LaserSharp Multiphoton Microscope (last updated 11/27/2012)

Transcription

1 LaserSharp Multiphtn (last updated 11/27/2012) **All users need t sign up fr EHS laser safety training. Refer t last page (6) fr device diagram. Refer t page 5 fr filter pack (FP) diagrams. Start Prcedure Overhead shelf: Turn n Pckels Cell pwer supply (PSPS) Turn n PMT pwer supply (PPS) Shut Dwn Prcedure Turn ff PMTs and exit PMT cntrller sftware. Lg ut f LaserSharp sftware. (D nt exit.) Turn ff laser by clicking the OFF buttn nce. (There is a slight delay.) Turn ff Pckels Cell Pwer Supply bx (PCPS). Turn ff PMT pwer supply (PPS). CLEAN micrscpe bjective with LENS PAPER nly. These shuld remain n, d nt turn them ff: Windws Cmputer (WC, Webbgrup/drbi) OS2 Cmputer (OS2C) Scanner pwer supply bx (SPS) Laser chiller (LC) Turn n multiphtn laser The multiphtn laser is an ultrafast Spectra Physics Ti:Sapphire (Mai Tai, <100 fsec utput pulses at 80 MHz). There are n buttns, switches r knbs n the laser head. All functins are cntrlled by the sftware n the Windws Cmputer (WC). Ensure that the Laser chiller (LC) is n. Duble click n Mai Tai Cntrl icn n Windw s Cmputer (WC) desktp. Chse Cm Prt 1 (click k). The laser cntrl bx appears. RS232 interface is active when green buttn is lighted. If System Status is n Standby, Click and hld the ON buttn until status changes t warming up. Wait until System Status reads Ready t Turn n (a few minutes). Switch n Laser by pressing and hlding ON buttn until Emissin (in red) appears Mve slider t desired wavelength (r type in and enter value): 780 r 880nm fr beginners. Make sure laser shutter is pen: Hld Shutter buttn until it pens (yellw when pen, grey when clsed). It is a gd idea t keep this clsed when yu are nt imaging. Set up The micrscpe is an Olympus BX-61WI interfaced t an lder BiRad 1024 laser scanner. Make sure the scanner pwer supply (SPS) and the micrscpe cntrller (MC) are n. Set up sample n stage Chse apprpriate bjective. Chse apprpriate filter pack (FP, see page 4) On micrscpe, hit ESC buttn n fcus cntrl t tggle bjective between escape and fcusing psitins; click n F/C t tggle between fine and carse adjustments. (If the fcus knb is tight, dn t frce. Smene prbably left the fcus cntrl buttn clicked in the LaserSharp sftware.) - Turning fcus knb twards yu bjective mves dwn - Turning fcus knw away frm yu bjective mves up

2 Imaging thrugh the eyepieces Mve slider (S) IN Mve turret (T) t psitin 1 Turn n transmitted light if needed (TL) and adjust it using the TL-adj. Turn n fiber ptic illuminatr if needed using the reflected light illuminatr cntrl bx (RL). Switching between eyepieces and scanning: When sample in fcus, and yu are ready t scan: Mve turret (T) t psitin 2. Pull slider OUT. TURN OUT ALL LIGHTS. Turn n PMTs as needed (see Detectin cntrl). Hit Space bar n lwer keybard t start scanning. Detectin Cntrl: External PMT Detectrs Duble click n PMT Cntrller icn n windws cmputer (WC). TURN OFF ALL LIGHTS. Click n black rectangular bx (PMTs Off) t turn n. Bx turns t red when PMTs are n. Check HV n under needed PMTs needed (1 t 3). Set PMT vltage/gain (HV) t ptimize imaging fr each channel. All PMT s can be switched n and ff using the large bx in the lwer right-hand crner. ENSURE THAT ALL LIGHTS ARE OFF WHEN BOX IS RED. Image Acquisitin Duble Click n LaserSharp flder. On ppup windw, duble click n LsrSharp.exe. The system will prceed with an initializatin phase which may take a few minutes t cmplete. After initializatin, the user interface is displayed. Since the instrument is perating in multi-user mde, the sftware will prmpt yu fr yur Lgin name and passwrd. The main menu bar and tl bars will appear in the tp left hand crner f the screen. This will allw access t ther cntrl menus frm the varius icns and pull dwns. The instrument cntrl panel is divided int three sectins: 1. Tp: Image Cllectin scanning speed, zm, cllectin filters, image size, bjective 2. Middle: Fcus mtr 3. Bttm: Mixer cntrl (which PMT s are directed t which panes) In the Image Cllectin panel, set the speed (usually Nrmal r Fast fr perusing yur sample), the cllectin filter t Direct, and N t 0. Fr frame averaging, change cllectin filter t Kalman. N=0 will average until stpped. N f sme ther number will average a specified number f frames. Fcus Mtr turn ff when adjusting fcus manually turn n when setting up fr a z-series acquisitin when n, make sure fcus knb (F/C) n micrscpe is in fine adjustment.

3 Mixer determines which PMT signal shws up in each quadrant n the screen. Suggested setting is 100% PMT3 in Mixer A, 100% PMT2 in Mixer B, and 100% PMT1 in Mixer C if using all 3 detectrs. The merge will autmatically clr Mixers A, B and C red, green and blue, respectively. Laser intensity can either be mdulated using the Pckel s Cell Cntrller (PCC) r the Neutral Density drpdwn menus. Fr beginning users set bth switches n the Pckel s Cell Cntrller t the up psitins and use the ND drp-dwn menus. 3-30% laser intensity is a gd starting pint. Start scanning, by clicking n the Laser buttn (turns red when n) r hitting the spacebar n the OS2C keybard. Yu can stp the scan by clicking n the laser buttn again r by pressing the spacebar. Saving Files Make a directry \User\yurname using the file manager. File Save as (saves image n screen int yur directry). Files will be.pic (Yu can save in ther frmats by using the Exprt as ptin. Files saved in ther standard file frmats will nt cntain imprtant calibratin and system infrmatin. It is advised that yu keep a.pic riginal f any file that yu exprt fr future reference.) Save specific mixers as needed Keep file names shrt. Yu can have 8 characters; n slashes, symbls, spaces. A multiclr Z-series will save as separate files, ne per clr. In this case, yu can nly use 6 characters because the sftware appends 01,02 r 03 t yur filename fr red, green and blue, respectively. Z-Series Acquisitin Determine the upper and lwer edges f yur specimen as fllws: Make sure yu are n fine fcusing using the F/C tggle buttn. Fcus twards yu (bjective mves dwn) t get t the upper edge (clsest t cver slip) f specimen Turn fcus mtr n. This sets the current mtr psitin t 0. Set the Z-step t a suitable increment (e.g. 0.5 um, 1 um). Make sure fcus cntrl is in Fine adjustment. While scanning, step deeper int yur specimen by clicking n the dwn psitin arrw (g in a negative directin). Adjust Z-step if desired. Stp n the last desired plane and stp scanning. Click n Z-Stp t assign this fcal plane t the stp psitin. T cllect: Set Kalman averaging and N if desired). Click n Cllect n the menu bar and chse Z-Series/time Series. A dialg bx will nw appear which will shw the chsen start and stp psitins, the z-step, and the number f sectins t be cllected based n these values. In the dialg bx, click n Save As... Click in the filename bx and enter a name. It is a gd idea t relate the name f the z-series file t the sample type, but prefix it with Z.

4 Click n OK. Yu will be returned t the Preview dialg bx; click n OK. Click n OK fr the riginal menu. The z-series will nw start and be saved autmatically. A Cllectin Infrmatin panel will infrm yu f the prgress f the z series and will disappear n cmpletin. The sectins will be cllected and the mtr will advance the image plane by the specified z step after each image. T stp cllecting images hit cancel. Images cllected will be saved. Always turn OFF Fcus Mtr befre fcusing with the micrscpe! T view z series: Click n 3-D icn t display the LaserSharp Prcessing menu. The current image will be transferred directly t the screen. If nt, yu can pen a saved file by selecting File/Open frm the menu bar. Duble click n the filename yu wish t pen. The specified set f images will be laded int memry and appear n the screen. Optimizing Image Cllectin 1) A gd image uses the full range f intensities between black and white. Yu can lad the LUT called Lad SETCOL in all panes t make sure that the full range f intensities are being emplyed in the image (Display Open LUT SETCOL). It is best t avid high laser intensities that will cause bleaching. Typically start with 3% t 30% befre cntinuing. 2) Mre phtns (higher laser pwer, mre averages r slwer scans) gives better, smther images, but ften mre phtdamage. Yu must determine the ptimal balance fr yur specimen. 3) Use Geg LUT fr dim images (Display Open LUT GEOG). Retrieving Files Files can be retrieved using the cmputer lcated at the entrance t the facility. G t Cmputer and type in the fllwing in the address line: ftp:// Username: nbtcc Passwrd: nbtcc Yur files are in BiRad.pic frmat (76 byte header fllwed by raw bytes). They can be read using ImageJ r a variety f ther image analysis packages. Please delete yur data ff f the acquisitin cmputer as there is very little space n this machine!! We reserve the right t delete any data at any time when this machine is nt in use.

5 MPP Standard filter packs UV/Blue/Vis 670DCXR iii 500DCXR ii BGG22 CH3: nm CH2: nm 400DCLP i UG11 + IR BLOCK CH1: nm Ex 670UVDCLP Em 670DCXR iii D605/90 CH3: nm RGB 550DCLP ii D525/50 CH2: nm 490DCXR i BGG22 CH1: nm Ex Em 670UVDCLP Ind 670DCXR iii 515DCLP ii HQ510LP D495/20+BGG22 CH3: nm CH2: nm UV440LP i D390DF65+BGG22 CH1: nm Ex Em 670UVDCLP

6 Pckel s Cell Pwer Supply (PCPS) Laser (L) Laser Scanner Cntrller (MC) Laser Chiller (LC) Reflected Light Illuminatr (RL) Windws Cmputer (WC) PMT Pwer Supply (PPS) OS2 Cmputer (OS2C) Slider (S) Turret (T) Filter Pack (FP) Scanner Pwer Supply (SPS) ESC F/C TL-adj TL Fcus