Fig 1.
Optics and light beampaths for microscope setups.
(A) Widefield illumination optics create field stop plane, whose light distribution is projected to focal plane in sample via objective. Camera images emission light from focal plane. (B) Introduction of SLM at field stop masks illumination light and allows arbitrary illumination patterns at focal plane. (C) Confocal microscope optics scan illumination beam and utilize pinhole to exclude out-of-focus emission light.
Fig 2.
Illumination setup and resolution.
(A) SLM on 3D-printed mount on 6-axis fine-positioning stage. SLM inserted into field stop of microscope. (B) Transverse illumination resolution determined using fluorescent dye thin film. (C) Approximate widefield axial illumination beam and intensity profiles. Illumination spans FOV (red). (D) Approximate scanned axial illumination beam and intensity profiles. Beam is scanned across FOV (red line). (E) Measured intensity along optical axis for widefield and SLM-scanned illumination beams. SLM-scanned illumination (black) has significantly better resolution than widefield illumination (gray).
Fig 3.
Illumination strategies and postprocessing.
(A) Dot array transmitted by SLM illuminates sample. Array is raster scanned across FOV. (B) Expansion of inset in (A). Raster scanning moves single-element illumination through 36 locations of 6 × 6-unit cell. Sub-image of entire FOV is acquired for each of 36 illumination patterns. (C) Line drawing of ASJ neuron imaged. Dorsal-ventral view in xy direction (top), lateral view in xz direction (bottom). (D-F) Fluorescence images, obtained by maximum projections of 3D image. (D) Conventional widefield image. (E) SLM-max image. Pixel value in 3D image is maximum pixel value of 36 subimages. (F) SLM-pick image. Pixel value is in 3D image is pixel value when corresponding sample location is illuminated. Note significantly improved optical sectioning in the max and pick strategies, mostly visibly above and below the cell bodies. Fluorescent image normalization saturates pixels near cell body to visualize dim neuronal fibers.
Fig 4.
SLM confers sectioning capability of diffuse fluorescent source.
(A) Intensity of light at axial positions around fluorescent thin film at z = 0. Dotted line at 50% maximum intensity. (B) FWHM of data in (A). n = 20 locations. Note optical sectioning capability conferred by SLM.
Fig 5.
SLM improves sectioning capability of localized fluorescent source.
(A) Average transverse intensity profile of 6-μm fluorescent beads. Dotted line at 10% maximum intensity. Insets show maximum projection of average bead images with profile line. (B) Average axial intensity profile of 6-μm fluorescent beads. Insets show maximum projection of average bead images with profile line. (C) Transverse full width at 10% maximum intensity in (A). (D) Axial full width at 10% maximum intensity in (B). Note improved confinement of fluorescence to true bead extent in SLM imaging compared to widefield, particularly in axial direction. n = 85 (widefield), 90 (SLM-pick), and 63 (confocal) beads.
Fig 6.
In vivo imaging of fluorescent neurons in C. elegans.
(A) Line drawing of amphid neurons imaged. General location of cell bodies and neuronal fibers shown for simplicity. (B-D) Fluorescence amphid neuron images taken by widefield (B), SLM-pick (C), and confocal (D) microscopes. Insets show 3x expanded view of axons (i) and dendrites (ii) with line profiles. (E) Intensity profile of axons (i) and dendrites (ii). Peaks correspond to clear fibers in fluorescence images (B-D). Note correlation of peaks in SLM and confocal profiles and large background obscuring fibers in widefield profile. (F-H) Fluorescence images of FLP neuron taken by widefield (F), SLM-pick (G), and confocal (H) microscopes. (I) Intensity profile in images (F-H). Note correlation of peaks in SLM and confocal profiles and large background in widefield profile, particularly near cell body. Excluded peak in SLM image indicated by *. Fluorescent image normalization saturates pixels near cell body to visualize dim neuronal fibers.
Table 1.
Signal-to-background ratios of neuronal fibers in Fig 6F–6H.