Fluorescence flow cytometry device and method
A flow cytometer including a laser, indexing structure, adjustment structure, and sensor structure. The cytometer is conventionally used with a removable microfluidic cassette, which is installed at a first position that is enforced by the indexing structure. The adjustment structure changes a relative position between an interrogation aperture of the cassette and the laser beam. Feedback from the sensor structure is used to optimize propagation of the laser through the interrogation aperture to reduce (and hopefully eliminate) autofluorescence caused by beam impingement onto the cassette.
1. A microfluidic interrogation apparatus, comprising:
indexing structure effective to hold a microfluidic device at an installed position such that an interrogation aperture of said microfluidic device is urged near to a desired location, said microfluidic device being of the type arranged to urge particles of interest through said interrogation aperture in a substantially single-file arrangement;
a source of stimulation radiation structured to emit radiation as a beam oriented for propagation of radiation in a particular direction;
adjustment means to refine alignment of said interrogation aperture, from an initially installed position, and with respect to said beam, to maximize beam propagation into said interrogation aperture;
sensor means disposed to provide steering feedback to said adjustment means; and
a first photodetector disposed to detect Stokes-shift emission radiation from a particle disposed inside said interrogation aperture, wherein:
said microfluidic device comprises a plurality of stacked layers forming a first channel segment disposed on one side of an interrogation layer, said interrogation aperture being structured and arranged to communicate fluid from said first channel segment to a second channel segment disposed on the opposite side of said interrogation layer, and said first channel segment and said second channel segment are structured to urge fluid flow therethrough in a direction generally parallel to said interrogation layer.
2. The apparatus according to claim 1 , wherein:
said adjustment means is operable to maximize beam propagation through said interrogation aperture in a direction congruous with a centerline of said interrogation aperture.
3. The apparatus according to claim 1 , wherein:
a blocking element associated with said microfluidic device is structured to resist propagation of radiation from said source of stimulation radiation toward said first photodetector, other than along a path having a cross-section bounded by a perimeter of, and passing through, said interrogation aperture.
4. The apparatus according to claim 3 , wherein:
said blocking device comprises said interrogation layer, said interrogation layer being made from a semi-transparent film.
5. The apparatus according to claim 3 , wherein:
said blocking device comprises a coating carried on one side of a stacked layer, a void in said coating being sized and disposed in agreement with said perimeter.
6. The apparatus according to claim 5 , wherein:
said coating is carried on one side of said interrogation layer.
7. The apparatus according to claim 5 , wherein:
said coating comprises an electrically conductive element carried on one side of said interrogation layer.