Interferometric scattering microscopy

Abstract

An interferometric scattering microscope is adapted by performing spatial filtering of output light, which comprises both light scattered from a sample location and illuminating light reflected from the sample location, prior to detection of the output light. The spatial filtering passes the reflected illumination light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures. This enhances the imaging contrast for coherent illumination, particularly for objects that are weak scatterers.

1 . A method of quantifying the mass of an object, wherein the mass of said object is quantified by interferometric light scattering, and wherein said mass is quantified with up to 5% mass error. 
     
     
         2 . A method according to  claim 1 , wherein said mass is quantified with equal or less than 2% mass error. 
     
     
         3 . A method according to  claim 1 , wherein said mass is quantified within lkDa of the sequence mass of the object. 
     
     
         4 . A method according to  claim 1 , wherein said object is 19 kDa or greater in size. 
     
     
         5 . A method according to  claim 1 , wherein said object is a weak scatterer of light. 
     
     
         6 . A method according to  claim 1 , wherein said object is a nucleic acid molecule. 
     
     
         7 . A method according to  claim 1  wherein said object is a virus-like particle. 
     
     
         8 . A method according to  claim 1 , wherein said object is a single protein. 
     
     
         9 . A method according to  claim 1 , wherein said object is a glycoprotein. 
     
     
         10 . A method according to  claim 1 , wherein said object is in solution. 
     
     
         11 . A method of measuring or quantifying a change in the mass of an object, wherein the change in mass of said object is measured or quantified by interferometric light scattering. 
     
     
         12 . A method of  claim 11 , wherein the mass of the object changes due to one or more events selected from the group consisting of single molecule binding/unbinding, phase transition, clustering, assembly/disassembly, aggregation, one or more protein/protein interactions and/or one or more protein/small molecule interactions. 
     
     
         13 . A method of  claim 11 , wherein the mass of the object changes due to oligomeric assembly or glycoprotein cross-linking. 
     
     
         14 . The method of  claim 11 , wherein the change in mass of the object is time-resolved, optionally at a specific position and/or local concentration of said object. 
     
     
         15 . The method of  claim 11  wherein the change of mass of the object is measured in a position and local concentration sensitive manner. 
     
     
         16 . The method of  claim 11 , wherein one or more interactions resulting in change in the mass of the object are quantified. 
     
     
         17 . The method of  claim 11 , further comprising determining thermodynamic and/or kinetic parameters influencing the change in the mass of the object or of one or more interactions resulting in change in the mass of the object. 
     
     
         18 . The method of  claim 11 , wherein the mass of said object is from 10 kDa to 5000 KDa. 
     
     
         19 . The method of  claim 11  wherein the change of mass of the object is measured over time in a localisation-dependent manner, optionally wherein the localisation is precise at the level of sub-diffraction. 
     
     
         20 . The method of  claim 11  wherein the change of mass of the object is due to binding and dissociation of binding partners. 
     
     
         21 . The method of  claim 11  wherein the change of mass of the object allows the correlation of binding events to single molecule fluorescence localization measurements, preferably to identify specific binding partners labelled with fluorescent molecules. 
     
     
         22 . The method of  claim 11  wherein the change of mass of the object is due to an interaction with a binding partner of known specificity to determine the identity of the object. 
     
     
         23 . An interferometric scattering microscope comprising:
 a sample holder for holding a sample in a sample location;   an illumination source arranged to provide illuminating light;   a detector;   an optical system being arranged to direct illuminating light onto the sample location and being arranged to collect output light in reflection, the output light comprising both light scattered from the sample location and illuminating light reflected from the sample location, and to direct the output light to the detector; and   a spatial filter positioned to filter the output light, the spatial filter being arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures.   
     
     
         24 . An interferometric scattering microscope according to  claim 23 , wherein the predetermined numerical aperture is the numerical aperture of the illuminating light reflected from the sample location that is comprised in the output light. 
     
     
         25 . An interferometric scattering microscope according to  claim 23 , wherein the spatial filter is arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to 10 −2  of the incident intensity or less. 
     
     
         26 . An interferometric scattering microscope according to  claim 23 , wherein the spatial filter is arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to 10 −4  of the incident intensity or more. 
     
     
         27 . An interferometric scattering microscope according to  claim 23 , wherein the predetermined numerical aperture is less than 1, preferably less than 0.5. 
     
     
         28 . An interferometric scattering microscope according to  claim 23 , wherein the illuminating light is spatially and temporally coherent. 
     
     
         29 . An interferometric scattering microscope according to  claim 23 , wherein the optical system comprises a beam splitter arranged to split the optical paths for the illuminating light and the output light, the spatial filter being part of the beam splitter. 
     
     
         30 . An interferometric scattering microscope according to  claim 23 , wherein the spatial filter is transmissive. 
     
     
         31 . An interferometric scattering microscope according to  claim 23 , wherein the spatial filter is reflective. 
     
     
         32 . An interferometric scattering microscope according to any of the preceding claims wherein the sample holder incorporates a solid immersion lens. 
     
     
         33 . An interferometric scattering microscope according to  claim 32 , wherein the solid immersion lens is hemispherical or superhemispherical. 
     
     
         34 . An interferometric scattering microscope according to  claim 23 , wherein the optical system includes an objective lens and the spatial filter is positioned directly behind the back aperture of the objective lens. 
     
     
         35 . An interferometric scattering microscope according to  claim 23 , wherein the optical system includes an objective lens and the spatial filter is positioned at a conjugate focal plane of the back focal plane of the objective lens. 
     
     
         36 . An interferometric scattering microscope according to  claim 23 , wherein the sample holder holds a sample comprising objects having a mass of 5000 kDa or less. 
     
     
         37 . An interferometric scattering microscope according to  claim 36 , wherein the sample holder holds a sample comprising objects having a mass of 10 kDa or more. 
     
     
         38 . An interferometric scattering microscope according to  claim 23 , wherein the sample holder holds a sample comprising objects having a scattering cross section with respect to the illuminating light of 10 −1  m 2  or less. 
     
     
         39 . An interferometric scattering microscope according to  claim 36 , wherein the sample holder holds a sample comprising objects having a scattering cross section with respect to the illuminating light of 10 −26  m 2  or more. 
     
     
         40 . An interferometric scattering microscope according to  claim 23 , wherein the microscope is arranged to operate in a wide-field mode and detector comprises an image sensor that is arranged to capture an image of the sample. 
     
     
         41 . An interferometric scattering microscope according to  claim 23 , wherein the microscope is arranged to operate in a confocal mode, and the microscope further comprises a scanning arrangement arranged to scan a region of the sample to build up an image. 
     
     
         42 . An interferometric scattering microscope according to  claim 23 , wherein the sample holder comprises a surface for holding the sample thereon. 
     
     
         43 . The method according to  claim 1 ,  11  or  19  to  22 , comprising use of an interferometric scattering microscope as defined in  claim 23 . 
     
     
         44 . A method according to  claim 43 , wherein the microscope is arranged to operate in a wide-field mode and detector comprises an image sensor that is arranged to capture an image of the sample. 
     
     
         45 . A method according to  claim 43 , wherein the microscope is arranged to operate in a confocal mode, and the microscope further comprises a scanning arrangement arranged to scan a region of the sample to build up an image. 
     
     
         46 . A method according to  claim 43 , wherein the sample holder comprises a surface for holding the sample thereon. 
     
     
         47 . A method of adapting an interferometric scattering microscope, the method comprising providing a spatial filter that performs spatial filtering of output light in reflection, which output light comprises both light scattered from a sample at a sample location and illuminating light reflected from the sample location, prior to detection of the output light, the spatial filtering passing the output light but with an intensity reduction that is greater within a predetermined numerical aperture than at larger numerical apertures, the method further comprising detecting the interferometric contrast to detect weakly scattering objects. 
     
     
         48 . A method according to  claim 47 , wherein the predetermined numerical aperture is the numerical aperture of the illuminating light reflected from the sample location that is comprised in the output light. 
     
     
         49 . A method according to  claim 47  or  48 , wherein the spatial filter is arranged to pass output light with a reduction in intensity within said predetermined numerical aperture 10 −2  of the incident intensity or less. 
     
     
         50 . A method according to any one of  claims 47  to  49 , wherein the spatial filter is arranged to pass output light with reduction in intensity within said predetermined numerical aperture to 10 −4  of the incident intensity or more. 
     
     
         51 . A method according to any one of  claims 49  to  50 , wherein the predetermined numerical aperture is less than 1, preferably less than 0.5. 
     
     
         52 . A method according to any one of  claims 47  to  51 , wherein the illuminating light is spatially and temporally coherent. 
     
     
         53 . A method according to any one of  claims 47  to  52 , wherein the sample holder holds a sample comprising objects having a mass of 5000 kDa or less. 
     
     
         54 . An interferometric scattering microscope according to  claim 53 , wherein the sample holder holds a sample comprising objects having a mass of 10 kDa or more. 
     
     
         55 . A method according to any one of  claims 47  to  54 , wherein the sample comprises objects having a scattering cross section with respect to the illuminating light of 10 −12  m 2  or less. 
     
     
         56 . A method according to  claim 55 , wherein the sample comprises objects having a scattering cross section with respect to the illuminating light of 10 −20  m 2  or more. 
     
     
         57 . A method according to any one of  claims 47  to  56 , wherein the microscope is arranged to operate in a wide-field mode and detector comprises an image sensor that is arranged to capture an image of the sample. 
     
     
         58 . A method according to any one of  claims 47  to  56 , wherein the microscope is arranged to operate in a confocal mode, and the microscope further comprises a scanning arrangement arranged to scan a region of the sample to build up an image. 
     
     
         59 . A method according to any one of  claims 47  to  58 , wherein the sample holder comprises a surface for holding the sample thereon.