Journal: Neurophotonics

Article Title: Two-dimensional electro-optical multiphoton microscopy

doi: 10.1117/1.NPh.11.2.025005

Figure Lengend Snippet: Design and implementation of a 2D KTN-EOD relay module. (a) Design overview of 2D KTN-EOD relay module with conjugated, orthogonally oriented KTN-EODs. Black arrows indicate laser scanning direction. λ / 2 , half-wave plate. (b) Projected views of beam propagation in the relay system. The first lens in the relay conjugates the scanning pivot points of both the KTNs. The second lens (cylindrical) collimates to make it a circular scanning beam. Dashed green lines are conjugate planes. (c) Photograph of the prototype microscope. Upper left inset: magnified image of first deflector during LED illumination. Superimposed red line illustrates the excitation path.

Article Snippet: The minor differences in frame rate from session to session reflected instability in the number of lines the microscope control software (ScanImage, MBF Bioscience) allotted for flyback.

Techniques: Microscopy

Journal: Neurophotonics

Article Title: Two-dimensional electro-optical multiphoton microscopy

doi: 10.1117/1.NPh.11.2.025005

Figure Lengend Snippet: Implementation and optical performance characterization of a 2D EO 2P microscope. (a) Layout of complete optical path including light source, beam conditioning optics, scanners, optical relays, and detection scheme. (b) Image of a single X − Y plane of 0.2 μ m diameter fluorescent beads when imaging via the 2D KTN-EOD scanners. (c) Vertical (cyan) and horizontal (red) line-outs of the intensity profile and FWHM determination of a selected bead. (d) Image of an X − Z slice of the bead, and (e) axial (green) line-out of the intensity profile and FWHM determination. EOM, EO modulator; λ / 2 , half-wave plate; KTN-X/KTN-Y, KTN-EOD crystals; RL1, x -conjugator; CL1, y -collimator; RL2, y -conjugator; CL2, x -collimator; RL3/RL4, relay lenses; GG, galvanometer scanning mirror pair; SL, scan lens; TL, tube lens; PMT, photomultiplier tube.

Article Snippet: The minor differences in frame rate from session to session reflected instability in the number of lines the microscope control software (ScanImage, MBF Bioscience) allotted for flyback.

Techniques: Microscopy, Imaging

Journal: Neurophotonics

Article Title: Two-dimensional electro-optical multiphoton microscopy

doi: 10.1117/1.NPh.11.2.025005

Figure Lengend Snippet: In vivo characterization of 2D EO microscope bleaching kinetics. (a)–(c) 2P images of three layer 2/3 pyramidal neurons expressing ASAP3 collected using standard scanning galvanometers. (d)–(f) 2D KTN-EOD scanning images (mean of 1000 frames) of the regions marked by dashed boxes in (a)–(c). Images were acquired at both [(d),(e)] 15.6 kHz and (f) 14.7 kHz frame rates. (g) Example single trials showing ASAP bleaching Δ F / F 0 for the three cells membrane regions indicated with the dotted lines in (d)–(f). SNRs ( σ ) are indicated in the corresponding legends. (h) Trial-averaged normalized fluorescence exponential decay bleaching kinetics (shading indicates mean ± standard error of the mean) were calculated for all three cells (cell 1 N = 106 trials; cell 2 N = 118 trials; cell 3 N = 117 trials; N = 1 mouse). Exponential decay fits to the trial averaged data are shown with the solid lines and time constants ( τ ) are provided for each of the three cells in the respective legends. The objective back-aperture was underfilled to an effective NA of ∼ 0.39 (with a theoretical PSF of lateral FWHM: 0.93 μ m ; axial FWHM: 10.3 μ m ).

Article Snippet: The minor differences in frame rate from session to session reflected instability in the number of lines the microscope control software (ScanImage, MBF Bioscience) allotted for flyback.

Techniques: In Vivo, Microscopy, Expressing, Membrane, Fluorescence