Patent GB-1389553-A (GB)
Title: Apparatus for investigating microscopic particles
IPC: G01N15/14
Filed: 1972-05-23
Abstract: 1389553 Investigating microscopic particles G LEGORRETA 23 May 1972 24062/72 Headings G1A and G1N [Also in Division G2] A microscope has photo-electric image analyzing means viewing its image plane arranged to investigate particles such as blood cells as they pass sequentially along a path including the image plane. In the arrangement of Fig.1, blood cells, suspended in a liquid, are introduced into the microscope through central conduit 22. Continuous staining of the cells is accomplished by passage of dye through outer conduits 23, the walls (24) Fig.2 (not shown) separating the conduits being of porous plastics membrane, which also serves to separate off some smaller cells in accordance with the pore size of the membrane. Cells pass one by one through the focal plane 18 of the microscope, and are illuminated via annular mirror 38, and objective at 16. The light source may be an incandescent lamp, a high pressure spark lamp, an arc lamp or a laser. Light reflected or scattered by the cell at 18 is directed via objective and eyepiece 17 to image plane 30. An image analysis may be initiated by detection of the presence of a cell at plane 18 by capacitor 35, which may also be used to sense cell size or volume, and inhibit analysis if the cell is too small or too large (e.g. a clump of cells). Image analysis is by an array of fibre optic guides 40 leading to corresponding photo-detectors 42, connected to respective amplifiers 43, amplitude discriminators 44a, 44b &c., which may classify the light intensities into e.g. four levels of grey (background, cytoplasm, normal nucleus, cancerous nucleus). A circuit 45 is used to determine characteristics of the cell from the discriminator outputs. A count or summing of spots of a particular intensity may be used to determine the area of a nucleus, cytoplasm &c. Display of counts on a C.R.T. is suggested. In dependence on an evaluation of the cell at plane 18, it may be diverted from the normal direction (along conduit 34) to conduit 31, by opening valve 32. An alternative sorting arrangement comprises altering the charge on collar 51, so that droplets of liquid formed by means of Piezoelectric vibrator 47 are deflected by plates 52 into different receptacles 53-55. In an alternative arrangement, a linear array of optical fibres monitors passage of a cell moving parallel to the optical axis, so as to generate a sequence of signals which provide indications of texture density and shape of parts of the cell. In the embodiment of Fig.3, a linear array of optical fibres is provided to allow investigation of a cell moving along the focal plane, the sample being carried on a glass slide 60 or transparent tape. The output ends of the the fibres are connected to an arrangement of three dichroic mirrors and associated photo-cells for colour analysis of the cell. In the embodiment of Fig.4, inlet and outlet conduits lead a sample flow in and out of the microscope substantially at right angles to the optical axis. The apparatus includes platinum electrodes 75,76 to which a D.C. supply is connected, to operate on the Coulter principle and allow volume sensing of cells, and initiation of the optical measurement by an array of detectors 42 e.g. in an integrated circuit. The optical system of Fig.4 is modified in the arrangement of Fig.5 (not shown) in which sample enters along the optical axis. In the embodiment of Fig.7, a sample is fed from container 85, via valve 88, into the microscope and cells pass through a focal plane aperture defined by knife edges, Fig.8 (not shown). A first sizing test is carried out by means of a device applying an electric field or, as shown, rods 117 applying a magnetic field, associated with pick-up electrodes 121. The output is amplitude discriminated by 127, 128 operating on valves 102, 103 to define a size range outside which cells pass to container 90. To decide which of 95 and 96 a cell is diverted to, the photo-electric system of three photo-detectors 42, viewing through sector apertures, includes an anti-coincidence circuit and further pulse height discriminators. The optical system for this embodiment is detailed in Fig.6 (not shown) but may be modified as detailed in Fig.9 (not shown), in which illumination by direct or tangential light is provided. The light source for the peripheral (tangential) lens system may be operated independently of that for the axial (direct) system, under the control of the  photo-detectors. Particles for analysis may be suspended in air. Besides absorption and reflection, fluorescence measurements may be made. The number of cells of certain types (red, white &c.) or sizes may be counted, e.g. in Fig.7, by connecting counters to amplifiers 130, 131.