Patent Application
20220236188
The application relates to a device for reducing the intensity reduction of the fluorescence dye by laser light when determining the fluorescence and the number of antibodies on exosomes.
A device for determining the fluorescence and the number of antibodies on exosomes is known from DE 20 2018 005 287 U1.
Vesicles in biology are intracellular, very small, round to oval bubbles, which are enclosed by a single or double membrane or a reticulated envelope made of proteins. The vesicles form separate cell compartments in which different cellular processes run. Their size is approximately one micrometer. Vesicles are responsible for the transport of many materials in the cell.
The mechanisms which result in the occurrence of extracellular vesicles have not been completely explained up to this point. Three types of extracellular vesicles are differentiated on the basis of their origin or size.
In this case, exosomes are small vesicles having a size of approximately 50 to 150 nm.
According to claim 1 of this document, it relates to a device having the following features:
Multiple measurements in succession using the same sample to achieve a statistically good result cannot be carried out in the case of the described prior art in DE 20 2018 005 287 U1, since the fluorescence dye used here fades too strongly due to the long laser action time and requires a replacement of the sample in the measurement cell after measurement.
If the action time of the laser on the laser dye (fluorochrome) were shortened, the fading could be reduced and multiple measurements using the same sample could be enabled.
The present application is therefore based on the object of shortening the action time of the laser light on the fluorochrome (fluorescence dye).
The object was achieved by the device as claimed in claim 1
and the method as claimed in claim 4
The figures of the application have the following content.
The number of the lasers is five instead of four in the prior art. The liquid filter 6 in the prior art is replaced by a notch filter 6 having an associated change device 7, for the notch filter 6 and a quartz glass 16. The quartz glass 16 is used to set and secure the same optical path lengths for the fluorescence light and the laser scattered light in the convergent beam path through the objective to the light sensor 12 during the adjustment of the measurement points 26 (see
The video camera 15 in the prior art has the number 13 here and has a graphene-based light sensor 12 having a controller 11 for the light sensor 12 and controls, by means of the camera optical unit 14 via an optical unit controller 8, a liquid lens 15 which in turn via the notch filter 6 is incident in the fluorescence plane 20 in the sample 18 of the measurement cell 17 and the optical passage window 19 in the measurement cell 17 on a beam path 24 of one of the lasers 1 to 5 and one of the collecting prisms 21.
The overall controller 10 corresponds to the overall controller 20 in the prior art, wherein the display 9 corresponds to the corresponding display 19.
The illustration of the measurement cell 17 in
The filling opening 25 of the measurement cell 17 can be seen on the front side.
The reduction of the intensity of the dye after the laser action in seconds can be seen well in percentage in
The illustration of the measurement positions in the measurement cell 17 and the measurement locations 26 for the focusing points (the laser scattered light plane and the fluorescence light plane) of the optical unit to the beam path of the laser 24 can be inferred from the various measurement positions 1 to 4 shown in
For example, the 4 positions shown in
The measurement cell 17 is filled using test liquid, wherein this test liquid contains polystyrene particles or certified exosome standards instead of the exosome sample.
The individual lasers are then guided in the respective measurement point 26 of the measurement cell 17 (see
That is to say, all 5 lasers are each guided into the measurement position 1, and the optical path lengths are ascertained, focused, and then stored. Furthermore, all 5 lasers are then guided into the next measurement position, focused, and then stored.
This is also carried out with the position 3 and the position 4 and all 5 lasers.
The lasers are then switched off.
As the next step, the calibration liquid is replaced with a sample liquid having exosomes in the measurement cell.
As the next step, the notch filter 6 is pushed between the liquid lens 15 and the measurement cell 17 and moved into the convergent beam path, wherein the quartz glass 16 is used for the fixing in conjunction with the device 7.
The actual measurement begins at measurement position 1 in that the five lasers are switched through in succession on their stored focus points and the images are recorded by the light sensor and the data are passed on to the pattern recognition. The time required for this purpose is less than two seconds. The lasers are then switched off.
The lasers move further to measurement position 2. The measurement follows as in the preceding step. The lasers are then switched off.
The lasers move further to the measurement position 3. The measurement again takes place as in the preceding step. The lasers are then switched off.
The lasers move to the measurement position 4. The data are evaluated.
The particles can form high or low concentrations of particles of different levels at various points in the measurement cell. For example, due to the filling. Multiple measurement positions are thus necessary in the measurement cell to obtain a good quality and a high statistical certainty of the measurement.
In the newly approached measurement positions in the measurement cell, the fluorescence on the antibodies is still fresh and unused.
The fluorescence is further preserved by switching off the laser when moving to the next measurement position, since no laser light can cause fading. This is only possible due to the prior storage of the individual measurement positions and focus points and the high-precision rapid approach to the positions again. (Rapid stepping motors, precision movement carriages without play at the lasers and liquid lens in the objective).
The multi-band pass filter 6 (multi-notch filter) enables the rapid switching through of the individual lasers. A time saving thus results for preserving the fluorescence, since no filter change is necessary.
By way of the careful measurement method shown, not only four, as in our example, but up to 100 or more measurement positions having the measurement locations 26 can be approached and measured using five lasers in the same exosome fluorescence sample. This ensures a very high quality of the measurement.
In the camera, a high-sensitivity sCMOS or an eCCD or a graphene light sensor has to be used, since the multiband pass filter 6 (notch filter) reduces the light intensity.
The method requires a complex control of the described method and movement sequences by a special control and analysis program.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2019 004 870.9 | Jul 2019 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/DE2020/000149 | 7/6/2020 | WO | 00 |
