Patent Publication Number: US-2022236188-A1

Title: Device and method for reducing the fading of a fluorescence dye by laser light when determining fluorescence and the number of antibodies on exosomes

Description:
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:
         a) the beams of multiple different lasers ( 1 ,  2 ,  3 ,  4 ) are each directed by means of a separate collecting prism ( 14 ) separately on a beam path ( 21 ) in a measurement cell ( 22 ) having a sample ( 9 ) containing particles, wherein the focusing of the laser beam ( 21 ), in interaction with the sample ( 9 ), forms the center of a convergent beam bundle, consisting of light from the fluorescence plane ( 5 ) and the scattered light plane ( 8 ), which, after passing through a liquid lens having an optical unit controller ( 18 ), is registered in a video camera ( 15 ),   b) the convergent beam path passes through a color filter ( 16 ), which is moved by means of a change wheel ( 17 ) and a controller ( 26 ),   c) a display ( 19 ) having a touchscreen ( 19 ) and an overall controller ( 20 ) having a particle tracking program are used to operate a video camera ( 15 ).       

     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 
         A device for reducing the quality reduction of the fluorescence dye by means of laser light when determining the fluorescence and the number of antibodies on exosomes having the following features:   a) means for storing various measurement points  26  of various different-colored lasers in a measurement cell ( 17 ) at certain measurement positions, wherein the focusing of the respective laser beam ( 24 ) in interaction with the sample ( 18 ) is registered as a center of a convergent beam bundle in a video camera,   b) a change device  7 , for the notch filter  6  and a quartz glass  16  for setting and securing the same optical path lengths for the fluorescence light and the laser scattered light in the convergent beam path between the liquid lens ( 15 ) and the measurement locations  26  in the measurement cell ( 17 ),   c) a display ( 9 ) having a touchscreen and an overall controller ( 10 ) having a particle tracking program are used to operate a video camera ( 13 ), and the video camera ( 13 ) has a graphene-based light sensor ( 12 ) having an associated controller ( 11 ) for the light sensor ( 12 ) and has rapid stepping motors and precision movement carriages free of play and the camera is a CMOS or an eCCD.       

     and the method as claimed in claim  4 
         A method for reducing the intensity reduction of the fluorescence dye by laser light when determining the fluorescence and the number of antibodies on exosomes having the following features:   a) the measurement cell ( 17 ) is filled with test liquid to detect the various positions of the laser used, its focusing, and storage,   b) the calibration liquid is replaced with sample liquid having exosomes to be measured, and the cell ( 17 ) is checked for freedom from bubbles, all lasers are moved back to position  1 ,   c) the lasers used are moved in succession to their stored focus points and switched on at the beginning of the analysis, the images are recorded by the light sensor ( 12 ), and the corresponding data are passed on to the pattern recognition, and a   computer program having a program code for carrying out the method steps as claimed in claim  4  when the program is executed in a computer, and a   machine-readable carrier having the program code of a computer program for carrying out the method as claimed in claim  4  when the program is executed in a computer.       

    
    
     
       The figures of the application have the following content. 
         FIG. 1 : shows an illustration of a special NTA nanoparticle tracking method. 
         FIG. 2 : shows an illustration of the measurement cell. 
         FIG. 3 : shows an illustration of the reduction of the intensity of the dye after laser action in seconds. 
         FIG. 4 : shows an illustration of the measurement positions in the measurement cell and the focusing points of the laser (measurement points). 
     
    
    
       FIG. 1  corresponds in essential parts to the illustration of FIG. 1 from DE 20 2018 005 U1. 
     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  FIG. 4 ) in the measurement cell  17 . The quartz glass simulates the same optical path lengths as the notch filter. 
     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  FIG. 2  shows, vertically from the top, the beam path also shown in  FIG. 1 —laser  24  which passes through the laser passage window  23 , wherein laterally the focus of the fluorescence plane  20  tapering to a point passes through the quartz glass  16 , out of the liquid lens  15  and the camera optical unit  14 . 
     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  FIG. 3 . The laser light is to act at most 2 seconds on the fluorescence at the measurement location  26 . The intensity of the fluorescence dye then becomes too weak and does not supply usable measurement results. 
     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  FIG. 4 . The camera optical unit  14  is also shown here. 
     For example, the  4  positions shown in  FIG. 4  are carried out hereinafter using the arrangement according to  FIG. 1 . 
     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  FIG. 4 ) and stored. 
     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. 
     LIST OF REFERENCE NUMERALS 
     
         
         
           
               1  laser 375 nm 
               2  laser (violet=405 nm) 
               3  laser (blue=488 nm) 
               4  laser (green=520 nm) 
               5  laser (red=640 nm) 
               6  notch filter (multiband pass filter) 
               7  change device for the notch filter and the quartz glass 
               8  optical unit controller 
               9  display having touchscreen 
               10  overall controller having particle tracking program 
               11  controller for light sensor  12   
               12  graphene-based light sensor 
               13  detector or video camera eCCD, sCMOS 
               14  camera optical unit 
               15  liquid lens having settable focus 
               16  quartz glass 
               17  measurement cell 
               18  sample 
               19  optical unit passage window of the measurement cell  17   
               20  fluorescence plane 
               21  collecting prisms (guide all lasers into one beam path) 
               22  scattered light plane 
               23  laser passage window of the measurement cell  17   
               24  beam path laser 
               25  filling opening of the measurement cell  17   
               26  measurement location (measurement point having the focus planes for laser scattered light and fluorescence light)