Journal: bioRxiv

Article Title: Parallel frequency-multiplexed aberration measurement for widefield fluorescence microscopy

doi: 10.1101/2025.10.11.681535

Figure Lengend Snippet: ( a ) Schematic of PFAM implemented in a widefield fluorescence microscope. A deformable mirror (DM, inset) is placed at a plane conjugate to the objective’s back pupil for both aberration measurement and correction. Inset: (Left) Enlarged view of the image plane at the widefield (WF) camera, showing N probe beamlets (gold) scanned around a stationary reference focus (gray) by controlling tip and tilt of their corresponding DM macro-segments; (Upper right) At each tip/tilt configuration, phase of probe beamlet is modulated at a unique frequency ωs ( ω 1 , ω 2 , …, ω N ) by controlling the piston of its corresponding macro-segment, modulating the fluorescence signal resulting from the interference between the probe beamlet and the reference focus at frequency ω s ; (Lower right) Interference strength between each probe beamlet and the reference focus is extracted by performing a Fourier transform (FT) on the fluorescence signal trace and identifying the Fourier magnitude at ω s . ( b ) DM grouping configuration. Modulated regions (gold) consist of macro-segments formed by grouping adjacent DM segments with piston, tip, and tilt control. Remaining segments (gray) are held static to generate the reference focus. To increase sampling density of phase gradient measurement, three DM groups are typically used sequentially (see other groupings in Supplementary Fig. 1 ). ( c ) Tip/tilt interference maps for macro-segments across three DM groups, from which phase gradients are measured. Inset: Example interference map (scaled to the sample plane) of a macro-segment. ( d ) Corrective wavefronts for system aberration measured using PFAM from a 0.5-μm-diameter fluorescent bead (left) and phase retrieval from a 0.2-μm-diameter fluorescent bead (right). Both PFAM and PR were iterated twice. ( e ) Maximum intensity projection (MIP) images in XZ of a 0.5-μm-diameter fluorescent bead acquired without adaptive optics (AO, left), with corrective wavefront from PFAM (middle), and with corrective wavefront from phase retrieval (right). ( f ) Axial fluorescence intensity profiles along dashed lines in ( e ).

Article Snippet: Fluorescent beads with diameters of 0.2 μm, 0.5 μm, 1 μm, 2 μm (FluoSpheresTM Carboxylate-Modified Microspheres, yellow-green 505/515; ThermoFisher Scientific) or 4 μm (ThermoFisher Scientific TetraSpeck Microspheres, fluorescent blue/green/orange/dark red) were diluted 1:1000 to 1:3000 in deionized water and pipetted onto a microscope glass slide (12-550-12, Fisher Scientific).

Techniques: Fluorescence, Microscopy, Control, Sampling

Journal: bioRxiv

Article Title: Parallel frequency-multiplexed aberration measurement for widefield fluorescence microscopy

doi: 10.1101/2025.10.11.681535

Figure Lengend Snippet: ( a ) (Left) Schematic of structured illumination (SI); (Right) Simulated illumination pattern at the back pupil plane of the microscope objective and the periodic hexagonal SI pattern at the objective focal plane. Yellow dashed circle: cutoff frequency; ( b ) (Left) Example image of a fluorescence film of 0.8 μm thickness under SI; (Middle) Power spectrum (PS) of the Fourier Transform (FT) of the image in logarithmic scale; (Right) Profile along the dashed white line in PS image. Yellow dashed circle: 0.5k 0 ; k 0 : cutoff frequency; Red dashed lines: spatial frequency of SI. ( c ) Same as in ( b ) but for a 2-µm-diameter fluorescent bead. ( d ) PFAM-SIFT calculates the average PS values within regions containing modulated in-focus signals (red boxes) in each frame to obtain the PS trace, which is then Fourier transformed and Fourier magnitudes corresponding to the interference strength between each probe beamlet and the reference focus are extracted for phase gradient measurement. ( e ) Example tip/tilt interference maps obtained using PS values with two-group DM configuration. ( f ) (From left to right) Ground-truth (GT) corrective wavefronts for astigmatism applied to DM, corrective wavefronts from PFAM-SIFT (top) and PFAM (bottom) from 1 μm, 2 μm, and 4 μm fluorescent beads, and 0.8 μm fluorescence films, respectively, at a camera frame rate of 50 Hz. Each method was iterated three times. RMSE: RMS errors between GT and measured wavefronts. ( g ) RMSEs for PFAM and PFAM-SIFT from different fluorescent structures.

Article Snippet: Fluorescent beads with diameters of 0.2 μm, 0.5 μm, 1 μm, 2 μm (FluoSpheresTM Carboxylate-Modified Microspheres, yellow-green 505/515; ThermoFisher Scientific) or 4 μm (ThermoFisher Scientific TetraSpeck Microspheres, fluorescent blue/green/orange/dark red) were diluted 1:1000 to 1:3000 in deionized water and pipetted onto a microscope glass slide (12-550-12, Fisher Scientific).

Techniques: Microscopy, Fluorescence, Transformation Assay

Journal: MethodsX

Article Title: Microinjection quality control in zebrafish model for genetic manipulations

doi: 10.1016/j.mex.2021.101418

Figure Lengend Snippet: Microinjection setup for needle and droplet calibration . A . The length of the pulled needle before cutting. B . Needle selection: cutting the tip of a pre-pulled needle with a sharp blade on a scale micrometer slide wrapped by parafilm under the microscope to produce a slant needle with similar opening. C . Injection with a slant needle backfilled with phenol red to produce a droplet. Volume determination by injecting into mineral oil placed on a micrometer. D . Fertilized embryos aligned into the ramps of pre-casted injection plate.

Article Snippet: Resource availability: , Materials and reagents • Phenol red (Sigma, cat#P0290, USA) • Petri dish • Phenyl Thio Urea (PTU) (P7629) • Egg water (E3): NaCl (Sigma, cat#S7653, USA), KCL (Sigma, cat#P9333, USA), MgSO4-7H2O (Sigma, cat#63138, USA), CaCl2-2H2o (Sigma, cat#C1016, USA) • Agarose (Sigma Aldrich, cat# A9539, USA) • Glass thin w/filament injection capillaries (World Precision Instrument, cat#TW100F-4, USA) • μTip Micropipette (World precision instruments cat#Tip05TW1F, USA) • Adhesive putty (tac patafix) • Mold (Eppendorf, Germany or Adaptive sciences tools, Cat # TU-1) • Micrometer - stage graticules S16 (VWR, cat#100499-308, USA) • Loading tips (Eppendorf, 20ul, cat#5242956.003, Germany) • Mineral oil (Sigma, cat#M5904, USA) • Blade • Parafilm • microscope slide • transfer pipette (VWR, cat#6124545) • DNA plasmid (Addgene, pCS2+8NmCherry, cat#34936, pSGEM-1 eGFP#44) • Subcloning efficiency chemi-competent cells: E.Coli (DH5α) (Thermofisher Scientific,18265017, USA) • Purelink Quick PCR purification kit (cat#K310002, Thermofisher Scientific, USA) • Mmessage Mmachine T7 transcription kit (Ambion, cat#Am1344, USA) • Mmessage Mmachine Sp6 transcription kit (Ambion, cat#Am1340, USA) • Restriction Enzymes: EcoRI (Thermofisher Scientific, cat#ER0271, USA), Nhel (cat#ER0971, Thermofisher Scientific, cat#ER0271, USA) • Rhodamine injection dye (Thermo Fisher Scientific, cat# D1816, USA) • Fluorescein injection dye (Thermo Fisher Scientific, cat# D1820, USA). Equipment's • Hood (Thermo scientific,1300 SERIES A2, USA) • Needle puller (Narishige, PC-10, Japan) • Picoliter injector (Harvard apparatus, Warner instrument, PLI-100A, USA) • Air source • Microscope (ZEISS, Stemi 2000-C, Lumar V.12, Germany) • Camera (ZEISS, Axiocam ERc 5s, USA) • ZEN blue Software (lite2.5, Germany).

Techniques: Microinjection, Selection, Microscopy, Injection