IVIS Spectrum User Guide
Contents
1. Password UCSFspectrum Sign in on Planner pop up window Start the Living Image software icon on desktop Returning users select your User ID New users type in your first and last initials Initialize the system It will take several minutes for the check up of the system and for the camera to cool down to 90C You can check the current temperature by clicking on the red bar at the bottom of the control window Once the system is initialized and the camera is at the proper temperature the red bar will turn green Service 13 4 Subject height 150 cm Sequence Setup Focus use subject height v Tenperature IIB ooea Start the anesthesia isoflurane system a Turn ON the O2 supply at the tank Check that you have enough oxygen for your imaging time on the tank manometer Check the level of isoflurane It should be full If not write down on planner Turn ON the oxygen supply oxygen ON OFF Turn ON the pump Pump ON OFF Close the switches to the induction chamber Chamber ON OFF and imaging box IVIS Flow ON OFF by putting the switches vertical Line the bottom of the induction chamber with paper towel and place the metal grid on it g Place your mice for anesthesia in the induction chamber Close it tightly h Turn the isoflurane knob to 2 i Open the switch to the induction chamber Chamber ON OFF the switch is ON when horizontal and OFF when vertical j For epi fluoresc2. FStop Excitation Emission Lamp Level Structure FOV Height Transilumination De 1 DE ww Medium 2 710 800 High No c 1 50 15x23 19 6 2 DE A Medium 2 710 800 High No c 1 50 15x23 19 7 3 DE v Medium 2 710 800 High No c 1 50 15x23 19 8 4 DE wr Medium 2 710 800 High No c 1 50 15x23 19 9 5 DE v Medium 2 710 800 High No c 1 50 15x23 19 10 PaE E W system ees 6 DE o Medium 2 710 800 High No c 1 50 15x23 18 6 Tale Acquire Sequence 7 DE o Medium 2 710 800 High No c 1 50 15x23 18 7 sevice 28 bia s DE Auto Medium 2 710 800 High No c 1 50 15x23 18 8 3 lt a Subject height 1 50 r Jom Image Setup Focus use subject height v Temperature SEEEEEB sasi me Number of Segments 1 Delay 0 0 min Apply to All J X Remove gt Update Insert Add Select desired Image Mode and Parameters Click Add This should add to the sequence list Changes can be made by double clicking Select desired Image Setting s on list b Check the box next to Number of Segments C Press Acquire Setting Insert desired number of segments and time delay Transillumination Settings Select Fluorescent and Transillumination Select desired Image Parameters Click Setup If you are prompted to acquire a subject photograph click Yes selecting the purple squares one image check Raster Scan Press Acquire In the Transillumination Setup box choose desired locations for transilluminati
3. a Acquisition gt gt Autosave b Navigate to your directory or create new folder c Click OK Set Field of View Enable Alignment Grid to see the field of view inside the imaging box Alignment Grid automatically turns off after 2 minutes a A 1 mouse cannot be used with Fluorescence mode b B 1 mouse c C up to3 mice d D up to 5 mice Select subject height The average mouse can be set at 1 5cm If unsure select scan midrange and the imaging system will automatically determine the subject height using a laser scan Select Acquisition settings a For single acquisition select preferred Imaging Mode and Parameters b For time lapse or acquisition of different Mode Parameters use Sequence Setup Press Acquire In Edit Images Label pop up enter relevant information Page 4 of 9 Acquisition Settings A IVIS Acquisition Control Panel Exposure Tme Binning Fistep ExctationFiter Emission Fiker puo S v wan vF wus vyso 7 meim wle v System Status Ide i Acquire om Subject height 1 50 cm Sequence Setup Focus use subjactheight Ternperature TD sas Intialze Imaging Modes You may select either Luminescent or Fluorescent as your imaging mode with an option for a photograph overlay For Fluorescent imaging there is an additional settings for transilumination Exposure Time This parameter affects the sensitivity of the camera If image acquired comes out saturated
4. lower the exposure time Dimmer samples require longer exposure time Do not surpass 5 minutes for in vivo imaging Binning Binning improves the signal to noise ratio for read noise but also reduces spatial resolution From the drag down list are binning options small 4 pixel medium 8 pixel and large 16 pixel F Stop F stop controls the lens aperture size adjusting the amount of light received by the CCD Smaller F stop means larger aperture and thereby increased sensitivity Larger F stop means smaller aperture gives less sensitivity but better resolution Typically f 1 is used for low light resolution luminescence f 2 or f 4 is used for brighter Emission and Excitation Filters There are a total of 18 emission filters with 20nm bandwidth For fluorescent imaging there are a total of 10 excitation filters with a 35nm bandwidth Excitation Filters Emission Filters Transmission transmission os 88h SBS88 400 440 480 520 560 600 640 680 720 760 Wavelength nm wavelength nm Page 5 of 9 Sequence Acquisition 1 Click Sequence Setup The Control Panel should open up like so IVIS Acquisition Control Panel Exposure Time Imaging Mode Binning F Stop Excitation Filter Emission Filter category to change on the list For time lapse a Display Photographic Settings Subject None m Probes m Mode Exposure Binning
5. B ragno W UCsE evelopment University of California enter San Francisco IVIS Spectrum User Guide Contents Rules of the Spectrum seimeni ninnan eieaa aaa aa aaa aaa aaa aaa aa aausaa 2 Setup ProCeduresu u u a Penns aa aaa in went iaaa aaa aAa A aa 3 IMAGING AGUISIHON U E E E AEE A AA 4 Workflow OV erVieW Ua awas esti a a a a i a E aa 4 Acquisition Settings uU eile leis enable 5 Seq ence Setting Ssrnr menesiena a asas a gasas a A a A et a aa 6 Transil mination Settings uuu taenn niaaa aaa aaa aaea aa aaa aa 6 Mage AMAlYS OS sea aae EN aS A ANE SA S ER O E A askalsahankustakalalassa 7 Shutdown PFoCged fes uuu u asi aaea anana an aa aa aaaea a aaeoa idee aa aa 8 Page 1 of 9 Rules of the Spectrum 1 Training All users must be trained by the BIDC NO SECOND HAND TRAINING is allowed If second hand training happens the whole lab may lose access to the Spectrum 2 Booking Reservation for the Spectrum must be done through the BIDC scheduler pathology ucsf edu bidc 3 Overtime Overtime will be charged to the lab If someone is booked after you and you need to go overtime be ready to leave as soon as they arrive 4 Supplies Isoflurane Each lab provides its own isoflurane and should REFILL THE ISOFLURANE AFTER USE If the isoflurane is not full when you start you should record it in the planner Repeating offenders may be denied access to the Spectrum Oxygen Oxygen tanks are available in the room and s
6. ales b Image Information Tools that provide intensity measurements Can give point grid or graph information c Corrections Filtering Enables corrections background subtraction smoothing and soft binning to image data d ROI Region of Interest Tools Tools to select of and take measurements of Regions of Interest i The first four icons are selection tools for either automated or manual selection of one or more regions of interest H The pen and ruler icon creates a table with information of all ROI information Page 7 of 9 lil iv Checkbox Apply to sequence to put the same ROI on every image in your sequence Copy and pasting from one image to another will paste on the same location Set Auto ROI parameters for auto selection Note It is better to overestimate the region of interest and gain miniscule noise rather than to underestimate and lose signal Page 8 of 9 ad Oe ONS Ns oe 10 11 12 Shutdown Procedure Remove animals from imaging box Turn the isoflurane dial to zero Open both switches and let pure oxygen run through the anesthesia machine for a few minutes Close the oxygen tank Allow the oxygen to run out on the machine check the flow meters Turn the pump OFF and the anesthesia machine OFF Clean the induction chamber with Nolvasan blue cleaning solution supplied Clean the bottom of the imaging box with Nolvasan DO NOT spray directly inside imaging box Spr
7. ay on paper towel and wipe clean Refill the isoflurane with the black violet connector located in the drawer Weigh the two purple cartridges and change them if the increased weight is more than 100g Ask for the scale in PSB 518 and get replacement cartridges in PSB516 Quit Living Image and log off the computer Write down any comments on pop up window ie Low supplies Mess left by last user System and computer are to be left ON at all times Page 9 of 9
8. ence place the black mat on the bottom of the imaging box matte side up For trans illumination remove the black mat k Open the isoflurane switch to the imaging box IVIS Flow ON OFF Allow time for the anesthesia gas to reach the manifold nose cones prior to removing animals from Induction Chamber The typical time is two to five minutes 1 Close the switch to the induction chamber Chamber ON OFF before transferring anesthetized animals m Quickly remove the animals and place them in the nose cones If not done already seal off unused manifold ports with Modified Black Rubber Stoppers n To reduce exposure to isoflurane keep the Induction Chamber lid closed and latched when not removing or replacing animals sao SF a Page 3 of 9 Image Acquisition The Living Image software on the PC workstation that controls the IVIS Imaging System includes both the acquisition and analysis features Imaging Analysis Station 1 located in S1126 has the Living Image software with only the analysis features Reservation for the Imaging Analysis Station should be done through the BIDC scheduler http pathology ucsf edu BIDC This User Manual is a brief overview of basic features Caliper s official Living Image Software Manual can be found at Caliper s website http www caliperls com support software downloads htm as well as BIDC s resource page Workflow Overview 1 2 3 4 5 6 Set Auto Save
9. hould be changed if in the red zone Cartridges The charcoal cartridges are available in the room or in PSB516 and should be changed when their weights show that they are full Cleaning solution Diluted Nolvasan blue solution is available in the room and should be the solution used to clean the spectrum Notify Jessica if the cleaning solution is running low or leave a comment at logoff 5 When you are done CLEAN the inside of the imaging station the induction chamber and the desk with diluted Nolvasan DO NOT directly spray inside the imaging station spray onto paper towel and then wipe down the bottom of the chamber Do not allow Make sure the ISOFLURANE DIAL IS OFF The PUMP IS OFF the 02 TANK IS CLOSED and the PC IS LOGGED OUT REFILL THE ISOFLURANE in the anesthesia machine WEIGH THE CARTRIDGES and record their weight on both Change them if there are above their maximum weight 100g more than the starting weight If there are no empty cartridges in the room you can find more in PSB516 Dispose of full cartridges in PSB516 6 Good practices At logoff there is a popup window for you to write down any problems with the system anything unusual or any bad practices For anything requiring immediate attention please notify Jessica Contact information Jessica Wong Jessica Wong ucsf edu Sebastian Peck Sebastian Peck ucsf edu Page 2 of 9 Setup Procedure Log into the computer Username IVISuser
10. on by To acquire each point as a different image leave Raster Scan unchecked To bring into Stem recat niet Qo on base button to proceed Single location mode acquires one image at the location marked by a green square 8 Page 6 of 9 Image Analysis After the image is successfully acquired and OK is pressed on Edit Images Label popup an Image Window and Tool Palette should appear The Tool Palette provides many data analyses tools me e vm ee hem ee SBA BRAS W uon Dawes AEL 310 fe fx _ xm A Image Window a Display You can choose which of the images to acquired to view For example if you chose Luminescent and Photograph Settings for Acquisition you can view them separately or as an overlay b Units Selects the measurement units for the image display There are calibrated physical units that automatically compensates for different settings Such that three luminescent acquisitions of different binning exposure and or f stop will give the same data in calibrated physical units which are photons per seconds for luminescence c Info Click to display or hide image label d Image Label Contains information entered in Edit Image Labels pop up box and other information automatically recorded by the software e Color Scale Reference for pixel intensities shown on image B Tool Palette a Image Adjust Tools to navigate the image as well as adjust color settings and sc
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