custom diffraction method routine in  (MathWorks Inc)

Journal: Frontiers in photonics

Article Title: Incoherent color holography lattice light-sheet for subcellular imaging of dynamic structures

doi: 10.3389/fphot.2023.1096294

Figure Lengend Snippet: The ICHLLS system. (A,B) Schematics of the ICHLLS systems with (C) one diffractive lens (ICHLLS 1L) of focal length f SLM,488nm = 400 mm or f SLM,561nm = 415 mm at the phase shift θ = 0; (D) The axis orientation in the ICHLLS 1L system; (E) The top-view of the lattice beams inside the ICHLLS 1L; (F) The OpticStudio schematics used to calculate the focal distances for the ICHLLS 1L; (G) Schematics of the ICHLLS systems with two diffractive lenses (ICHLLS 2L) with focal lengths f d1,488nm = 220 mm , f d2,488nm = 2356 mm, f d1,561nm = 228 mm, and f d2,561nm = 2444 mm, at the phase shift θ = 0, superposed with a slight defocus to bring the objects in focus in the middle of the camera FOV; (H) The z-galvo positions in the ICHLLS 2L system; (I) The top-view of the lattice beams superposed with Fresnel diffractive lenses inside the ICHLLS 2L; (J) The OpticStudio schematics used to calculate the focal distances for the ICHLLS 2L; The system consists of a water immersed microscope objective MO (Nikon ×25, NA1.1, WD 2 mm), lenses L 1 = L 4 with focal lengths 175 mm, L 2 = L 3 with focal lengths 100 mm; mirrors M 1 , M 2 , M 3 ; polarizer P oriented at a 45° angle ( ) to the active axis of the SLM ( ); band pass filters BPF 1 centered at 519 nm (Chroma Tech, 26 nm bandpass width) for the excitation wavelength λ = 488 nm, and BPF 2 centered at 575 nm (Chroma Tech, 23 nm bandpass width) for the excitation wavelength λ = 561 nm; phase spatial light modulator SLM (Meadowlark Inc.). The light propagates through either pathway LLS emission (dotted black line in A) for the original LLS or pathway ICHLLS (red and green line in A) for ICHLLS, depending on the orientation of sliding mirror. A collimated 30 Bessel beam is focused by an excitation objective lens (A,B) which generates a lattice light sheet. While the z-galvo (light sheet) and z-piezo (detection objective) are moved along the z-axis to acquire stacks in LLS and IHLLS1L, (D,E) , in IHLLS 2L only the z-galvo is moved at various z positions (B,H,I) . For IHLLS, the size of the beam coming out the objective is diminished in half by the relay lens system, L 1 and L 2 , to fit the size of the SLM. The SLM plane is optically conjugated with the objective back-focal-plane. The diffraction mask in the original LLS system was positioned for all experiments on the anulus of 0.55 outer NA and 0.48 inner NA. The CMOS camera, tube lens, filter, and detection objective lens are used for fluorescence detection. The detection magnification is 62.5. The width of the light sheet in the center of the FOV is about 400 nm. x -axis is the direction of the x-galvo mirror motion, z -axis is the direction of the z-piezo mirror motion, and s-axis is the direction of excitation light propagation. For values of distances d1 to d8, see .

Article Snippet: Each hologram processing can be sub-divided as follows: 1) Image pre-processing in which a background correction is performed by subtracting an average background level obtained by measuring the mean intensity of each stain outside the cells; 2) Hologram reconstruction in which the complex hologram is propagated and reconstructed at the best focal plane using a custom diffraction method routine in MATLAB (MathWorks, Inc.); and 3) Object feature extraction performed on the reconstructed amplitude and phase images using FIJI (ImageJ).

Techniques: Microscopy, Fluorescence

Journal: Frontiers in photonics

Article Title: Incoherent color holography lattice light-sheet for subcellular imaging of dynamic structures

doi: 10.3389/fphot.2023.1096294

Figure Lengend Snippet: The ICHLLS system diffraction. (A) The 9 z-galvo position hologram intensity images: z galvo = ± 40μm± 30μm; ± 20 μm; ± 10 μm,; 0 μm; (B) Hologram intensity images at θ = 0, for each z-galvo level; (C) Example of hologram reconstruction at different z-galvo positions; (D) The reconstructed amplitude and phase at two z-galvo positions; z galvo = −40 μm and 0μm. The FOV is 208 μm 2 × 208 μm 2 and pixel size 0.101 μm.

Article Snippet: Each hologram processing can be sub-divided as follows: 1) Image pre-processing in which a background correction is performed by subtracting an average background level obtained by measuring the mean intensity of each stain outside the cells; 2) Hologram reconstruction in which the complex hologram is propagated and reconstructed at the best focal plane using a custom diffraction method routine in MATLAB (MathWorks, Inc.); and 3) Object feature extraction performed on the reconstructed amplitude and phase images using FIJI (ImageJ).

Techniques: