====== Detailed description of 2P1 ====== * This page documents the current state of 2P1, since [[http://flybrain.mrc-lmb.cam.ac.uk/dokuwiki/doku.php?id=alex:projects:multiphoton:start|Alex's 2-photon page]] became obsolete. To follow the step-by-step history of 2P1 see [[http://flybrain.mrc-lmb.cam.ac.uk/dokuwiki/doku.php?id=equipment:multiphoton:2p1_log|2P1 log]] * Before planning any changes it's worth visiting the most recent version of [[http://scanimage.vidriotechnologies.com/display/SI2019/Supported+Microscope+Hardware|supported parts]] list on Vidrio's website ===== Lasers ===== * Coherent Chameleon Vision (A, below). Specs are {{:alex:projects:multiphoton:chameleonvision_ds_final.pdf|here}}. Tunable wavelength (680-1080nm) Ti-sapphire laser, primary purpose is GCaMP imaging * Spark Lasers - [[https://spark-lasers.com/compact-femtosecond-laser-biophotonics-alcor/|ALCOR]] series 1040nm, 2W. Single wavelength laser, primary purpose is targeted optogenetic excitation ===== Core Microscope ===== * Sutter [[https://www.sutter.com/MICROSCOPES/mom.html|MOM]] (Moveable Objective Microscope), see detailed [[https://www.youtube.com/watch?v=hLmTZQFQU9o|explanation]] and [[https://www.youtube.com/watch?v=hwCFtQ3WHoo|alignment]] tips from Sutter * Secondary beam path follows the basics shown in this [[https://www.youtube.com/watch?v=DLTaqzz8hJM&t=200s|video]], beams merged by polarising beam splitter cube ([[https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=739|Thorlabs PBS255]]) ===== Beam paths ===== {{:equipment:multiphoton:istvan_beam_paths.png?800|}} * Chameleon path * Chameleon laser (A) - internal shutter operated from software or via panel on power supply * Iris - blocks reflected light from re-entering the Chameleon (B) * Thorlabs (WPH05M) rotating half-wave plate (C) - controls beam intensity * Fixed (GL10-B) Glan-Laser polariser (D) - controls beam intensity, rejected beam goes into beam trap next to it (Thorlabs BT610/M) * Shutter Thorlabs SH05 (E) - This shutter is normally opened by ScanImage during a run. Additionally, it is connected to an interlock on the shroud door, forced closed when the door is open. * Conoptics Pockels cell / EOM (F1, F2) - controls beam intensity rapidly based on input voltage (0-2V) * Beam sampler and photodiode, reflected portion is angle dependent (g1, g2) - used for calibrating Pockels cell, also displayed on a power meter, relationship with power after objective is linear * Fixed half-wave plate (H) - controls beam intensity * Variable beam expander (Thorlabs BE02-05-B) (I) to control objective (over/under) filling * Sutter MOM periscope mirror (J) * ALCOR path * ALCOR laser (a) - manual shutter on the side, normally open * Shutter Thorlabs SH05 (b) - operated by ScanImage trigger when this beam is operated * AOM from AA Opto-electronic MT110 (c ) - controls beam intensity rapidly based on input voltage (0-5V) * Beam sampler and photodiode, reflected portion is angle dependent (d1, d2)- used for calibrating AOM, also displayed on a power meter, relationship with power after objective is linear * 3x beam expander (not on picture, placed before e) - to fill objective back aperture * Second path periscope mirror ==== Scan Systems ==== * Scanner for Chameleon (imaging beam): Fast scan 6mm Galvo-Galvo mirrors (Cambridge 6215) and scanner driver electronics (includes two Cambridge Technologies 671XX-HP Micromax scanner driver boards) (K) * Scanner for ALCOR (stimulation beam): LSKGG4/M - 4 mm Galvo-Galvo Scanner and Controller (f) * Scan lenses (L, g) * polarising beam splitter cube (M) ([[https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=739|Thorlabs PBS255]]) {{:equipment:multiphoton:galvos.png?600|}} ==== PMTs ==== Hamamatsu H7422 P40-MOD unprotected GaAsP PMTs (Hamamatsu part number, H10770PB-40 SEL) and Hamamatsu C3719 pre amps. The power supply is labeled "Sutter PS-2" but it looks suspiciously like two Hamamatsu model C3830 supplies piled into a Sutter box. I can't find any info on the PS-2, but the Hamatsu specs are [[http://www.google.co.uk/url?sa=t&source=web&cd=1&ved=0CBcQFjAA&url=http%3A%2F%2Fwww.sales.hamamatsu.com%2Fassets%2Fpdf%2Fparts_C%2FC3830-22.pdf&rct=j&q=C3830-22.pdf&ei=8e2aTYrpCtSahQf04vDXBg&usg=AFQjCNGDNivxZS6CuiCZA-Scu2JU-hqQrA&sig2=Ux5G2eGF8ECaxZyhX-JLzw&cad=rja|here]] or {{:alex:projects:multiphoton:c3830-22.pdf|here.}} This is just a guess on my part (Alex), so if you are really fussed about these specs, it would probably be best to contact Sutter. ===== Other Parts ===== * Piezo for fast z movement (Z stacks) * PI725, 1CD PIFOC * controlled by PI E665 * Filters/dichroic for detection * Chroma filter cubes housed in Olympus BX51 filter wheel * Table * T1525P; 1.5 m x 2.5 m x 210 mm Sealed Holes Metric Optical Table with PTS603 700mm High Set Of Active Legs from Thorlabs * Luigs-Neumann micromanipulators Mini 23 x 2, controlled by SM-5 * Computers and Data Acquisition system * PC controlling the imaging system, see hardware details at [[http://flybrain.mrc-lmb.cam.ac.uk/dokuwiki/doku.php?id=equipment:multiphoton:2p1_log|2P1 Log]], hosts all data acquisition systems * Dev1: NI PCIe 6323: * Dev1/AO 0 : Stim Galvo X * Dev1/AO 1 : Stim Galvo Y * Dev1/AI 0 : Stim Galvo X position feedback * Dev1/AI 1 : Stim Galvo Y position feedback * Dev1/AO 2 : Stim AOM control * Dev1/AI 16: Stim power meter * PFIO: Trigger IN - Acquisition start * Dev2: NI PCI 6110 - this one's pretty old, at the next DAQ upgrade it should be changed for compatibility reasons * Dev2/AO 0 : Imaging Galvo X * Dev2/AO 1 : Imaging Galvo Y * Dev2 AI 0: PMT A - red * Dev2 AI 1: PMT B - green * Dev3: NI PCIe 6321 * Dev3/AO 0 : Imaging Pockels * Dev3/AI 0: Imaging power meter * Dev4: NI PCIe 6321; Piezo input voltage and feedback * Dev4/AO 0: Piezo Control * Dev4/AI 0: Piezo position feedback * Dev4 could be used alternatively to be paired with MultiClamp 700B * Mac: * PointGrey Grasshopper CMOS camera controlled via MicroManager for wide-field view * ITC-18 DAQ board current stimuli: * Sync Trigger acquisition trigger sent to Dev1 PFIO (5V TTL) * 617nm LED controlled via Thorlabs LED driver LEDD1B with analog input (0-5V) * FLIR BlackFly S USB 1MP Camera recording trigger (3.3V, 15ms) - used to record behaviour with Computar MLM3X-MP 0.3-1X zoom lens * Custom ODD valves (5V TTL) - controller made by Electronics Workshop, Andy Howe is best to ask if they need fixing * Picospritzer (5V TTL) {{:equipment:multiphoton:2p1_overview.jpg?700|}} ===== Objectives ===== * [[https://www.micro-shop.zeiss.com/?s=393578527f2544&l=en&p=uk&f=o&a=v&m=s&id=421452-9900-000|Zeiss 20x]] 1.0 NA M27 (70 mm parfocal, 75 also avail w more DIC) - Currently used (25. 01. 2020.) * [[http://www.olympusamerica.com/seg_section/uis2/seg_uis2_xlumplflnw_20xw.asp#|Olympus 20x]] 1.0 NA 25 diam * [[http://www.olympusamerica.com/seg_section/uis2/seg_uis2_lumplfln_40xw.asp|Olympus 40x]] 0.8 NA (RMS mount) * Olympus 25x 1.05 NA (? mount) * [[http://www.nikoninstruments.eu/en_GB/Products/Optics-Objectives/Apochromat-Objectives/Lambda-S-Objective-Series/%28specifications%29|Nikon 25x water 1.1 NA with correction collar]] ====== Software ====== * ScanImage 2020 Premium - Vidrio, license activated in November 2020, comes with 1 year support * [[http://scanimage.vidriotechnologies.com/display/SI2019/Concepts+Guide|Concepts page]] * [[http://scanimage.vidriotechnologies.com/display/SI2019/Machine+Data+File|Machine Data File]] * [[http://scanimage.vidriotechnologies.com/display/SI2019/Scanner+Alignment|Scanner alignment]] * [[http://scanimage.vidriotechnologies.com/display/SI2019/Pockels+Cell+Concepts|Pockels cell]] * [[http://scanimage.vidriotechnologies.com/display/SI2019/Photostimulation|Photostimulation]] * [[http://scanimage.vidriotechnologies.com/pages/viewpage.action?pageId=28377257|Online ROI integration]] * MATLAB 2019A * NI DAQmx 18.0 * SpinView - for FLIR camera * Chameleon GUI * Spark Lasers GUI * MPDS - software to interact with AOM control wave modulator * MultiClamp 700B * [[https://wavesurfer.janelia.org|wavesurfer]] ====== Filters for PMTs ====== Alex set up the filter cube for the PMTs as below: RED CHANNEL *-------------* | HQ605/75 \/ | *---*-------------* G | |\* R | E | \* E | T | \* E | 5 | \* N | 2 | \* | 5 | \* C | / | \* H | 7 | \* A | 0 | \* N | | \* N | | \* E | > | \* L | | \* *---* The filters are by Chroma and the arrows point towards the coated side.