Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

Abstract

Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ≈90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

Mirror-enhanced super-resolution microscopy

“…No experimental data using mirror reflection were obtained until the I 5 M microscopy 14 , where two opposing objectives were used to interfere two coherent incident beams and the fluorescent signal. Most recently, by employing a low numerical aperture (NA) objective, a mirror, and a plan-convex lens to form Newton rings, standing-wave multiplanar excitation was demonstrated for axial imaging 9 .…”

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…If a planoconvex lens is placed on the mirror or the mirror is replaced with a planoconvex reflector, and a high‐NA objective lens is used, the interference of the incident and reflected light produces Newton rings. The number of rings represents the axial position of the object and can be utilized to map the contour of the surface plasma membrane of red blood cells with ≈90‐nm axial resolution, for example …”

Widefield standing wave microscopy of red blood cell membrane morphology with high temporal resolution

“…Uncoated silica plano-convex lenses, with a focal length of 30 mm and a diameter of 6 mm (Edmund Optics), were cleaned using deionized water and then blow dried with compressed air to remove any contaminants. We amended the lens preparation protocol described by Amor et al [ 15 ], by replacing the APTMS coating with a solution of 0.01% mass concentration poly-L-lysine in H 2 O (Sigma Aldrich) to allow the binding of 1,1'-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (DiI) to the lens surface. The specimens and poly-L-lysine solution were placed on a platform rocker for 45 - 60 minutes to evenly coat the curved surface of the lenses in the solution, after which the lenses were thoroughly washed in deionised H 2 O and blow dried.…”

“…Amor et al [ 15 ], previously reported the use of confocal laser scanning SW microscopy to image the red cell membrane. By placing the specimen on a mirror at the specimen plane they simultaneously imaged multiple anti-nodal planes to create a contour map of the membrane structure.…”

“…By placing the specimen on a mirror at the specimen plane they simultaneously imaged multiple anti-nodal planes to create a contour map of the membrane structure. They were able to achieve this in both healthy and unhealthy red blood cells and clearly observe the topography of the red blood cells biconcave section with an axial resolution on the order of 90 nm though the use of confocal microscopy limited their acquisition time to 40 seconds per frame [ 15 ].…”