Figure 1.
Schematic diagram of the complete imaging system.
The illumination sub-system employs spatial filter at the back-focal plane of the cylindrical lens for generating multiple light-sheet pattern. The detection sub-system is essentially a theta-detection system (orthogonal detection) with a fine scanning ability along axis. Position
and
indicate two scan positions of the detector sub-system.
Figure 2.
Illumination PSFs obtained from computational simulation.
The study was carried out for two configuration, small numerical aperture (aperture angle, , stop angle,
), and large numerical aperture (aperture angle,
, stop angle,
). For comparison, the illumination PSF of the state-of-art SPIM system (aperture angle,
, stop angle,
) is also shown. Scale bar is
.
Figure 3.
Experimental generation of multiple light-sheets and characterization.
(A) Schematic diagram of the experimental setup used for characterizing the illumination PSF. (B) Experimentally obtained transverse profile of the multiple light-sheet system. (C) Intensity plot of the experimentally obtained illumination PSF along y−axis. The inter sheet separation is found to be 15 whereas the FHWM of each sheet is 7.5
. The dotted line indicate
prominent light-sheets with relative intensity
.
Figure 4.
Fluorescently coated yeast cells, encaged in gel matrix, imaged by multi-sheet excitation and orthogonal detection.
(A) Cartoon depicting various positions (a–n) of a particular yeast cell as the sample is translated with respect to the detection focal region and the multi-sheet system. (B) Image sequence captured from the orthogonal detection arm. Each frame is spatially separated by along the y−axis. The primary region of interest is marked by the number ‘1′ and enclosed by an orange square in frames (d)–(n) and the cell within it exhibits variation in overall intensity, indicating the presence of multiple light-sheets. This can be understood clearly when compared with the cartoon in B. Additionally, the images of the cell defocus far from the detection focus. The cells ‘2′ and ‘3′ encircled in blue and green respectively, also exhibit similar intensity variations and defocusing but are situated at different
-planes.
Figure 5.
Demonstration of multi-plane imaging capability.
(A) Schematic depiction of the experimental setup (not to scale). The near focal region of the cylindrical lens is magnified to show the multiple light-sheets. The orthogonal detection arm is traversed along the axis to focus on the different light-sheets. The numbers
figuratively depicts five light-sheets from which experimental images were obtained. (B) Experimental images corresponding to different planes illuminated by the light-sheets. The sample used was fluorescently coated yeast cells encaged in agar gel matrix. Each image is parallel to the
plane and contains images of yeast cells in focus and defocussed images of cells illuminated by other light-sheets. Some of the in-focus cells are marked by the orange boxes and those regions are zoomed and presented below each such image. The distance between each image (
) is approximately
and the scalebar represents
.