Random collection of notes from training session. Not intended to be that informative, just an aide memoire perhaps.
Objectives: Extreme care, don't knock- misaligns even if put down hard Extreme care, don't scratch - very small scratches show up badly White ring at top means immersion Comp ring on 20x/IMM is for oil/glycerol/water/coverslip thickness Comp ring on 63x 1.2 W: SW=salt water T=temperature Middle=coverslip thickness Iris on 63x/1.4 oil should be fully OPEN for max resolution Detector slit 5nm to 400 nm width range Lower numbered detectors must be shorter wavelengths AOBS needs 1 hour warmup for brightest stablest intensity image. Must launch software to warm up AOBS Init stage only if need stitching/point visiting Galvo Z stage 100g max load, accuracy 40 nm, max travel 165 um Set focus point (top) with up arrow on scope - press and hold (says "set") , release, press once more. Step on scope changes step size for focus S0 (finest control) ... S3 (coarsest) Set top button also saves step size. Do not press first 3 buttons - scope seriously out of whack Best to lower objective manually before changing Prism wheel under objectives should be BF for best resolution Auto gain - looks at middle strip of sample. Good starting point. QLUT - Blue = saturated Green=zero (black level) Click on overlay display and gain/offset knobs cotroll all active PMTs at once. Z series. ----------- Find one end, press BEGIN Find other end, press END Click SECT to look at number of sections - best step size is half the axial resolution of the objective. Averaging. -------------- Integration. (frame/line average) Set average to be same as first digit of PMT voltage - gets ball park. Line average - faster, best for highest resolution. Have to balance averaging number with bleaching. Do not use Gallery until data saved to disk. Save each new series to a new folder. R click on saved series, export as TIF/AVI. Cinepak ?best compressor .lei - leica file. Contains all scan params, instrsettings etc. .txt - text version of .lei R click file, choose properties - can anotate. R click, save as XML file, print XML file R click, properties, apply to set instrsettings to old file Tools, settings, Instr param settings to choose what can be set. Save image: R click Send To, Experiment, All (snapshot) - saves to memory folder. Control knobs - R click, can set explicitly or set sensitivity. Higher zoom - more bleaching Do not zoom past half of XY resolution of objective (e.g. 70 nm for 63x Oil) Process, change gamma to enhance dimmer objects in image 3D: 3D Visualization, Projections. Animation: 4 degrees/frame is good. Lambda scan. ------------------- 1 channel. Set to 20 nm wide. Averaging off Open pinhole Set levels Set lambda begin, end, step as for Z series. Quantify,Lambda Stack Profile. Make ROI. R click graph, export to excel Send to, Document to print Process, Dye Finder, Spectral dye separation. Select ROI, save emission spectrum Tools, Stains - can delete user defined stains Sequential scan: --------------------- Have to use if using DAPI as too broad emission spectrum. Procedure: Off all channels except one Set intensity - must not saturate Save beam setup for first channel Set up for next channel, save beam setup Repeat for all channels. In BEAM window, click SEQ, add saved beam setups (or drag to window) Set mode - line (best temporal res, if sample moves)/frame/stack GOTCHA - line mode will not work for UV as no AOTF for 405 beam. Frame avg - most flexible. Can have different pinhole sizes for different detectors Colocalization - do sequentially (no chance of crosstalk) Go faster: ------------- Change to 512x64 i.e. strip. Set scan speed higher (dimmer) Bidirectional (phase problems) (Process, enhance, phase correction for offline) If scanning too fast for PC display, Tools, Customize, Basic Scan: BURST mode Process, Editing, Merging, select "ch", check "append"