm and 488 nm, the wavelength 488 nm being in the region of the second absorption maximum of MG, at a power of 225 mW. A
constant power of 7.2 W/cm2 was used for in vitro inactivation of ~-galactosidase. A molar ratio of about 20:1 for MG-coupled antibody (200 ug/ml) to ~-galactosidase target molecule (10 ug/ml) was employed. The coupling ratio for the antibody was about 2.8 MG molecules per antibody. The half-maximum inactivation time of ~-galactosidase under these conditions was 8.5 s at a wavelength of 620 nm and 13 s at a wavelength of 488 nm. A weaker continuous-wave argon ion laser having a wavelength of 488 nm and a constant power of 2 mW was used for CALI with FITC.
This corresponded to a constant power of 0.065 W/cmz. A
molar ratio of about 20:1 for MG-coupled antibody (10 ug/ml) to ~-galactosidase target molecule (5.5 ug/ml) was employed. The coupling ratio for the antibody was about 2.3 FITC molecules per antibody. The half-maximum inactivation time of ~3-galactosidase under these conditions was 18 s.
It is furthermore described in Jean, B. et al., Selective Laser-Induced Inactivation of Proteins (SLIP) by Labeling with Chromophores, 1992, Med. & Biol. Eng.
& Comput., 30, CE17-CE20, that an adjustable continuous-wave argon-pumped dye laser (Meditech dye laser with rhodamine 6G) was used for SLIP (selective laser-induced inactivation of proteins) with FITC at a wavelength of 488 nm. The samples were irradiated at a power of 3 W. In the case of SLIP, the dye was coupled directly to the molecule to be inactivated (antibody and enzyme horseradish peroxidase). The coupling efficiencies were between l:l and 2:1. 8 to 10 pulses with a duration of 1 s were selected. This resulted in a half-maximum inactivation of horseradish peroxidase at an energy density of about 7500 J/cm2. This value seems unusually high, since, for comparison, only about 480 J/cm2 act on the sample during, for example, a half-maximum inactivation time of 150 s using a high-power pulsed Nd:YAG laser.
Regarding the known equipment systems, the object of the present invention is to provide a flexible, inexpensive, highly reliable automated CALI
system/process which enables high throughput of the samples to be inactivated and is particularly flexible regarding the light source employed.
This object is achieved by an apparatus having the features of claim 1 or 10 and by a process having the features of claim 27.
Advantageous embodiments are defined in the respective dependent claims.
The apparatus for automated chromophore-assisted laser inactivation in accordance with the invention has a laser device for irradiation of samples by means of a laser beam for identifying the function of biological molecules, a control unit for controlling, for example, mechanical, optical and thermal parameters of the apparatus, and at least one sample holder. At least one drive is provided in order to generate relative movement between the laser beam emerging from the laser device and the sample holder in at least two, preferably mutually perpendicular directions. By means of this relative movement, the samples are irradiated by the laser beam or can be scanned by this laser beam. In accordance with the invention, the laser beam can be matched in shape and/or intensity to the sample size and/or sample type and/or sample number by means of a beam modification device. In order to achieve this matching, it is possible for a beam expander and/or a beam splitter to be used as beam modification device. By means of the beam splitter, the laser beam is split in such a way that, in the interests of high throughput of samples to be treated, a number of samples can be irradiated simultaneously. The control unit is designed in accordance with the invention in such a way that the drive for the relative movement between the laser beam emerging from the laser device and the sample holder and the drive for the beam expander or beam splitter can be controlled centrally by this control unit by means of system software.
By means of the apparatus according to the invention, the automated CALI procedure is considerably accelerated and optimized with respect to the samples to be treated in a time unit, its costs are significantly reduced, and at the same time its setting accuracy with respect to the exclusion of human sources of error and thus the reliability and reproducibility of the results are increased. Through the use of a single piece of software for control of at least the essential parts of the CALI apparatus, a considerable simplification in the function and operation of the apparatus is achieved.
The use of a beam expander makes it possible to match the intensity and the cross section of the light beam not only to the particular sample, but also to different dimensions of the wells for the individual samples in the sample holders. High flexibility of use for a wide variety of sample sizes and a wide variety of analytical purposes is thus achieved.
On use of a beam splitter or on modification of the beam expander in such a way that the light beam is split into a plurality of individual light beams, as is the case on use of a beam splitter likewise known per se, it is possible to subject a plurality of samples in parallel to the protein inactivation, which can further increase the throughput of the CALI apparatus according to the invention.
The beam expander preferably expands the laser beam one-dimensionally in cross section, in particular as a straight line, or two-dimensionally as an area of various configuration, the cross-sectional area of the laser beam preferably being rectangular, circular or elliptical. This flexibility in cross section enables use for various sample-accommodating cavities provided in the sample holder.
In accordance with a preferred embodiment, the beam expander is designed in such a way that at least two samples can be irradiated simultaneously. This is possible either through a correspondingly expanded laser beam or through splitting of the laser beam into two separate beams after it leaves the beam expander.
However, it must be noted here that the number of beams leaving the beam expander or beam splitter is dependent on the power of the laser, with higher requisite powers of the laser employed being associated with higher costs of the apparatus.
According to a refinement of the invention, the control unit is designed in such a way that it varies the beam leaving the laser device in cross section, intensity, irradiation duration and dimension as a function of the movement of the sample holder and of the number and/or type of the samples. Agreement between the parameters of the laser beam and the -relative movement between the laser beam and the sample holder likewise serves to increase the throughput of the CALI apparatus and to increase the accuracy and reproducibility of the results owing to precise timing.
According to a preferred embodiment, the light-source device is a laser device, preferably a pulsed laser, in particular an Nd:YAG infrared laser. In order to operate an optical parametric oscillator (infinity-XPO, Coherent Radiation) for wavelength modification, the third harmonic wavelength of the pulsed infrared laser is used.
According to a further embodiment, the laser device is a continuous laser, in particular an adjustable argon-pumped dye laser. However, it is also possible to use an argon ion gas laser or a helium-neon laser.
According to a further embodiment of the invention, the irradiation of samples for identifying the function of biological molecules is carried out by means of light emitted by a light-source device on the basis of automated chromophore-assisted light inactivation (CALI). The apparatus has a control unit for central control of all elements/assemblies of the apparatus which are to be controlled, and at least one sample holder. In accordance with the invention, the light-source device is a lamp in which at least part of the wavelength of its emitted light is in the visible region. The lamp used is preferably an incandescent lamp, quartz lamp, fluorescent tube or light diode, or a combination thereof, where the lamp is, in particular, a white-light source. This means that, for example, a commercially available incandescent lamp is capable, using suitable chromophores, of achieving the inactivation of biological molecules otherwise necessary in accordance with the first embodiment without the need to use a laser device.
The light emitted by the light-source device can be matched to various parameters of the samples in its emitted shape and/or intensity by means of a beam _ g _ modification device. These parameters are, in particular, the sample size, the sample type and the sample number, where the emitted light can be matched to one, more or all of the sample parameters mentioned.
The flexibility of the apparatus according to the invention is thus increased enormously, since a very wide variety of samples can be analyzed with high throughput, high reliability and very large band width.
Furthermore, according to a refinement of the invention, a control unit is preferably provided by means of which a drive for moving the sample holder and the beam modification device can be controlled centrally via system software. The control unit is provided in order to control all the controllable components of the apparatus according to the invention by means of the central system software. The beam modification device preferably has masks, in particular having variable apertures/diaphragms. The masks having variable apertures or diaphragms serve to direct the emitted light specifically onto a plurality of samples arranged on the sample holder at the same time as the irradiation, it preferably also being possible for all the preferably 96 samples arranged on a standard sample holder in the form of a microtiter plate to be irradiated simultaneously, which facilitates extremely high throughputs through the apparatus according to the invention.
The light is preferably at least one light beam. According to a refinement of the invention, however, it is also possible, in accordance with a particularly preferred embodiment of the invention, for the light to be diffuse light. On use of diffuse light, optical elements between the light-source device and the samples are generally unnecessary, but instead the light emitted in a diffuse manner is utilized for the simultaneous irradiation of a very large number as possible of samples arranged on the sample holder. On use of a diffuse white-light source of this type, individual beam splitting is unnecessary; if it is g necessary to restrict the irradiation to certain samples in order to avoid having to irradiate all the samples simultaneously, the use of suitable hole masks is provided. This means that particularly high parallelization of the sample irradiation can be achieved using a hole mask diaphragm if a diffuse white-light source is used. This parallelization of the sample irradiation is particularly high compared with the focused laser beam in the first embodiment, in which the individual samples arranged in wells on a microtiter plate on the sample holder are scanned successively.
On use of a white-light incandescent lamp known per se, the apparatus according to the invention can be particularly simple in construction and, in particular, also inexpensive to produce. On use of a white-light incandescent lamp, either the diffuse, simultaneous irradiation of a large number of samples is possible via a hole mask, or it is possible, by means of groups of optical elements of relatively simple construction, to re-split the light emitted by the light-source device after appropriate bundling in such a way that the largest possible number of samples can be irradiated simultaneously. Irradiation even without groups of optical elements is likewise possible. A
further advantage of the use of white-light incandescent lamps for irradiation is, inter alia, that sources of risk for operating personnel are considerably reduced compared with the use of a laser device as light-source device.
For the respective samples, taking note of the fact of whether a beam expander and/or a beam splitter is provided in connection with a laser device or with a white-light incandescent lamp, i.e. in order to be able to adjust the powers or light yields of the light beams used in each case that are necessary for the respective samples, a power meter is preferably provided for determining the power emitted by the light-source device. A power meter of this type thus also provides higher reproducibility of the analysis conditions.
In order to ensure high flexibility with respect to the relative movement between light beam and sample holder and in order to ensure rapid irradiation of the microtiter plates, with the corresponding samples, mounted on the sample holder, the sample holder has a positioning stage which carries at least one microtiter plate, but preferably has two microtiter plates, and which can be positioned in the x,y directions. Positioning in the x,y directions is achieved by means of displacement mechanisms which are driven by stepping motors and which are arranged at right angles to one another. The stepping motors are based on full-step or half-step technology and allow very precise setting, which is also substantially play-free in connection with the corresponding displacement mechanisms, of precisely desired positions of the positioning stage with the microtiter plates. An x,y positioning stage of this type is particularly necessary on use of a laser device as light-source device.
The displacement mechanisms are preferably guided on rails, in which case, in order to transmit the movement from the respective stepping motor, provision is made for ball spindles, which achieve high positioning accuracy with respect to the movement caused by the electronically driven stepping motor.
The apparatus according to the invention represents the central part of a CALI system for identifying the function of biological molecules and, in accordance with a further embodiment of the invention, additionally comprises, in order to further increase the flexibility of the system as a whole, a ligand-binding partner (LBP) screening machine for generating specific LBPs aimed at specific target molecules/ligands, if desired a chromophore synthesis unit for the production of chromophores, an LBP-chromophore coupling unit for linking the selected LBPs and synthetic chromophores, a charging device for transferring LBP tags into wells arranged in the microtiter plate, a device for moving the microtiter plate into the CALI apparatus, and a device for reading the activity of the target molecules to be inactivated.
An apparatus containing these further components is thus an apparatus of high flexibility which facilitates substantially automated loading, irradiation, analysis and replacement of samples which have already been analyzed by new samples, and furthermore also automated reading and storing of the results obtained by the analysis. In particular the use of a central control system for all components of the system as a whole enables easy and clear handling and optimum operation of the apparatus as a whole.
In order to be able to compare newly obtained results with previous results in order to be able to detect trends in the analysis of certain samples in good time and also in order to have later access to analysis results which have already been obtained, the reading results are stored in a data base.
In addition to control of the screening machine, the LBP-chromophore coupling unit, the charging device and the device for moving the microtiter plate, a further drive for moving an optical lens which is arranged between the beam modification device and the sample holder is controlled centrally by means of the control unit via the system software. This further increases the flexibility of the system as a whole and simultaneously increases its reliability.
According to a preferred embodiment of the invention, a housing around at least one microtiter plate of the sample holder is arranged in such a way that it surrounds the plate, but is preferably arranged around the entire sample holder, including movement mechanism. The housing is connected to external supply systems in such a way that certain preset, desired state conditions can be controlled in the housing.
These state conditions should include, in particular, state conditions of the atmosphere surrounding the respective sample or microtiter plate. These include, in particular, the carbon dioxide content, the moisture content, the temperature, the pressure, etc., of the atmosphere. For the purposes of the present invention, the term "supply systems" is taken to mean supply via special central or decentral line systems with appropriate valves, where the valves or pumps provided in the supply systems can be controlled in such a way that the state conditions can be set to a certain value or adjusted in accordance with a desired change curve or set to certain state values. The corresponding pumps and valves in the supply systems can preferably likewise be centrally controlled by means of the control unit via the system software. In order to increase the flexibility of the apparatus as a whole, microtiter plates having wells of different dimensions are used, enabling samples of widely varying consistency and composition to be arranged as needed.
According to a further aspect of the invention, the process according to the invention for identifying the function of a ligand uses a lamp, in particular in the form of a white-light source, for the irradiation necessary for inactivation. In the process according to the invention, a sample containing a complex of a ligand and a ligand-binding partner (LBP)-light-activatable tag is irradiated with a light beam from the lamp. The lamp, in the form of a white-light source, has a conventional incandescent lamp, quartz lamp, fluorescent tube and/or light-emitting diode. The light emitted by the lamp is white light, where, for the purposes of the present invention, the term "white light" is taken to mean light having a wavelength spectrum which is at least partly in the visible region, but which is not light emitted by a laser device.
With respect to further embodiments of the process and individual process steps for achieving the identification of the function of a biological molecule during inactivation which contribute to the technical success of the invention and for which protection is granted, we refer to the German patent appplication by the same applicant which was submitted on November 24, 1998, under the Application No. 198 54 195.3, the disclosure content of this application, in particular all the features of claims 1-6, being incorporated herein in full by way of reference.
Further advantages, features and possible applications of the invention are explained in detail below with reference to illustrative embodiments and with reference to the attached drawings, in which:
Fig. 1 shows the principle of the structure of the CALI apparatus according to the invention;
Fig. 2 shows a plan view of a sample holder in the form of a positioning stage;
Fig. 3 shows a side view of the positioning stage according to Fig. 2;
Fig. 4 shows a support formed with Peltier elements on the positioning stage for accommodating microtiter plates;
Fig. 5 shows a perspective view of a housing in the form of an incubator around the CALI
positioning stage according to Fig. 2;
Fig. 6 shows a plot of the experimental results for inactivation of (3-galactosidase using MG;
Fig. 7 shows experimental results concerning the potency of MG-CALI and of FITC-CALI;
Fig. 8 shows experimental results concerning inactivation of (3-galactosidase using FITC
under light and under dark conditions;

Fig. 9 shows the effect of daylight on the inactivation of (3-galactosidase by means of a FITC-labeled antibody;
Fig. 10 shows the specific degree of inactivation of (3-galactosidase by means of FITC-CALI as a function of the irradiation duration with a diffuse white-light source;
Figs. 11.1 to 11.3 show the flow chart of the central system software used in the control unit; and Figs. 12.1 and 12.2 show the principle of the overall construction of the CALI apparatus according to the invention on use of a white-light source.
Fig. 1 shows the principle of the structure of a CALI apparatus according to the invention. The CALI
apparatus 1 employed in a fully automated laboratory analysis procedure has a laser device 2, from which a laser beam 3 emerges. The laser beam 3 serves to irradiate the samples in order to identify the function of biological molecules which are arranged in wells in microtiter plates 10, where the microtiter plates 10 are mounted on a sample holder 5. The sample holder 5 has a positioning stage 13 in the form of a cross-shaped stage with displacement mechanisms 11, 12, so that the assembly carrying the microtiter plate 10 can be displaced along the displacement mechanisms 11, 12 in the x and y directions. The displacement mechanism has two components provided with displacement rails: a first slide guide 11 serving for movement of the microtiter plate 10 in the x direction, and a second slide guide 12 serving for displacement of the microtiter plate 10 in the y direction. In order to execute a corresponding displacement of the microtiter plate 10 in the x direction or in the y direction, drives are provided for the respective slide guides, so that, in accordance with the displacement in the respective direction, the plurality of sample accommodation wells present in the respective microtiter plate 10 can be exposed successively, in the case of a laser beam 3 of essentially fixed location, to irradiation by means of the laser device 2. In order to increase the flexibility of the CALI apparatus, a beam modification device 6 is provided between the exit of the laser beam 3 from the laser device 2 and the entry into a prism 9 arranged in the region of the microtiter plate 10. By means of this beam modification device 6, the laser beam 3 is matched to the respective sample. This means that the laser beam 3 can be matched in shape to the sample or to the size of the well in the microtiter plate 10 and can also be matched with respect to intensity to the particular sample size.
Furthermore, the beam modification device 6 is designed in such a way that the laser beam can be matched to the sample type and/or also to the sample number. By means of a central control unit 4, which belongs to the CALI
apparatus and uses XCALIbur central system software 4.1, all the movements, powers and state conditions of the automated CALI apparatus 1 are controlled or set to certain predefined values.
In order to be able to carry out the analyses reproducibly and in order to be able to set and monitor the intensity of the laser beam 3, which intensity is matched to the respective sample type, a power meter 7 is provided at the exit of the laser device 2; this power meter indicates and/or records the exit power of the laser beam 3. The entire apparatus is arranged as a unit on an optical bench 8.
The pumped laser used is a pulsed Nd:YAG
infrared laser, for example from Infinity, Coherent, whose third harmonic wavelength of 355 nm is used in order to operate an optical parametric oscillator (Infinity-XPO, Coherent). The pumped laser and the XPO

are each centrally computer-controlled via two user-friendly menu programs (control modules 4.2 and 4.3) using XCALIbur via the control unit 4. Pulse energies of greater than 30 mJ/pulse can be generated, where the pulse repetition frequencies are freely selectable in the range from 1-30 Hz. The typical pulse length is 3 ns . The working signal beam wavelengths which can be generated extend from 420 to 709 nm and the unused, so-called "idler" beam wavelengths from 2300 to 709 nm.
In order to be able, in an on-line measurement with beam coupling out, reliably to control the laser output power during the experiment or irradiation by means of the power meter 7, about 2% of the beam emerging from the XPO are separated off from the laser beam 3 by means of a beam sampler (diameter 25.4 mm, Coherent, Catalog No. 44-2343) and passed to a pyroelectric sensor (LMP 10i, Coherent, Catalog No. 33-1173). The incident energy is displayed by a power and energy analyzer (such as, for example, a Fieldmaster-GSTM, Coherent, Catalog No. 33-0498). This power and energy analyzer is integrated into the "XCALIbur" central control software 4.1 via an RS-232 interface for documenting the measurements obtained on-line.
For laser beam diameter regulation, a precision beam expander (Coherent, Auburn Group, Catalog No. 31-2505) with a zoom expansion factor of from 1 to 8 times is employed. By means of an antireflection coating, the light transmission at 630 nm, i.e. in the vicinity of the wavelength used for the CALI analysis with MG, is optimized (>94%).
Before the laser beam 3 hits the respective sample, the laser beam 3 is passed through a right-angle prism 9 having a side length of 40 mm (Product No. OlPRA029, Melles Griot, Zevenaar, NL) whose hypotenuse is provided with a silver coating for total reflection and whose cathetus is provided with a HebbarTM broad-band antireflection coating (415-700 nm, Product No./078). The laser beam 3 is thus deflected from the horizontal through 90° into the vertical in the direction of the positioning stage 13, i.e. the well containing the sample in the microtiter plate 10.
In order to increase the sample throughput in the case of laser irradiation, a broad-band beam splatter (diameter 25.4 mm, beam splitting 50:50 at from 420 to 700 nm, Coherent, Catalog No. 44-2168) which removes half of the incident laser beam 3 or its energy at a 90° angle to the transmitted component of the beam, is introduced before the right-angle prism 9. Depending on the energy required for a particular sample in order to ensure CALI inactivation, and depending on the dye used, 3-fold, 4-fold or optionally 8-fold beam splitting is provided by insertion of a series of beam splatters having the suitable splitting ratio in each case. A corresponding combination of 1, 2, 3, 5 and 9:1 beam splatters from Coherent is possible here.
Fig. 2 shows a plan view of the sample holder 5 designed as a cross-shaped stage, for accommodating the microtiter plate 10. The sample holder 5 has a slide guide 12, on which the actual positioning stage 13 which carries the microtiter plate 10 is mounted on a corresponding guide. The drive for moving the stage in the y direction takes place by means of a stepping motor 14.
The slide guide 12 itself is arranged on a further slide guide 11 arranged perpendicular thereto, for implementing the movement in the x direction. The drive of the slide guide 12 for its movement on the slide guide 11 in the x direction likewise takes place by means of a stepping motor 14.
Fig. 3 shows a sectional side view of the positioning stage 13. It can be seen that the slide guides 11, 12 are arranged one above the other on the piggyback principle and are movable relative to one another, so that, through movement in the x direction and the y direction, the microtiter plate 10 arranged on the actual positioning stage 13 can be moved reliably within an area range. High accuracy of the movement of the respective slide guides 11, 12 and thus of the microtiter plate 10 is achieved by ball spindles 15.
The ball spindles 15 are in effective drive connection with the stepping motors 14, which are designed as 3-phase stepping motors, such as, for example, Berger VDRM 368 stepping motors. These stepping motors 14 are controlled via a CNC control module 4.4 in the form of an industrial CNC standard control device, such as, for example, SM300, SM
Electronics GmbH. This CNC control module 4.4 operates in command mode and is coupled directly to the control unit 4 via appropriate electronic connections, or is a part thereof.
The two microtiter plates 10 which have wells for accommodating the samples and are arranged on the positioning stage 13 each have 96 wells in a manner known per se, making it possible to analyze or irradiate 192 samples one after the other without the need for intervention by operating personnel. It is of course also possible to arrange other microtiter plates which have a number of wells other than 96 on the positioning stage 13.
The CNC control module 4.4 for the 3-phase stepping motors 14 of the ball spindle drives is connected to the RS-232 port of the control unit 4 via a serial cable.
Besides precise setting, the use of ball spindles 15 enables a repeatability accuracy of at least 0.1 mm. This is necessary in order that the laser beam 3 always hits the center of the microtiter plate well and the sample is thus irradiated completely and beam scattering at the edges of the well is avoided.
Fig. 4 shows the principle of construction of a support 16 or a receptor for microtiter plates 10. The support 16 has a sandwich structure with 6 Peltier elements 20, preferably from CONRAD ELECTRONIC, 7105, which are mounted between two black-anodized steel plates 17, 18. The entire sandwich structure is connected to the positioning stage 13 by means of screws 19. However, it is also possible to achieve another type of connection, for example by means of magnets, in order to enable or simplify automation of the entire process . The microtiter plates 10 should be kept independent at a moderate temperature with the aid of these Peltier elements 20 over a range from about 15°C to 45°C, the temperature being monitored by means of external or internal sensors (not shown).
In order to simplify the description, it has not been described in connection with Figs. 1 to 4 that the positioning stage 13 with the microtiter plates 10 (CALI cross-shaped stage) is surrounded by a housing (not shown in Fig. 4), which, owing to the fact that a defined atmosphere is maintained or set within the housing for each sample, is also referred to as an incubator. The principle of this incubator 25 is shown in perspective view in Fig. 5. In its side walls, the incubator 25 has an aperture 24 for the irradiation optics and apertures 21 for supply lines, such as, for example, electrical supply lines, air lines, gas lines, etc. Furthermore, the incubator 25 has a larger sealable opening in the form of a flap 22, through which, for example, the microtiter plate 10 can be introduced into the incubator 25. This can take place, for example, using a robot arm. In the sealed state, the sealable flap 22 is sealed in a gas-tight or temperature-insulated manner, depending on the state conditions in the interior of the incubator 25. In the base of the incubator 25, further openings 23 are provided in the form of attachment openings which serve for attachment of the incubator 25 to the optical bench 8.
Not shown are corresponding valves in the supply lines, by means of which the atmospheric state conditions in the interior of the incubator 25 are adjusted.
The control unit 4 represented in Fig. 1, which operates with the central system software 4.1, is designed in such a way that the valves (not shown) of the supply lines can be controlled and actuated in such a way that the atmospheric state conditions in the interior of the incubator 25 can be adjusted. Equally, it is possible to provide the opening flap 22 with a drive which can be actuated via the central control unit 4, so that charging of the incubator 25 with samples, in particular arranged on the microtiter plate 10, can also take place in the automated CALI
procedure.
The central control software 4.1 (XCALIbur) forms the user interface for all movement and control operations throughout the CALI apparatus 1. While the control of the positioning stage 13 takes place via a serial interface (RS-232) of the system control unit 4, which is a PC, control of the laser device 2 is carried out by means of Windows DDE servers, which are integrated into the software for the laser control module 4.2 and the XPO 4.3, thus excluding incorrect operation of the laser device 2 by the user. The XCALIbur control software 4.1 also enables operation of the incubator 25, including the atmospheric conditions to be set therein.
When using the XACLIbur software, a user can select each individual well and thus each individual sample in the microtiter plate 10 and assign all laser parameters or light-beam parameters to the sample arranged in the respective well in a differentiated manner. The sequence of irradiation can likewise be pre-set simply by the user by means of a mouse click.
Figures 6 to 10 show the most important results of an experimental protocol for sample preparation and performance of in vitro CALI using the example of (3-galactosidase. The dye was coupled to the effector molecule (antibody) using malachite green isothiocyanate (MG) or fluoroscein isothiocyanate (FITC). The specific antibody used was purified polyclonal rabbit antigalactosidase IgG antiserum. The negative control used was purified IgG of non-immunized rabbits. The antibodies were first dialyzed against PBS, pH 7.4, for 3 hours with a molecular weight exclusion of 3500 Da. 600 ug of antibodies together with the same amount of bovine serum albumin (BSA) in the case of MG and about 400 ug of antibodies without bovine serum albumin in the case of FITC were then employed for the coupling. MG and FITC were dissolved in dimethyl sulfoxide in a concentration of 10 mg/ml.
For MG, the coupling was carried out in 600 ul of 0.5 M
NaHC03, pH 9.5, with a maximum of loo antibody contribution to the volume. MG was added to the antibody in small aliquots on ice, namely 5 times 1 ul every minute, 2.5 ul after a further 5 minutes and then a further 2.5 ul after 2.5 minutes, 5 ul after 5 minutes and a further 5 ul after a further 10 minutes, and finally incubation for 30 minutes, i.e.
a total of 20 ul of MG with a coupling time of about 1 hour. Un-coupled MG was subsequently separated off by gel filtration. This was carried out by means of a PD 10 column (not shown) containing Sephadex G25 matrix, for example from Pharmacia. To this end, the column was first equilibrated in PBS, pH 7.4 (4 mM
KHZP04, 16 mM Na2HP04, 115 mM NaCl) . The coupling reaction was made up to 1 ml with PBS, charged onto the column and then washed with 2 ml of PBS, and the MG-coupled antibody was subsequently eluted in 1.5 ml of PBS. The coupling ratio of the sample was determined by absorption measurement at 620 nm (MG component) and protein concentration determination by means of Bradford assay (antibody component). For the coupling with FITC, the manufacturer's protocol was followed.
400 ug of antibody in 180 ml of PBS were mixed with ul of 0.5 M NaHC03, pH 9.5, with a volume, calculated as a function of the protein concentration, 35 of 3.2 ul of FITC being added. The coupling was carried out for 1 hour at room temperature with exclusion of light, the coupling reaction likewise being purified via a gel filtration column. The coupling ratio of the sample was determined by absorption measurement at 494 nm (FITC component) and 280 nm (protein concentration determination) taking into account the corresponding correction factors (in accordance with the Molecular Probes formula).
The sample preparation and the laser or light irradiation were carried out as follows:
The samples were first made up in conical reaction flasks, with a total volume of 15 ul per sample, and were pre-incubated for 1 hour on ice in order to allow antibody-antigen binding. The desired amount of dye-coupled antibody, namely between 3.75 and 240 ug/ml, was added to a constant amount of purified (3-galactosidase (0.67 U/ml, 1.3 ug/ml; purity grade X, Sigma), virtually the maximum inactivation usually being achieved with about 60 ug/ml, which corresponds to an enzyme: antibody molar ratio of about 1:45.
Besides the addition of the desired amount of dye-coupled antibody, 250 ug/ml of BSA as support were added in order to prevent non-specific absorption of the enzyme at the reaction flask. The mixture was then made up to 15 ul per sample using 1 x PBS, pH 7.4.
After pre-incubation, the sample volume was made up to 50 ul using 1 x PBS (dilution factor 3.3x), and the BSA
concentration in the batch was re-adjusted to 250 ug/ml. The samples were transferred, as independent triple determinations per treatment type (sample type), into the wells of a 96-well microtiter plate, known as a flat base microtiter plate, and subjected to laser irradiation under the desired conditions.
For the CALI process using a laser device 2, the laser was usually operated with an infrared energy of about 270 mJ/pulse with a wavelength of 1064 nm, which corresponded to an irradiation energy of about 13 mJ/pulse at 620 nm and 15 mJ/pulse at 494 nm, measured using the Fieldmaster energy analyzer via approximately 2o sampling of the beam. The laser beam 3 had a diameter of about 5 mm and covered the entire area of a well. The irradiation duration was usually between 1 and 5 minutes.

On use of FITC for CALI in combination with diffuse light, a white-light incandescent lamp was used and a standard desk shade lamp with a diameter of 18 cm was set up vertically above the samples at a distance of about 20 cm, and the samples in a 96-well microtiter plate on ice were irradiated for from 0 to 60 minutes.
After the irradiation, the degree of inactivation was determined. The degree of inactivation of (3-galactosidase was determined via the ONPG (ortho nitrophenylgalactosidase) assay method. To this end, 50 ul of reaction buffer A (100 mM NaH2P04, pH 7.5;
10 mM KCl, 1 mM MgS04) and 50 ul of ONPG solution (4 mg/ml in 100 mM NaH2P04, pH 7.2) were added to the 50 ul sample. This mixture was incubated for a defined time (usually 30 minutes) at 37°C, and the reaction was then terminated using 90 ul of 1 M Na2C03. The degree of reaction of the ONPG substrate (yellow coloration) was determined in the individual samples at 405 nm in a microtiter plate absorption spectrophotometer (Tecan), which is likewise not described directly.
The results shown in Figs. 6 to 10 can be interpreted as follows. In order to test the effectiveness of the novel CALI system by means of the Nd:YAG laser employed, the CALI inactivation was carried out using the example of (3-galactosidase. The different dyes MG and FITC, which have already been described as suitable for CALI, were employed. The irradiation conditions, the irradiation sequence and the irradiation intensity were programmed by means of the XCALIbur software, with the samples thus being scanned automatically.
Fig. 6 shows a titration of the concentration of the MG-coupled antibodies used (ONPG assay). All samples were treated in independent triple sets.
Standard error with the new automated CALI system was generally < 100. The half-maximum inactivation of (3-galactosidase was reached at about 5 ug/ml of specific anti-(3-Gal antibody (approximately 10-fold antibody excess), under the given conditions, which were 5 minutes, 620 nm, 30 Hz, 13 mJ/pulse. The saturation of the inactivation effect, which corresponds to a value of 600 of specific CALI-determined inactivation, occurred at 35 ug/ml (approximately 70-fold antibody excess). The polyclonal anti-(3-Gal serum used exhibited an inhibitory effect even without laser irradiation, but this was significantly increased by laser irradiation. The control IgG serum used caused no inhibitory effect with or without laser irradiation.
The use of FITC, which had an approximately 5 times better coupling to the antibody than MG (10:1 for FITC compared with 2:1 for MG), caused faster and stronger CALI-determined inactivation than MG, as can be seen from Figs. 7 and 8. After irradiation for 1 minute, 47o inactivation was achieved with FITC, compared with 20o with MG (Fig. 7). After laser irradiation at 494 nm at 30 Hz and 15 mJ/pulse for 2 minutes, 800 of the (3-galactosidase activity (ONPG
assay) was inactivated, with a 45-fold antibody excess (Fig. 8). This experiment was carried out as far as possible in the dark. When the same experiment was carried out under the effect of daylight, significant inactivation of (3-galactosidase was evident in the presence of the specific, FITC-coupled antibody even without laser irradiation (see Fig. 8).
In a further experiment, in which the effect of the duration of exposure to daylight was determined, it was found that the laser-independent degree of inactivation increased with increasing incubation duration in light, as can be seen from Fig. 9 (cf. in this respect the inactivation after 1 and 1.5 hours).
In order to work out this FITC-dependent effect more clearly, a light source on the basis of white light of defined strength (incandescent lamp with a power of 60 W) was employed. The specific degree of inactivation of (3-galactosidase was determined as a function of the irradiation duration. This is shown in Fig. 10. It is evident from Fig. 10 that half-maximum inactivation was achieved after from about 10 to 15 minutes, and saturation of the inactivation effect set in after about 30 minutes.
The XCALIbur flow diagram is described in Figs.
11.1 to 11.3.
a) Fig. 11.1 Step 100:
After the program is started, it is firstly tested whether an instance of the XCALIbur program is already active on this computer. If this is the case, a corresponding marker (AnotherInstance) is set which affects the remainder of the program execution. This procedure is necessary in order to ensure that only one instance of the XCALIbur program has control of the laser and the positioning stage.
Step 200:
Only executed if the started program is the first instance on this computer. The user is offered a reference run of the positioning stage for calibration. If the user agrees, the reference run is carried out (step 210).
Step 300:
The user is now in an entry mask in which the definition of the individual sample positions takes place. The sequence in which the user defines the samples determines the sequence of the later laser irradiation. The user has the opportunity to store the definition for the irradiation run for later or immediate use. The usual editing functions, such as copy, insert and cut, are available.

Step 400:
When the definition of the irradiation run is complete, the state of the marker AnotherInstance is evaluated. In the "TRUE" state, it is checked whether the user would like to end the program (step 410) or whether he would like to define further runs (back to step 300). It is thus possible to program a number of irradiation runs in advance, even if another instance of the program is carrying out a current irradiation run on the computer.
Step 500:
If the state of the AnotherInstance marker is not "TRUE", the user can start the irradiation run, define further runs or end the program.
Step 600:
If the user decided to start the run in step 500, the positioning stage is moved into the so-called load position at the front edge. In this position, it is very simple to secure the microtiter plates to the corresponding holders on the positioning stage without touching parts of the beam deflecting optics (beam modification device).
Step 610:
The optimum path for irradiation of the samples and the total run time are calculated.
Step 620:
A command is given to the CNC control module 4.4 according to which calibration runs can be carried out successively in the x and y directions by means of the positioning stage.
Step 700:
An error counter is set to zero.

b) Fig. 11.2 Step 710:
The laser is switched to standby mode via the DDE
interface of the Coherent laser control software.
Steps 720/730/740/750:
For safety reasons, it is now checked whether the laser really is in standby mode. If this is not the case, the error counter is incremented. If the error counter exceeds the value 3 (corresponds to 3 unsuccessful attempts to switch the laser into standby mode), the laser energy is set to zero (step 1500) and an error message is displayed ( step 1600 ) . The user then has the opportunity to remedy the problem and re-start the run or to end the program.
Step 800:
An error counter is set to zero.
Step 810:
The laser parameters (power, wavelength, etc.) for the first sample are set in a safety position (outside the irradiation area). The laser is set exclusively via the DDE interface of the Coherent laser software in order to exclude miscontrol of the laser by incorrect parameters, since these are collected by the Coherent software.
Steps 820/830/840/850:
It is tested whether the laser parameters set have been achieved. If this is not the case, the error counter is incremented and the check of the laser parameters is continued. If the parameters are not achieved after three attempts, the laser energy is set to zero (step 1500) and an error message is displayed (step 1600). The user then has the opportunity to remedy the problem and re-start the run or to end the program.
Step 900:
An error counter is set to zero.
Step 910:
The positioning stage is moved to the position of the sample to be irradiated.
Steps 920/930/940/950:
It is tested whether the positioning stage has reached the corresponding position. If this is not the case, the error counter is incremented and the position of the positioning stage re-tested. After three unsuccessful attempts, the laser energy is set to to zero (step 1500) and an error message is displayed (1600). The user then has the opportunity to remedy the problem and re-start the run (or another run) or to end the program.
c) Fig. 11.3 Step 1000:
The sample is irradiated with the set laser parameters and to the programmed radiation duration.
Step 1100:
It is tested whether further samples are intended for irradiation. If this is the case, the program returns to step 800.
Step 1200:
The laser energy is set to zero, and, for safety reasons, the laser is switched to "external triggering". Since no external trigger is connected, the laser cannot be switched on again, even by mistake.

' - 29 -Step 1300:
The positioning stage is moved back into the load position in order to enable safe removal of the microtiter plate(s).
Step 1400:
The program can now be ended or the user can return to step 300 in order to program a further run.
Fig. 12.1 shows a side view of the principle of the overall structure of the CALI apparatus using a lamp unit. Fig. 12.2 shows a plan view of the whole arrangement. Analogously to the illustrative example of the CALI apparatus with laser device in accordance with the first illustrative example, the apparatus in accordance with the second illustrative example has a control unit 4, a sample holder 5 with a positioning stage 13, on which one or more microtiter plates 10 are arranged and which can be moved in one direction, preferably the x direction, at least for loading of the CALI apparatus, and a slide guide 11, which is connected to a stepping motor 14 serving as drive. A
support 16 for the microtiter plate 10 is provided on the.positioning stage 13. Above the microtiter plate 10 is arranged an air-conditioned irradiation chamber 26, above which a lamp unit 27 is mounted as light-source device. The lamp unit 27 can have simple incandescent lamps, quartz lamps or conventional fluorescent tubes, or a combination thereof. If conventional incandescent lamps are used, they are arranged in an array, it also being possible to replace the incandescent lamps by light-emitting diodes, which can then be switched on corresponding to the samples to be irradiated.
The CALI apparatus according to the invention with lamp unit in the form of a white-light source has an arrangement of the light source such that the entire area of the microtiter plate 10 is covered. If, for reasons of the analysis sequence for the individual samples, individual samples arranged on the microtiter plate 10 have their irradiation included or excluded, it is possible to employ, for example, a ferroelectric LC shutter 30 (for example Scitec FLC 2436). The shutter 30 is likewise is controlled via a software module of the central XCALIbur software belonging to the central control unit 4. This control module can be programmed in advance on use of a plurality of microtiter plates 10, it being possible to store the individual programs until they are executed.
The individual components of the CALI apparatus according to the invention are arranged in a likewise air-conditioned housing 25, which can likewise be sealed from the environment and in which certain defined conditions can be produced. The charging of this housing 25, which is likewise referred to as an incubator, with a new set of samples to be analyzed takes place via an opening in the form of a flap 28 arranged in a wall of the incubator 25, through which the positioning stage 13 can be moved out of the incubator 25, loaded with fresh microtiter plates and moved back into the incubator 25 by means of the slide guide 11 driven by servomotors 14.
In order to increase the throughput, it is of course also possible to arrange and operate two or more lamp units (irradiation units) 26 in parallel, their control likewise being effected by the XCALIbur central software. This control software ensures coordination of the movement of the individual units, of the analyses of the individual samples and of the external automatic transport system.
The CALI apparatus according to the invention can, owing to its simple structure, easily be incorporated into existing laboratory automation, it being possible for the charging and emptying of the CALI apparatus to take place by the appropriate transport robots belonging to the overall laboratory automation equipment (for example CRS Linear Robot).

" - 31 -The CALI apparatus allows the maintenance of cell-culture conditions, such as, for example, carbon dioxide content, atmospheric humidity and temperature, throughout the irradiation time. For certain analyses, it is furthermore possible likewise to incorporate flexible changes to the cell-culture conditions over time. Furthermore, it is likewise possible for defined areas of the microtiter plate to be excluded from the irradiation or irradiated for different radiation durations. The flexibility of the analysis is thus increased further, and control experiments are readily possible.
Owing to the use of a lamp unit 27, it is thus possible, in a simple manner, without using a laser, to achieve an effective, inexpensive and reliable instrument for carrying out the CALI process.

List of reference numerals 1 CALI apparatus 2 Laser device 3 Laser beam 4 Control unit 4.1 Central system control software 4.2 Laser control module 4.3 Control module for optical parametric oscillator (XPO) 4.4 CNC control module Sample holder 6 Beam modification device 7 Power meter 8 Optical bench 9 Optical prism Microtiter plates 11 Slide guide in the x direction 12 Slide guide in the y direction 13 Positioning stage 14 Stepping motors Ball spindles 16 Support/receptor for microtiter plates 17, 18 Steel plates 19 Screws Peltier elements 21 Aperture for supply lines 22 Flap 23 Attachment openings 24 Aperture for irradiation optics Incubator 26 Irradiation chamber 27 Light source device/lamp unit 28 Flap 29 Supply lines Shutter

Claims (27)

Claims

1. Apparatus (1) for automated chromophore-assisted laser inactivation (CALI) having a laser device (2) for the irradiation of samples by means of a laser beam (3) for identifying the function of biological molecules, having a control unit (4) having at least one sample holder (5), where a relative movement in at least two directions between the laser beam (3) and the sample holder (5) can be produced by means of a drive for the laser device (2) and/or the sample holder (5) in such a way that the samples can be irradiated/scanned by the laser beam (3)and having a power meter (7) for determining the power emitted by the laser device (2), characterized in that the laser beam (3) can be matched in shape and/or power and/or intensity to the sample size and/or the sample type and/or the sample number by means of a beam modification device (6, 9), and the control unit (4) is designed in such a way that the laser device (2) and its drive and/or the drive for the sample holder (5) and the beam modification device (6, 9) can be controlled centrally by this control unit (4) by means of system software (4.1).

2. Apparatus according to claim 1, characterized in that the beam modification device (6, 9) expands/narrows/limits the laser beam (3) one-dimensionally in cross section, in particular as a straight line, or two-dimensionally, as an area.

3. Apparatus according to claim 2, characterized in that the beam modification device (6, 9) modifies the laser beam (3) in cross section in a rectangular, circular or elliptical manner.

4. Apparatus according to one of claims 1 to 3, characterized in that the beam modification device (6) is a beam expander.

5. Apparatus according to one of claims 1 to 3, characterized in that the beam modification device (9) is a beam splitter and/or a beam expander is additionally provided.

6. Apparatus according to claim 5, characterized in that the beam modification device (9) is designed in such a way that at least two samples can be irradiated simultaneously.

7. Apparatus according to one of claims 1 to 6, characterized in that the laser device (2) is a pulsed laser, in particular an Nd:YAG infrared laser, having a downstream parametric oscillator for wavelength modification.

8. Apparatus according to one of claims 1 to 6, characterized in that the laser device (2) is a continuous laser, in particular an argon ion gas laser, an adjustable argon-pumped dye laser or a helium-neon laser.

9. Apparatus according to one of claims 1 to 8, characterized in that the control unit (4) varies the laser beam (3) in cross section, intensity, irradiation duration and dimensions as a function of the movement of the sample holder (5), the number and/or the type of the samples.

10. Apparatus (1) for automated chromophore-assisted light inactivation (CALI), having a light-source device (27) for irradiating samples by means of light for identifying the function of biological molecules, having a control unit (4), having at least one sample holder (5) and having a power meter (7) for determining the power light yield emitted by the light source device (27), characterized in that the light-source device (27) is a lamp unit (27) in which at least part of the wavelength of its emitted light is in the visible region, and wherein the light yield measured with the power meter (7) is adjustable by means of the control unit.

11. Apparatus (1) according to claim 10, characterized in that the light can be matched in shape and/or intensity to the sample size and/or the sample type and/or the sample number by means of a beam modification device (6).

12. Apparatus according to claim 11, characterized in that a drive for moving the sample holder (5) and the beam modification device (6) can be controlled centrally via system software (4.1) by means of the control unit (4).

13. Apparatus according to one of claims 10 to 12, characterized in that the lamp unit (27) has at least one incandescent lamp, quartz lamp, fluorescent tube or light-emitting diode, in particular in the form of a white-light source.

14. Apparatus according to claim 13, characterized in that the beam modification device (6) has masks, in particular having variable apertures/diaphragms.

15. Apparatus according to one of claims 10 to 14, characterized in that the light is diffuse light.

16. Apparatus according to one of claims 1 to 15, characterized in that the light-source device (27) is designed in such a way that samples arranged on the sample holder (5) can be irradiated simultaneously.

17. Apparatus according to one of claims 1 to 16, characterized in that the light is at least one light beam.

18. Apparatus according to one of claims 1 to 17, characterized in that the sample holder (5) has a positioning stage (13) which carries at least one microtiter plate (10), in particular having wells of different dimensions, and can be positioned in the x, y direction by means of displacement mechanisms (11, 12) which are arranged at right angles to one another and which have stepping motors (14) as drive.

19. Apparatus according to claim 18, characterized in that the displacement mechanisms (11, 12) are guided on rails and have ball spindles (15) for transmission of the movement from the respective stepping motor (14).

20. Apparatus according to one of claims 1 to 19, characterized in that the sample holder (5) carries two microtiter plates (10).

21. Apparatus according to one of claims 1 to 20, characterized in that a ligand-binding partner (LBP) screening machine for generating specific LBPs aimed at specific target molecule ligands, a unit for the production/purification of BPs, and an LBP-chromophore coupling unit for linking the selected LBPs and the synthesized chromophores, a loading device for transferring LBP tags into wells arranged in the microtiter plate (10), a device for moving the microtiter plate (10) into the CALI apparatus (1), and a device for reading activity results are provided.

22. Apparatus according to claim 21, characterized in that the activity results read can be stored in a data base.

23. Apparatus according to claim 21 or 22, characterized in that the screening machine, the LBP-chromophore coupling unit, the loading device and the device for moving the microtiter plate (10) can be controlled centrally by means of the control unit (4) via the system software (4.1).

24. Apparatus according to one of claims 18 to 23, characterized in that an incubator (25) surrounds at least one microtiter plate (10) of the sample holder (5) and is connected to external supply systems in such a way that preset state conditions, in particular carbon dioxide, moisture, temperature and pressure, can be controlled.

25. Apparatus according to claim 24, characterized in that the supply systems can be centrally controlled by means of the control unit (4) via the system software (4.1).

26. Process for identifying the function of a ligand L, in which a sample containing a complex of a ligand and a ligand-binding partner (LBP)-light-activatable tag is irradiated with a light beam from a lamp, in particular in the form of a white-light source, such as an incandescent lamp, quartz lamp, fluorescent tube or light-emitting diode, the power of which is measured and controlled/set in a sample dependent manner.

27. Process according to claim 26, characterized by the steps:
a) selection of a ligand-binding partner (LBP) having specificity for the ligand L, b) coupling of the LBP to a light-activatable tag with formation of LBP-tag, if necessary after prior modification of the LBP in respect of efficient binding to the tag, c) bringing the ligand L into contact with at least one LBP-tag with formation of an L/LBP-tag complex, and d) irradiation of the L/LBP-tag complex while measuring/adjusting the power of a light source device, where the irradiated LBP-tag modifies the bound ligands selectively, where the sequence of steps b) and c) can be interchanged.