CUSTOM-BUILT LUMINESCENCE BOX USER GUIDE

Transcription

1 CUSTOM-BUILT LUMINESCENCE BOX USER GUIDE START-UP PROCEDURE: 1. Log on with your net id and password. 2. Turn on the camera by pressing the red on/off switch located at the top of the camera. 3. Open software: Double-click Andor Solis icon on desktop. 4. Wait a few minutes until the temperature indicator at bottom left of main display window turns blue (30 o C). 5. While waiting for the camera temperature to be ready for imaging, prepare to anesthetize animal. 6. Make sure clamps A and B are open, and C and D are closed. 7. Open oxygen supply valve. Set flow gauge to ~800 ul/min. Make sure vaporizer has isoflurane. 8. Set vaporizer value to ~3.5 4% isoflurane. 9. When animal is ready to transfer, open clamps C and D, and close clamps A and B. 10. Place the animal in the induction chamber, then close and lock the chamber. 11. Open luminescence box* and lower stage by depressing up/down buttons on Stack Shot control box. 12. Put animal/specimen on the stage. *The anesthesia flows through both the induction chamber and the luminescence box. SHUT-DOWN PROCEDURE: 1. Take animal off stage, and either put back in cage for recovery or sacrifice. 2. Turn off vaporizer. 3. Turn off oxygen valve. 4. Exit Andor Solis software. 5. Turn off camera. 6. Turn off LED lamp (if on). 7. Lower stage and wipe with disinfectant/ethyl alcohol. 8. Perform image analysis on data if desired, or copy data files to the Imaging shared folder. 9. Log-off computer.

2 1 1 Camera on/off switch (located at the top of camera) 2 2 Stack Shot control box for stage control & adjustment 3 LED switch for BF illumination 3 IMAGING: This luminescence box is designed to take Brightfield (BF) and luminescence (L) images. It is equipped with an Andor Neo scmos camera. 1. When camera is ready (-30 0 C temperature) and sample/specimen is set on the stage, you are set to start imaging. Camera not ready Camera ready 2. Open luminescence box and put sample/specimen on the stage.

3 3. View the sample under brightfield mode by flipping the LED switch (3) on. 4. Click on the Video icon in the main menu for continuous image acquisition. 5. Adjust the Exposure time (in sec) in the scmos Control Box located at the right side of the main display. If not there, this window can be opened by clicking on the Acquisition tab in the main menu, then Set up Acquisition. Generally, Brightfield images require ~0.2 sec exposure time. 6. Adjust stage height by depressing Up/Down buttons in control box (2). Be cautious when using these buttons. Make sure sample doesn t physically touch the optics located on the top of the box. 7. On the scmos control display, set your Acquisition Mode to either of the following: Single scan, Kinetic Series, or Accumulation.

4 8. Click on the Camera icon (Take Signal) for a single image. Save your image as a tif file in the following format: *_BF.tif. 9. To take a luminescence image, switch off the LED. Turn off room lights. Wait for about 30 seconds (not necessary, if LED light was not turned on prior to taking luminescence image). Adjust the exposure time. Typically, exposure times could range from 1 sec to 60 sec (for any light-induced phosphorescence to die down). 10. Click on either the Video or Camera icons to capture luminescence image. Progress of the acquisition can be monitored by looking at the acquisition % located at the bottom of the screen (beside the temperature display). 11. Save luminescence image in the following format: *_L.tif. 12. If you wish to cancel your acquisition, click on the Abort Acquisition icon (red when acquiring image). SAVING DATA: 1. Save files as either *_BF.tif or *_L.tif in C:\Users\BRC-Imaging\Data\netid\file name 2. Files in the above directory need to be transferred into the Imaging Share file. Under Scan Range, you can choose to save the Currently displayed scan (for a single image), Entire Series (for a kinetic series, saved as a stack), Series to individual files (for Accumulation or kinetic series)

5 FOCUS CONSIDERATIONS You can focus either by using the focus knob on the camera lens or by using the automated stage. Remember that your effective detection efficiency depends not only on the f# of the lens (1.4 in this system), but the distance of your object from the lens. If you have dim samples, get them up close to the lens, but please keep them from smudging or (even worse) scratching the lens. ANDOR SOLIS MAIN DISPLAY WINDOW PIXEL BINNING - 1x1; 2x2; 3x3; 4x4 - Increasing binning = increasing sensitivity IMAGE AREA - 128x128; 512x512; 1392x1040; 1920x1080; 2048x2048; Custom; Full Image

6 IMAGE WINDOW MAKING OVERLAYS IN IMAGEJ 1) Open ImageJ 2) Open two macros from C:/Users/Public/Macros. 3) Tif2JpgBatchScale_.ijm makes scaled jpgs placed in a jpgs folder in the directory you specify as indir. First you must decide if you want all your luminescence images scaled to their respective maxima or to a defined maximum (for comparison to others). Line 4: In the first case, Scale = 0;

7 In the second case, set Scale to whatever value is maximum in the images to be compared. (Find the brightest image and read off the intensity in the ImageJ cursor.) 4) Line 5: Set whatever the working directory is (indir). 5) Run the macro. 6) LuminescenceOverlayBatchJpgs_.ijm overlays the resulting images in the jpgs file. These images are placed in a merge folder in the jpgs folder. Line 4: Select LUT of choice (See Image - > Look Up Tables or in C:\Users\Public\Luts). 7) Line 5: Set the jpgs directory to be the working directory. 8) Line 7: Set Luminescence threshold. 9) Run the macro.

8 Preparation and Injection of Luciferin for In Vivo Bioluminescent Assays Procedure 1. Prepare a fresh stock solution of Luciferin at 15 mg/ml or 30 mg/kg in DPBS. Filter sterilize through a 0.2 μm filter. 2. Inject 10 μl/g of body weight. Each mouse should receive 150 mg Luciferin/kg body weight. e.g. For a 10 g mouse, inject 100 μl of 15 mg/ml to deliver 1.5 mg of Luciferin. 3. Inject the Luciferin intra-peritoneally (i.p.) 5-15 minutes before imaging. A Luciferin kinetic study should be performed for each animal model to determine peak signal time after Luciferin administration. After luciferase ip injection, the bioluminescence signal gradually increases and reaches a plateau at 10 min, which lasts for min, so the best time window of imaging is min after luciferin administration. Intraperitoneal (i.p.) Injection of Luciferin Preferred Site Animal s lower left abdominal quadrant. Needle Size 25 gauge, usually used with 1 cc syringe Volume 100 μl of Luciferin (15 mg/ml stock) per 10 grams of mouse body weight. Note: 1 ml i.p. injection of a nonirritating solution is easily tolerated. Position of Animal Manually restrained, dorsal recumbency (abdomen side up), with cranial (head) end of animal pointed down. Injection Needle should be bevel-side up and slightly angled when entering the abdominal cavity. Penetrate just through abdominal wall (about 4-5 mm). The tip of the needle should just penetrate the abdominal wall of the animal s left lower abdominal quadrant.