The Leica Confocal Microscope System B46 Weill Hall (revised 4/01/10)

Transcription

1 The Leica Confocal Microscope System B46 Weill Hall (revised 4/01/10) Startup Turn ON 3 red buttons on far right: 1. PC ON 2. Scanner ON 3. Fan ON Turn Laser Power Level to lowest setting (Min) Turn argon laser KEY to Start briefly, and then back to ON Turn Green and Red HeNe Lasers ON with keys if needed Turn on microscope must be on to start software Turn on Mercury lamp If microscope says SET? Press and hold upper stop button until 0 appears Login to PC with your firstname space lastname (or netid or whatever we agreed on) Password is Confocal1 (note capital C) Domain (3 rd line) is Biotech, Click on Leica icon to start software When software is loaded, Always click on Beam Icon to set lasers, channels, etc. Adjust Power Level of Argon Laser to 9 o clock (most stable) Note: Z-galvo stage moves down, then up ~80 um as software loads. Always lower stage before starting software If you get a request to initialize microscope, do it See Troubleshooting pages 3-4 if you have a problem Read this handout if you forget how to do something Shutdown If another user within 1 hour Save images Exit Leica software Copy files to mifserver Log off Windows Wipe oil or water off objectives Shutdown If another user >2 hours As above plus: Mercury lamp off HeNe lasers off Laser power to lowest setting Total Shutdown If no appt within 3-4 hours Argon laser to min Turn off all lasers with keys Save images Exit Leica software Copy files to mifserver Shut down Windows Mercury lamp off (must cool before restarting) Scope and brightfield lamp off Scanner and PC off (2 red buttons) Wipe oil or water off objectives Cover microscope **Leave fan on for 15 min to cool Fan off REMEMBER: after hours, always shut everything off if the next person is not there. When in doubt, shut down. 1

2 Microscope (DMRE-7) Light path control / Laser Interlock upper left out for scan --- in for eyepieces -- do not use middle position (Polarizer, just below, should be out completely from microscope) Fluorescence filter cubes Position 1 DAPI or CFP fluorescence Position 2 Green Fluorescence Position 3 Red Fluorescence Position 4 Scan or brightfield Wollaston Prism use BF for confocal Mercury Lamp Light Path Control--upper right 1 st position is field stop, 4 th is aperture stop 2 nd is Heat Filter up=in, down=out Keep up. 3 rd is Hg shutter, marked with orange. up=closed, down=open Transmitted Light Halogen lamp switch on lower right, next to on/off switch ON for eyepieces OFF for scanning Transmitted Light Detector (TLD) (in front of on/off switch) ON for scanning =IN OFF for eyepieces =OUT (push in to pop it out) On left of base: Back controls: Field stop (lower) and Condenser Aperture (upper) Front controls: Neutral density filter and Rheostat On Base: Lower polarizer out gives a better bright field image Objectives - See Specs at end Focus Motor 2 button group in back upper to move stage up, lower to move stage down Single button above focus knob adjusts coarseness of focus knob 1 = fine, 2 = medium, 3 = coarse Turn focus knob away to raise stage Finding Specimen Plane For oil or water immersion, raise stage with motor until close, then use knob to focus manually until fluid joins objective. Continue up with focus knob while looking through eyepieces. Wiggle stage as you move up. DO NOT USE MOTOR WHILE LOOKING THROUGH EYEPIECES!!! For dry lenses, get close, then move down with focus knob while looking thru eyepieces. Stage Stop Press and hold upper button on front to delete previous stop, release Press and hold again to set to zero at your focal plane Do not leave in SET? When exiting Leica software Stage Rotation Galvo stage only Loosen thumbscrews in front and rotate center, tighten screws 2

3 Basic Procedure See Startup system page 1 Choose your objective Choose your fluorescence filter (usually 2 or 3) for viewing in the microscope. If no light, see troubleshooting, below. Focus on sample When ready to scan, switch filter to position 4 Pull out knob on upper left of microscope (laser interlock) Software Always click on Beam Icon Double click on desired setting Check control panel settings at bottom of screen (for the dials) Check that pinhole is at Airy 1, especially if you have changed objectives Check Objective icon and make sure the one you are using is checked. If not, see Things to Remember, p.5. Hit Continuous to start scanning Adjust gains and focus When you have a nice image, set Line Averaging and use Single Scan to collect an image To save it, click on Save icon This asks for the name of the experiment folder To return to viewing with microscope Change filter to 2 or 3 Push in knob on upper left Troubleshooting Problem: No light thru eyepieces with fluorescence: Set filter wheel to 2 (green) or 3 (red) Push in laser interlock knob (upper left side of microscope) Open shutter in fluorescence light path (upper right side of scope, see orange tape) Turn on mercury lamp (white box on table labeled Mercury lamp) Focus specimen in microscope Problem: Light thru eyepieces with fluorescence seems very dim: Check settings on upper right of microscope, the fluorescence light path Two levers slide up and down. Up is fully open for max light. Check for polarizer just below laser interlock knob on upper left. If there is a polarizer here (it has a knob and an arrow on it, take it out.) Problem: No light thru eyepieces with brightfield (transmitted light) Turn on lamp, switch is next to scope on/off switch Turn off TLD, in front of on/off switch, push in to pop out Adj brightness with wheel on lower right side of scope Set filter wheel to 4 Push in laser interlock knob 3

4 Problem: Image on screen all black Set filter wheel to 4 Turn up gain for PMT in use (check settings for control panel, bottom of screen) Adjust focus Did you select a setting with the Beam Icon? Do that first. Check that lasers are on-yellow light at each laser key should be on Problem: software can t initialize microscope Turn scope on If scope says SET? Press and hold upper stop button (front base of scope, hold until it says 0) to set a stop temporarily Click on initialize Problem: stage won t go up far enough Press and hold upper stop button (front of scope) until it says del, then SET? when in focus, press and hold again to set position = zero Problem: Image on screen not as bright as it should be Are the proper lasers on? Yellow light should be on at key Is the Pinhole set at Airy 1? Is the sample very bright thru the eyepieces? Try exiting the program, turn scanner (middle red button) off and on, wait 30 sec, restart the program Common Error Messages: Error: Laser Interlock: pull out knob (rod) on upper left of scope Error: Can t initialize microscope or lost microscope initialization or other microscope error: make sure scope is on make sure stage is not in SET? (see above) Go to Tools-Microscope, hit Initialize (our scope is the default, DMRE-7) You can always turn the scope off and on again. Other Errors: Any kind of scanning error, device error, system freezes or won t stop scanning: Exit software* and turn scanner off (middle red button). Then turn scanner on, wait 30 sec and restart Leica software. You don t have to reboot the computer. When in doubt, turning the scanner off and on again and restarting the software is a good idea. *When software not responding, exit by: Ctrl-Alt-Del Task Manager Highlight Leica software End Task Repeat if necessary, you should return to Windows Error: Not enough memory: Delete some of your files (the program needs space to make temporary files on the D: drive. If there is less than 1 GB free, the program won t run) 4

5 Things to Remember: Lasers Turn on the lasers you need to use (don t assume they are on check!) Adjust laser power to 9:00 position (this is for green only) Do not turn up laser power beyond 9:00 unless 488 laser line is at 100% in software (or 458, if that is what you are using) Laser power adjustment knob does not affect the Green He Ne (543nm) or the Red He Ne (633nm) lasers. It only affects 458, 476, 488, 514 laser lines. Brightfield For transmitted light thru the eyepieces, lamp must be on but Transmitted Light Detector (TLD) must be off. Turn off TLD by pushing in and pulling out so the button pops out. To collect a transmitted light image, lamp must be off and TLD on. Click on box in software to activate TLD When scanning, the brightfield lamp must be off. Whenever using fluorescence, the bright field lamp must be off. Polarizer on upper right should be out. If you see a little knob where there usually is not one, pull it out. Objective changes If you remove an objective and put in a different one, tell the software Check the Obj icon and make sure the lens you are using is checked. Also check that the Pinhole is at Airy 1 To change objectives in software: Tools-Objective Sort by number- see list on right of scope for numbers Drag desired objective to desired position. Close window. Remember to put everything back when you are finished If you forget to change the objective back and you have already exited the software, leave a note for the next person. After Hours If you have a problem after hours, reread these two pages. If you still have a problem, feel free to call Carol at home:

6 Control Panel This is the set of knobs (dials) next to the keyboard used to control gain, offset, zoom, focus, etc. At the bottom of the screen are tiny diamonds representing this control panel and next to them the actual function each knob controls. Just above on the right are tiny icons to load and save new configurations. The most common ones are red-green and smart. Smart gain and offset will control the highlighted channel and is useful if you have more than 2 channels. Red-Green sets PMT2 and PMT3 gains and offsets. Each knob can be configured by right-clicking on its function at the bottom of the screen. The sensitivity of the knob rotation should be kept fine. The second option is generally good. A new configuration can be saved by clicking on the tiny disk icon. Right click on the name in the list to save it as the default. Software Acquire Options Beam icon always click on this and choose a setting Move box to center of screen Choose setting from Leica list Adjust to your needs: Laser power and lines needed Emission ranges and PMTs needed Dichroic beam splitter must match laser lines LUTS (Look-up tables provide colors) Save with new name. You can customize what is included in this setting by going to Tools Settings Instr. Parameter Settings. Other Defaults (adjust as desired) Obj should have the correct objective use this to see what obj is where Bit 8 bits or 12 bits. Use 12 bits for quantitative work or if you have low signal. Expan use 6 always Mode xyz mostly. xyt for time course, xzy for cross-sections Format 512 x 512, go to 1024 x 1024 for higher resolution. Use narrow window (512 x 256) for faster scanning. Speed 400 MHz, (400 lines/s) 200MHz is slow but gives a cleaner image. Faster scans require a slight increase in gain and a zoom factor is automatically set. 800MHz=1.7x 1000MHz= 3.0x 1400 MHz=6.0x Scan -> single direction (for bidirectional must set phase while acquiring) Pinh pinhole, 1 Airy disk (AU) recommended. Use AE scale when adjusting. Field rotate field being scanned. 2 to 90 degrees Zoom set a zoom Z In zoom to region drawn on image Pan arrows move image when zoomed Continuous Scan to start scanning Creates a Preview that cannot be saved Single Scan to do one scan or averaging Creates an Image file that can be saved Li A line average does each line as it scans This is faster than Frame average Aver Frame Ave goes frame by frame Accumulate Integrates (adds) images together to increase signal. These images can be line averaged but not frame averaged. Very good for low signals. 6

7 View Options -- Next to the displayed image QLUT This calls up the glow scale with upper and lower saturation limits displayed. The glow scale is a non-linear color range that highlights bright objects. Saturated pixels will be blue and pixels that are too black will be green. Proper settings for gain and offset will show just a few blue and green pixels (none if you want to quantify). However, these are only guidelines and your image may require different settings. This button has 3 settings: glow scale, black and white, and original colors. LUTs=Look-up tables This opens the list of Look-up tables so you can change the color of the image without reacquiring it. If you save again, the image will be saved with this color. Zoom, Z-IN Use Z-in to draw a box around the area you want to zoom in on. First click highlights the channel, second click draws box. OVL=Overlay Click on the OVL button next to image for a view of the overlay. To get an overlay image without the grey transmitted light image, unclick the channel containing the transmitted light image. To save the overlay image, see Saving images Gallery - To see all the planes in a Z-series or Time series. Unclick to undo. X-sec - Use with a Z-series to get a cross section wherever the lines are on the image. Z-Series Acquisition Small series button to design Z-series with nice graphic **To get the most accurate starting plane, set the end plane first. Use Z position (=focus) knob to find end plane, Click on End. Note: Clockwise moves deeper into your specimen Use Z position knob to find first plane, Click on Begin. When you look at the graphic of the box when setting your begin and end points, moving the Z knob clockwise makes the yellow line go up. This is the stage is moving up, and you are moving deeper into your specimen. Sect 123 to choose # of sections or step size You must stop scanning to set this Choose Others to set step size, then click on calculate # sections -- to leave step size unchanged or calculate step size -- to leave height unchanged cubic voxels is recommended for highest resolution and 3D rotations get voxel size from list on screen Intensity compensation Designed to increase signal as you go deeper into your sample Can use this to go to top or bottom Check top before starting Large Series Box to begin collecting Z-series, will use frame or line averaging if set If you forget how you collect your series and want to know later on: For top (cover slip) to bottom, the depth size in the.txt file or the physical length of Z in properties, will be a negative value. For bottom to top, these values will be positive. 7

8 Sequential Scanning Between Lines This is the easiest to use but the only things that can change are which laser is on and which channel is active. You cannot change emission collection ranges or beamsplitter filters. 1) Set up a program for each color separately and save each, eg green and red. Remember, the only differences between them will be which laser is on and which PMT is active. You can change gains anytime. Changes in laser power must be resaved. 2) Click on Sequential in the beam window, between lines is the default. Activate the first channel (green), click Add. Activate the second channel (red), click Add. You can do as many as you like and can collect a transmitted light image with any color. 3) Keep the sequential window open and start scanning. A continuous scan will actually do one green line then one red line sequentially. It is slower than simultaneous scanning but still very fast. Work as usual. You can change gains without resaving the program. Between Frames This allows you to vary all of the parameters checked in the list. The checked parameters will use the saved settings in the separate green or red program. If you want to change the gain between samples without resaving each time, uncheck gain/offset. Set up separate programs as above and Add each. Keep the sequential window open use the Series button to collect. If a Z-series is set up, including the number of sections, it will be active also. Lambda Scan Only one laser line and one channel can be active Mode=xyλ The λ buttons will highlight Example: 488nm excitation Set your channel range to be and hit λbeg Set your channel range to be and hit λend Set λsteps to be 20. This will collect every 10 nm between 500 and 700. If you set the number to 40, this will collect every 10 nm with 5nm overlap, giving better resolution. Start collecting with Series button. If frame or line averaging are set they will be used. Results Quantify menu, choose Profile in Z. Choose an area of interest, you may want to keep it very small and specific. Click on Graph to see results. You can export the data or it can be entered back into Leica to create an emission scan like the ones supplied by Leica. Optimizing Image Quality Increasing Signal Check focus choose brightest plane Turn up gain this increases noise, line or frame average to reduce noise Turn up laser power this can increase photobleaching, watch for it Widen your PMT regions to collect more wavelengths to avoid laser lines, you may have to do sequential imaging Increase the pinhole (use Airy Unit scale) this can reduce your Z-resolution, compare to pinhole = 1 Airy. Image may be slightly fuzzy. Accumulate use with line averaging, lower the offset to darken your background Collect 12-bit images and adjust LUT (contrast stretch) Use the LUT called P4 to collect dim images 8

9 Increasing signal to noise ratio Keep Gain below ~700; Offset ~ 0 Use QLUT in the view menu to determine best gain and offset settings (see above) For quantification, avoid all blue (saturated) and green (too black) pixels Always average (frame or line average) to decrease noise. The higher the gain, the more averaging is needed. Scan Speed at 200 will also decrease noise Increasing Resolution For maximum resolution, use the maximum zoom on the table posted. Use objective lens with higher Numerical Aperture (NA) This usually means an oil- or water-immersion lens and higher mag Change format to 1024 x 1024 or higher to maintain large field Increasing Speed Use a faster scan speed. 400 Hz is 400 lines/sec. Faster speeds require zooming. See Software defaults. Collect fewer lines, eg 512 x 256 Use bi-directional scanning, adjust Phase so image looks normal Types of Overlays Default=Fast Bitwise On the far left is a small icon that says Display. If you have an overlay image in the view, there will also be an icon that says Overlay. Click on it. For RGB fluorescence images, the Fast bitwise, which is the default, works fine. If you have other colors like yellow or cyan, and for brightfield images, the fast bitwise default is very bad. You should switch to True Color with or without dynamic adjustment ie scaling. The grey will not be as bright as the original, for DIC you can try brightening by adjusting the Wollaston prism or increasing the gain. To brighten without saturating the fluorescence, adjust the gamma and black level in Photoshop (see below). The result will be more or less the same. Save your overlays with the proper setting and switch back to Fast for scanning. True color slows down scanning considerably. You can adjust the overlay and resave in Leica Lite. Process Menu 3D Visualization and Animations Projections use Maximum option Or try color-coded projection Projections with animations to do 3D rotations Click on rotation, choose angle (eg. half is +/- 90 degrees) Choose # frames (eg 10 with half is 18 degree jumps) Essentially makes a projection at each angle and you play them for the 3D effect Enhancement use to adjust Brightness, Contrast and Gamma not very good Editing use for Image Separation (eg separate red and green Z-series) Use to remove planes Use for file merging (make 2 separate images into one file for overlay) Quantify Menu To see a histogram of a line or a profile through a series or across an image.etc. 9

10 Saving Files and File Management All images are saved in an Experiment folder. You cannot name or save an Expt folder until you acquire an image using single scan. After acquiring an image, click on the Save icon Select the D:\ALL USER IMAGES HERE folder and your personal folder. Then name your expt. Folder where it says File name. You image will go into this folder. This folder name CANNOT be easily changed. Keep it short. Always save (use icon) every image after collecting. If the software crashes, your images can be recovered but they go into a new folder and it gets confusing. Right click on image name to Rename or Delete images. Images are saved as.tif files. Each channel is saved as a separate file. If you collect the green and red channels, you will have 2.tif files. If you save a Z-series, each plane and channel will be a separate.tif file. If you save in color, they will open in color in Photoshop and other places. Naming Images will have VERY long names, eg. foldername_imagename_plane00_channel00.tif Do not rename or delete anything outside of the Leica software. This will confuse the.lei file and you will never be able to open that folder in the Leica software. The.lei file tells the Leica software all of the hardware settings under which all the images in the expt. folder were taken. You may want to put every Z-series in a separate folder (experiment.) Include in that folder any projections, overlays, screen shots, etc related to that series. More than one large Z-series in an experiment folder is a lot of files and they can get confused. Image Properties To view the conditions under which an image was acquired, click on the file and choose Properties. The Apply button will apply the properties listed to the system. You can also get this information from the.txt file, which is in the expt folder. New Folder To make a new folder, click on the New icon. Do NOT try to name it with rename! First put an image in it and hit the Save icon. Now you can name the folder. Check the path before saving so it does not become a subdirectory of the preceding experiment folder. Saving Overlays Right click on the overlay image, choose Send to Experiment Selection Raw is best. The Snapshot option saves it as a bitmap image. Use snapshot to save a Gallery or an image with a scale bar. Saving all vs selection saves the entire screen. Saving Overlay of Z-Series Click on gallery to see entire series. Right click, Send to Experiment raw. This saves a series of.tif images just like the red or green series. Saving as.avi Right click on series name. Choose Export, name it and save as type AVI. Scale Bar On the left side of the screen, click on display icon and check the scale bar box. Then click on the scale bar button at the bottom. Change size and hit Apply. Move if desired. Click on Auto-rescale to keep the same value at all zooms. Must save as Snapshot. 10

11 Transferring your files to the MIF Server Drag files from your Leica folder (D:\All User Images Here) to your folder In MIF netapp1 (M:). Both the Leica hard drive and the server have limited space. Please download your files promptly and delete the files from both places when you have them stored safely. After 1 month, your files may be deleted. Retrieving files from the mifserver see Carol for instructions Leica Lite Free version of the Leica software for viewing your images and doing simple processing, like projections. You can get it from the mifserver. Look under Leica Lite. Use the Installation folder to install. Does not work with Vista or Windows 7. Projections, go to View Fix Max send to expt to save each channel You can project a time series this way. Overlays, go to view To change the colors of your image with Leica Lite, double-click on the color bars at the right of the image. This will bring up the LUT options and you can change the color of the image and resave. Image Editing you can do separations, merges, convert to 8 bit. To change the color, click on the color bar on the right side of the image Scale Bar you can put a scale bar on your image but you cannot adjust it. Leica Software We have a full version of the Leica software now on the MetaMorph computer in MIF. Does all the 3D reconstructions, etc. It is free to use anytime the Metamorph system is not being used. You may want to signup on the Metamorph calendar. Login as Leica-user, Confocal1, so you do not get charged for the time. 11

12 Transmitted Light ( Brightfield ) Introduction You can collect a transmitted light image at the same time as the fluorescent image to aid in localization of your fluorescence. The brightfield image can be overlaid (merged) with the fluorescent image(s). **Note: Adjusting the microscope for an optimal brightfield image is more complicated than fluorescence. It is highly recommended that you ask for help the first few times. Kohler Illumination Focus on your specimen in the microscope. Close the field stop diaphragm on left side of microscope base. Lower back wheel Adjust the height of the condenser until the aperture is sharply focused. Silver knob on left. Center the aperture with the centering screws. Open the field stop until the light just fills the field and aperture is no longer visible. Open the condenser diaphragm for desired contrast. Upper back wheel. Wide open is best. Remove the polarizer at the base of the scope if you have annoying video lines. Differential Interference Contrast (DIC) -- Nomarski DIC is a method of enhancing the contrast of a brightfield image. It has optical sectioning qualities and is very good for viewing surfaces. The effect looks similar to a scanning EM image, with a black shadow on one side and a white shadow on the other side. The shadows are formed by differences in refractive index or by differences in height. Microscope setup for DIC First follow procedures above for Kohler Illumination Need upper polarizer in for eyepieces-- out when scanning Find in upper drawer and put in slot below laser interlock knob, removing plug first. Rotate lower polarizer into place Wollaston Prism (below filter wheel) set for objective: C=20x D=63x/100x E=40x Condenser --Set for objective: H-brightfield 10/ oil 40/63 The condenser diaphragm should be open. Wollaston Prism The Wollaston Prism has a thumbscrew, which can be adjusted for desired contrast. The full range of the screw is such that the image is darkest in the center (extinction) and brighter near the ends. The difference between the two sides is the direction on which the shadows fall. The optimum setting is usually somewhat off center on either side. Note that this prism can cause a slight doubling of your image at very high magnification. Switching between the scope and scanning Scope Halogen lamp on Transmitted Light Detector (TLD) off Scanning Halogen lamp off TLD on To increase contrast, lower offset to -10 to -20 and increase gain 12

13 Photoshop The most valuable feature in Photoshop is: Image--Adjustments--Levels choose RGB or individual color use the histogram to adjust white level, black level, and gamma Save with a new name. This does a contrast stretch and is permanent. Coloring Images Your Leica images should open in color To make a Red/Black or Green/Black image from greyscale. Open image Image--Mode choose RGB Color Image--Adjust--Levels Choose colors not wanted Drag left triangle under histogram all the way to the right to eliminate this color Repeat with other color not wanted. You will be left with the desired color. Adjust as above. Save image (You can get rid of the color by changing Mode back to Grayscale) Saturated pixels will turn white with this method. To avoid this, try the following method: Image--Mode--RGB Color Image--Adjust--Hue/Saturation Check Colorize Saturation at 100% is probably best Adjust Hue to ~120 for green, red is the default Lower Lightness to color white areas and darken background Save image Specifications for Leica Confocal SP2 3 Laser system 4-line argon laser, lines at 458nm, 473nm, 488nm, 514nm. 458 for CFP, 514 for YFP, 488 for all green dyes Green Helium Neon laser at 543nm for all orange / red dyes eg. rhodamine, Cy3 Red Helium Neon laser at 633nm for far red dyes eg. Cy5, TOPRO 4 fluorescent detectors 1 transmitted light detector 5 channel simultaneous collection Confocal Beamsplitter Filters Beam Splitter Laser Lines Range 1 Range 2 Range 3 RSP , 476, / , /543/ , 543, / , RT 30/70 any reflects 30% transmits 70% 13

14 Microscope Leica Upright DMRE-7 Objectives (new arrangement 9/1/08) Position ID# Description x dry HC PL APO 10x / 0.30 WD 12mm x Imm HC PL APO 20x / 0.70 Corr WD 250um can also be used with oil or water Adjust collar for oil or water and cover slip x oil HCX PL APO 40x / 1.25 oil WD 100um x oil HCX PL APO 63x / 1.32 oil WD 70um x oil HCX PL APO 100x / 1.40 oil WD 90um All oil lenses: keep aperture open x water HCX PL APO 63x / 1.20 Corr WD 220um Adjust collar to.13 for #1 cover slip x dipping HCX APO L 40x / 0.8 W WD 3.3mm In Drawer 32 5x dry HC PL FLUOTAR 5x / 0.15 WD 12mm In Drawer 52 40x dry HCX PL APO CS L 40x / 0.85 Dry, WD 240um Adjust collar to.13 for #1 cover slip Filters on Microscope UV Excitation BP 360/ nm (position 1) Dichroic 400 Emission BP470/ nm GFP (green) Excitation BP 480/ nm (position 2) Dichroic 505 Emission BP 527/ nm Red Excitation BP nm (position 3) Dichroic 580 Emission LP nm CFP Excitation 436 / nm (position 1) Dichroic 455 Emission 480 / nm Green-Red Excitation BP nm (in drawer) Dichroic 510 Emission LP nm 14