Stimuli were programmed in C++ using OpenGL, and projected using a LightCrafter 4500 DLP (Texas Instruments, Dallas, TX, USA) with only blue LED illumination. Stimulus light was attenuated with a 482/18 bandpass and ND1 filters, before reaching a rear projection screen. The screen measured 8 cm × 8 cm, subtending about 60° × 60° (azimuth × elevation) of the right visual field of the fly. Stimuli were displayed at 6-bit pixel depth and at a frame rate of 300 Hz, but the stimulus frame was updated at 100 Hz. The stimulus frame parameters, including timestamps, were saved to disk together with the imaging data frame timestamps to allow for stimulus-imaging time alignment during data analysis. The stimulus and data acquisition computers were linked via a NI-DAQ USB-6211 device (National Instruments).
Dlp lightcrafter 4500
Manufactured by Texas Instruments
Sourced in United States
The DLP Lightcrafter 4500 is a high-performance digital light processing (DLP) evaluation module from Texas Instruments. It features a 0.45-inch DLP chip and a powerful LED illumination system, enabling precise and flexible control of light projection.
Automatically generated - may contain errors
Lab products found in correlation
Orcaflash2, by Hamamatsu Photonics (3 mentions)
Band pass filter, by IDEX Corporation (2 mentions)
Nd1.0 neutral density filter, by Thorlabs (2 mentions)
Sp5 2, by Leica camera (2 mentions)
Hcx apo l 20x na1.00 water dipping lens, by Leica camera (2 mentions)
Nd1 filter, by Thorlabs (2 mentions)
Usb 6001 daq, by National Instruments (1 mentions)
Pda36a ec, by Thorlabs (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Te2000 u, by Nikon (1 mentions)
15 protocols using dlp lightcrafter 4500
1
Real-Time Visual Stimulation in Flies
2
Visual Stimulation Setup for Fly Recordings
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Visual stimuli were presented on a 9 cm–by–9 cm rear projection screen in front of the fly covering a visual angle of ~80° in azimuth and ~55° in elevation. To cover a larger part of the horizontal visual field of ~168°, we rotated the fly with respect to the screen two times by 45° and recorded each fly at three positions relative to the screen (fig. S1A). In total, we thus stimulated an area of the visual field ranging from −34° to 134° in azimuth and −17° to 36° at the closest point of the screen to the fly in elevation (fig. S1A). Note that results are just plotted in a range between −23° and 120° in azimuth, as no neuronal responses were measured to the stimulus beyond that visual area. Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (54 (link)). Visual stimuli were generated using custom-written software using C++ and OpenGL. To correct for distortions due to the fly’s viewing position relative to the screen, stimuli were drawn on a virtual cylindrical surface and perspective-corrected using frustum.
3
Two-Photon Imaging of GCaMP6f Responses
4
Two-Photon Imaging of GCaMP6f Responses
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Imaging and delivery of visual stimuli followed Leong et al. 201638 (link). Fluorescence was monitored in vivo using two-photon microscopy. We used a Leica SP 5 II equipped with the HCX APO L 20X/NA1.00 water dipping lens (Leica, Wetzlar, Germany). GCaMP6f was excited at 920 nm, and the power was ~5–8 mW at the stage. Recordings lasted ~3.5 minutes. GCaMP6f fluorescence signals were acquired with a bandpass filter (525/50m), at ~20 Hz (bidirectional scanning at 1.4 kHz, across a FOV of 128 pixels x 256 pixels, rows x columns). Pixels measured ~290 x ~290 nm. The stimulus screen subtended ~ 60° x 90° (azimuth x elevation) of the left visual field. Visual stimuli were delivered with a LightCrafter 4500 DLP (Texas Instruments, Dallas, TX, USA) using a 100 Hz frame rate. The LightCrafter was configured to use only the blue LED, then the stimulus was filtered with a 447/60 bandpass filter (Semrock, IDEX Health and Science, Rochester, NY, USA), and a ND1 filter (Thorlabs, Newton, NJ, USA). The mean radiance was ~0.04 W sr−1 m−2.
5
Visual Stimuli Presentation for Fly Neuroscience
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Visual stimuli were presented on an 8 cm x 8 cm rear projection screen in front of the fly covering a visual angle of 60° in azimuth and elevation. To cover a larger part of the horizontal visual field of 150°
we rotated the fly with respect to the screen two times by 45° (Extended Data Fig. 1a). Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (46) . All visual stimuli were generated using custom-written software using C++ and OpenGL.
we rotated the fly with respect to the screen two times by 45° (Extended Data Fig. 1a). Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (46) . All visual stimuli were generated using custom-written software using C++ and OpenGL.
6
Optogenetic Perturbation Using DMD
7
Optogenetic Perturbation Using DMD
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LEXY perturbation was achieved using a digital micro-mirror device (DMD; DLP 4500 LightCrafter, Texas Instruments, USA) to project spatial patterns and to rapidly change light levels (Rullan et al., 2018 (link); Wilson et al., 2017 (link)) (see Figure S3A ) through a parallel light path using a long-pass 473 nm dichroic mirror and a combination of color and interference filters to attenuate the DMD’s blue LED wavelength (445 ± 8 nm). To synchronize two-photon image acquisition and DMD blue light activation cycles, an external trigger mode in DLP LightCrafter control software was used. The software controls the LED light wavelength, pulse duration, pulse duty cycle, the number of pulses, and the type of spatial image pattern to project on the imaging sample (see details in Table S3 ). The optimum blue light level for optogenetic perturbation was determined by optimizing the maximum protein export with minimal light scattering to neighboring nuclei (see Figures 1B and 1C ). After scanning the range between 50 and 250 μW/cm2 of blue-light on/off pulsatile cycles of LEXY-tagged protein nuclear signal (data not shown), 100 μW/cm2 (pulse duration = 40 ms, pulse duty cycle = 100 ms, number of pulses = 5) was determined for all optogenetic perturbations performed in this study.
8
Dynamic Dark Field Microscopy
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Dark field imaging is performed using a custom inverted optical microscope equipped with a ×4 magnification objective (Nikon; NA = 0.13) and a high-sensitivity CMOS camera (Hamamatsu Orca-Flash 2.8). Patterns of green light (520 nm) are generated using a digital light processing (DLP) projector (Texas Instruments DLP Lightcrafter 4500) coupled to the same microscope objective used for imaging through a dichroic mirror. A more detailed description of the custom setup can be found14 (link). The positions of the modulation disks in the the projected light patterns follow particle centers through an automatic feedback algorithm updated every dt = 0.5 s.
9
Retinal Light Stimulation with Customized DLP
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A Digital Light Projector (DLP) LightCrafter 4500 (Texas Instruments, Dallas, TX) coupled to a custom optical, two-lens system capable of 6 cycles per degree (cpd) resolution focused light stimulation onto the retina from below (i.e., presenting to the ganglion cell side of the retina, consistent with normal vision). The system provided 1280 × 800 pixels of spatiotemporally patterned stimulation over the area of the MEA with a refresh rate of 60 Hz and control of brightness and simultaneous RGB LED operation. The DLP projector was outfitted with three independently controlled RGB LEDs with a recorded maximum emission at 617, 509 and 455 nm for red, green, and blue, respectively, with an average half-width of ±30 nm. For all wt and BENAQ-treated experiments, regardless of isolated or compound use of LEDs, a total photon flux of approximately 5 × 1010, approximately 3.5 × 1013 and 5.5 × 1016 photons/cm2*s at 500 nm, respectively, was used as measured for each maximal emission regardless of stimulus color. For white light stimulations, equivalent photon flux per LED was used at photopic light levels. Total quantal catch for the MW- and UV-opsin, respectively was calculated as 1.7 and 5.5 × 10−5 R*/cone/s.12 (link)
10
Optogenetic Stimulation Setup for Microscopy
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Optogenetic stimulation was done with a DMD projector (DLP LightCrafter 4500, Texas Instruments) mounted on an optical table, together with the necessary optical elements to focus the emitted light at the focal plane of the microscope’s objective. A schematic of the setup, together with a list of components is provided in Figure S1 A and Table S3 , respectively. The light intensity at the specimen and the blue-light spectra is shown in Figure S1 B.
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