Optical arrangement for flow cytometer to facilitate large angle light-scattering measurement

• US 5,684,575 A
• Filed: 09/18/1995
• Issued: 11/04/1997
• Est. Priority Date: 03/18/1993
• Status: Expired due to Fees

• Current Assignee: Flowtech As
• Original Assignee: Harald Steen
• Inventors: Steen, Harald
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): VIERRA EISENBERG, JASON D
• Application Number:
  US08/522,396
• Time in Patent Office: 778 Days
• Field of Search: 356/73, 356/72
• US Class Current: 356/73
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1452 Adjustment of focus; Alignment

Abstract

An optical arrangement for a flow cytometer, wherein intense light is focused by a microscope objective having a numerical aperture Na_i, onto the cells carried by a flow of water through the focal plane of the objective, with another microscope lens situated opposite the objective, with an optical axis and object plane coinciding with the objective and with a numerical aperture, NA_O, which is significantly larger than that of the objective. The objective contains a circular field stop in, or close to, its secondary focal plane, with a diameter corresponding to a numerical aperture NA_df, which is slightly larger than NA_i, and much less than NA_O. The fluorescence and scattered light from the stream of cells are separated by a dicroic mirror on basis of their different wavelength, so that they give rise to separate images in separate image planes of the objective. A telescope is situated behind the image plane and creates an image of the field stop in a plane with two concentric mirrors of different diameters, which separate light scattered from the cells according to the different scattering angles and direct them onto separate light detectors.

Claims

1. An optical arrangement for a flow cytometer, comprising:

an intense light focused by an objective having a numerical aperture Na_i, onto a stream of cells carried by a laminar flow of water through the focal plane of said objective;

a microscope lens situated opposite to said objective, with its optical axis and object plane coinciding with those of said objective, and having a numerical aperture NA_O, which is significantly larger than that of said objective;

said microscope lens containing a circular central field stop disposed substantially in its secondary focal plane, said field stop having a diameter corresponding to a numerical aperture NA_df, which is slightly larger than NA_i, while being much less than NA_O, so that the illumination field of said objective falls entirely within said field stop, and hence so that the image of said stream of cells created by said microscope lens contains only fluorescence and scattered light from said stream of cells;

said fluorescence and scattered light from said stream of cells being separated by a dichroic mirror on basis of their different wavelength, so that said fluorescence and scattered light give rise to separate images of said stream of cells in separate image planes of said microscope lens;

two separate light detectors;

a telescope situated immediately behind said image plane and creating an image of said field stop in a telescope image plane and

two concentric mirrors, of different diameter, situated in said telescope image plane and separating light scattered from said stream of cells to different scattering angles and directing said scattered light of different scattering angles onto said two separate light detectors.

2. The arrangement according to claim 1, wherein:

said stream of cells coincides with said object plane of said objective and microscope lens; and

said stream of cells is illuminated through said objective in two adjacent foci of different wavelengths emitted by two separate light sources.

3. The arrangement according to claim 2, wherein:

first and second slits cover the image of each of said adjacent loci from said light sources in said object plane, so that fluorescence measured behind said first slit originates from only one of said foci, whereas fluorescence measured behind said second slit originates only from the other of said foci.

4. The arrangement according to claim 1, wherein:

said mirrors in said telescope image plane are flat, polished end planes that are cut at an angle of 45°

, of two concentric tubes having their common axis coinciding with the optical axis of said telescope.