MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION

Transcription

1 Olympus VS120-L100 Slide Scanner Standard Operating Procedure Startup 1) Red power bar switch (behind monitor) 2) Computer 3) Login: UserVS120 account (no password) 4) Double click: WAIT FOR INITIALIZATION TO COMPLETE Shutdown 1) Transfer data to external drive and delete from computer 2) Remove ALL slides from cassettes 3) Close VS-ASW FL software 4) Shut down computer 5) Switch off power bar MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION CONTENTS BRIGHTFIELD (BF) SLIDE SCANNING SINGLE SLIDE... 2 FLUORESCENCE (FL) slide scanning Single Slide... 4 BRIGHTFIELD (BF) slide scanning Batch Scans... 7 FLUORESCENCE (FL) slide scanning Batch Scans... 9 Troubleshooting FILTER SPECS Multi band (Switch channels before moving XY) Single band (Move XY before switching channels) DAPI-FITC-TRITC-Cy5 CFP-YFP-RFP** DAPI-FITC-TRITC-Cy5 Ex (nm) Em (nm) Ex (nm) Em (nm) Ex (nm) Em (nm) DAPI 391/32 435/30 CFP 436/28 474/24 DAPI 375/28 460/50 FITC 479/33 520/30 YFP 506/21 540/24 FITC 480/30 535/40 TRITC 554/24 594/32 RFP 578/24 642/80 TRITC 540/25 605/55 Cy5 638/31 694/60 Cy5 638/31 694/60 Notes: ** Filter change needed, please contact AMP staff (If you require a filter set suited to mcherry/texas Red, use the CFP/YFP/RFP multiband) Page 1

2 MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION BRIGHTFIELD (BF) SLIDE SCANNING SINGLE SLIDE 1) a. For first time users, click on the Scan Projects tab and choose VS120 Brightfield project. This will populate the parameters with the most common settings (20x detailed scan, VSI file format). You can modify settings during the scan setup. b. For all other users, you may apply your own Scan Project as above or click on: under Single Slide Scan column. 2) If you have more than one slide, carefully load them into the cassettes as instructed by staff. 3) Choose Manual Load or Automatic Load. If Manual Load is chosen, CAREFULLY lift stage access cover NO MORE THAN 90 DEGREES OR YOU WILL HIT THE CAMERA. Pull back round tab and place slide sideways/horizontal with label on the LEFT. 4) If Automatic Load is chosen, the sensor will automatically detect where your slides are loaded in the cassettes. Once the sensor has detected the slide positions, choose the slide you wish to scan. 5) Fill out Slide Properties left column as needed. 6) Set naming and saving parameters in Automatic Naming and Saving middle column. 7) Click Overview 8) Once overview image is completed, choose objective magnification for detailed scan. Z-Scan Mode should be set to Scan one Z-plane. If you require more than one Z-plane, please see a member of staff. 9) Click Edit Scan Area 10) Under Scan Area in the left column. You can either click on Create Scan Area button to allow the program to automatically detect and draw Scan Regions around your samples or you can choose `Remove All Scan Areas` to draw your own. Use mouse wheel to zoom. 11) Under Detailed Scan Settings, make sure Scan whole area and focus sample only (middle button) option is selected. 12) Next, click on to put down a focus map. Choose the Focus Grid Density in the left column. You can manually add more Focus points with a mouse click, move existing focus points or select and delete those not required. NOTE: The more focus points you put down the longer the scan will take. Flat (fold and bubble free) samples with good contrast do not need a large amount of focus points while samples with less contrast and/or uneven surfaces require more points. 13) When done click on 14) If ready to proceed, click on Scan Now. If you require more focusing options, click on Next Page 2

3 a. In the Focus Settings menu, you may choose the Focus Logic. Options are Automatic (Default), Semi-Automatic (user confirms automatic focusing at each point) and Manual (user manually sets each focus point). b. You can now click on the Scan Now button. 15) Once completed you have several options: a. You may Add another Detailed Scan to the existing slide b. Scan a new slide with the current parameters c. Remove/Unload current slide d. Save Project (HIGHLY RECOMMENDED*) e. Or finish the experiment. *To preserve your scan parameters, it is highly recommended that you Save your Project so you do not have to worry about accidentally using a previous users settings. 16) If not saving a projecty, Click Finish and save your scans if you have not done so. 17) To remove the slide, click on and choose Manual Load. Page 3

4 MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION FLUORESCENCE (FL) slide scanning Single Slide 1) a. For first time users, click on the Scan Projects tab and choose one of the following options to populate the parameters with the most common settings (20x detailed scan, VSI file format, typical exposure etc.). You can modify settings during the scan setup. i. VS120 4 Channel Single Band. This option uses separate discrete filters for each channel and moves the stage in XY before switching channels. Less crosstalk but slower. (See page 1 for filter specs) ii. VS120 4 Channel Multi Band. This option uses a single multiband filter for collecting 4 emission bands simultaneously. Best for speed but more crosstalk. (See page 1 for filter specs) iii. VS120 3 Channel FP Multiband. This option uses a single multiband filter for collecting 3 fluorescent protein (CFP/YFP/RFP) emission bands simultaneously. Best for speed but more crosstalk. FILTER CHANGE NEEDED SEE MEMBER OF STAFF. (See page 1 for filter specs) b. For all other users, you may apply your own Scan Project as above or click on: under Single Slide Scan column. 2) If you have more than one slide, carefully load them into the cassettes as instructed by staff. 3) Choose Manual Load or Automatic Load. If Manual Load is chosen, CAREFULLY lift stage access cover NO MORE THAN 90 DEGREES OR YOU WILL HIT THE CAMERA. Pull back round tab and place slide sideways/horizontal with label on the LEFT. 4) If Automatic Load is chosen, the sensor will automatically detect where your slides are loaded in the cassettes. Once the sensor has detected the slide positions, choose the slide you wish to scan. 5) Fill out Slide Properties left column as needed. 6) Set naming and saving parameters in Automatic Naming and Saving middle column. 7) Click Next 8) Under Overview Type, choose the method you want to collect your overview. a. BF (Brightfield): Quickest method but only if your sample has enough contrast. Click drop down arrow to choose Normal, Dim or Faint Sample to correspond to level of contrast. b. Tissue: To scan a fluorescence overview image on tissue samples. Click drop down arrow to choose filter set. c. Cells: To scan a fluorescence overview image on tissue samples. Click drop down arrow to choose filter set. 9) Choose objective lens for overview image (2x is usually sufficient). 10) If you chose option (a) or (b) above, click Start Live button under Exposure Time column to set exposure. Use joystick to navigate to sample. 11) Click Overview button. 12) Once overview image is completed, choose objective magnification for detailed scan. Z-Scan Mode should be set to Scan one Z-plane. If you require more than one Z-plane, please see a member of staff. Click Next button. Page 4

5 13) Click Edit Scan Area 14) Under Scan Area in the left column. You can either click on Create Scan Area button to allow the program to automatically detect and draw Scan Regions around your samples or you can choose `Remove All Scan Areas` to draw your own. Use mouse wheel to zoom. 15) Under Detailed Scan Settings, make sure Scan whole area and focus sample only (middle button) option is selected. 16) Next, click on to put down a focus map. Choose the Focus Grid Density in the left column. You can manually add more Focus points with a mouse click, move existing focus points or select and delete those not required. NOTE: The more focus points you put down the longer the scan will take. Flat (fold and bubble free) samples with good contrast do not need a large amount of focus points while samples with less contrast and/or uneven surfaces require more points. 17) Click Next. 18) If you chose one of the preset Scan Projects, the list of filters under Channel Settings should be filled with the default choices. 19) To add a channel, click and choose to add a single or multiband filter set. To remove a channel, click. 20) Select Channel 1 (this is the channel that will be used for focusing so it should be your brightest and most widespread) and click 21) In the Stage Navigator window in the far right, make sure the yellow square is in the middle of your Detailed Scan area. If not, click inside your region of interest to move the stage. 22) Refer to the histogram in the top right window. Change the Exposure Time so that you achieve at least 1500 counts in the mean intensity reading. The Orca Flash 4.0 fluorescence camera is a 16-bit camera, allowing for a high dynamic range (65536 bit depth). It is recommended to use as much of this range as possible (without excessive exposure times causing photo-bleaching) to capture as much data as possible for potential post-scan analyses. 23) If ready to proceed, click on Scan Now. If you require more focusing options, click on Next 24) In the Focus Settings menu, you may choose the Focus Logic. Options are Automatic (Default), Semi- Automatic (user confirms automatic focusing at each point) and Manual (user manually sets each focus point). 25) You can now click on the Scan Now. Page 5

6 26) Once completed you have several options: a. You may Add another Detailed Scan to the existing slide b. Scan a new slide with the current parameters c. Remove/Unload current slide d. Save Project (HIGHLY RECOMMENDED*) e. Or finish the experiment. *To preserve your scan parameters, it is highly recommended that you Save your Project so you do not have to worry about accidentally using a previous users settings. 27) If not saving a project, Click Finish and save your scans if you have not done so. 28) To remove the slide, click on and choose Manual Load. Page 6

7 MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION BRIGHTFIELD (BF) slide scanning Batch Scans 1) Carefully load slides into the cassettes starting from cassette #1 (closest to you) before populating cassette #2 (furthest from you). Take note of your slide descriptions and positions in the cassettes for your own references. Carefully return cassettes back into the machine in the same order, ensuring that they are seated correctly. 2) To scan multiple brightfield slides, click on: under Batch Scan column. A sensor will now detect the positions of your slides within the cassettes. Once complete the next screen shows the positions of your slides in the cassettes under the Slide Loader column. Use the dropdown menu to choose between cassette #1 and #2. 3) Highlight each slide and fill out its properties in the middle column Slide Properties then click Next when done. A template can be used to pre-fill these properties. Talk to staff if needed. 4) Under the Slide Loader column, you are now able to load one or more previously saved Scan Project(s) or use the provided VS120 Brightfield project for typical parameters. 5) In the Scan Settings column and Magnification and Image Document tab, select desired magnification and Z-scan mode. 6) a) Click the Apply settings to all slides of all cassettes button to save these settings to every slide in every cassette. All slides in the loader should now be highlighted green. b) If you only want to scan a selection of your slides, hold the Ctrl-key and click the positions in the cassette to select only the slides you wish to scan. Once the selection is complete, repeat step 5) and click the Apply settings to selection button to save settings to just the selected slides. The selected slides in the loader should now be highlighted green. c) If you want to scan different slide selections with different parameters/projects, repeat steps 4 6 for each slide selection. 7) Set naming and saving parameters in Automatic Naming and Saving column and click Overview. 8) If for whatever reason you need to scan a single slide NOT loaded in the cassettes, click on Priority Scan to interrupt the batch process. 9) Once overviews are completed, you can review each slide in the Batch Slide Selection column. With a slide selected, click on Edit Scan Area button. 10) Under Scan Area in the left column. You can either click on Create Scan Area button to allow the program to automatically detect and draw Scan Regions around your samples or you can choose `Remove All Scan Areas` to draw your own. Use mouse wheel to zoom. 11) Under Detailed Scan Settings, make sure Scan whole area and focus sample only (middle button) option is selected. Page 7

8 12) Next, click on to put down a focus map. Choose the Focus Grid Density in the left column. You can manually add more Focus points with a mouse click, move existing focus points or select and delete those not required. NOTE: The more focus points you put down the longer the scan will take. Flat (fold and bubble free) samples with good contrast do not need a large amount of focus points while samples with less contrast and/or uneven surfaces require more points. 13) When done click on 14) Select next slide in the Batch Slide Selection column and repeat steps ) If you do not wish to scan a particular slide(s) in the batch, select the slides in the list and click on Exclude slides from the batch scan button 16) If ready to proceed, click on Scan Now. to exclude from batch scan. Note: If you need to scan a single slide NOT loaded in the cassettes, click on Priority Scan to interrupt the batch process. Page 8

9 MAKE SURE YOUR SLIDES ARE CLEAN (TOP & BOTTOM) BEFORE LOADING DO NOT LOAD SLIDES DURING SOFTWARE INITIALIZATION FLUORESCENCE (FL) slide scanning Batch Scans 1) Carefully load slides into the cassettes starting from cassette #1 (closest to you) before populating cassette #2 (furthest from you). Take note of your slide descriptions and positions in the cassettes for your own references. Carefully return cassettes back into the machine in the same order, ensuring that they are seated correctly. 2) To scan multiple fluorescence slides, click on: under Batch Scan column. A sensor will now detect the positions of your slides within the cassettes. Once complete the next screen shows the positions of your slides in the cassettes under the Slide Loader column. Use the dropdown menu to choose between cassette #1 and #2. 3) Highlight each slide and fill out its properties in the middle column Slide Properties then click Next when done. A template can be used to pre-fill these properties. Talk to staff if needed. 4) Under the Slide Loader column, you are now able to load one or more previously saved Scan Project(s) or use the provided default projects (outlined on Page 4-1a) for typical parameters. 5) In the Scan Settings column and Magnification and Image Document tab, select desired magnification and Z-scan mode. You may also modify the Overview Settings, Illumination Settings and Focus Settings tabs. However, you will have a chance to modify these settings later. 6) a) Click the Apply settings to all slides of all cassettes button to save these settings to every slide in every cassette. All slides in the loader should now be highlighted green. b) If you only want to scan a selection of your slides, hold the Ctrl-key and click the positions in the cassette to select only the slides you wish to scan. Once the selection is complete, repeat step 5) and click the Apply settings to selection button to save settings to just the selected slides. The selected slides in the loader should now be highlighted green. c) If you want to scan different slide selections with different parameters/projects, repeat steps 4 6 for each slide selection. 7) Set naming and saving parameters in Automatic Naming and Saving column and click Overview. 8) Once overviews are completed, you can review each slide in the Batch Slide Selection column. With a slide selected, click on Edit Scan Area button. 9) Under Scan Area in the left column. You can either click on Create Scan Area button to allow the program to automatically detect and draw Scan Regions around your samples or you can choose `Remove All Scan Areas` to draw your own. Use mouse wheel to zoom. 10) Under Detailed Scan Settings, make sure Scan whole area and focus sample only (middle button) option is selected. Page 9

10 11) Next, click on to put down a focus map. Choose the Focus Grid Density in the left column. You can manually add more Focus points with a mouse click, move existing focus points or select and delete those not required. NOTE: The more focus points you put down the longer the scan will take. Flat (fold and bubble free) samples with good contrast do not need a large amount of focus points while samples with less contrast and/or uneven surfaces require more points. 12) When done click on 13) Select next slide in the Batch Slide Selection column and repeat steps ) In the Scan Settings column, set the parameters required for the Magnification and Image Document tab, Illumination Settings tab and Focus Settings tab if required. 15) If you do not wish to scan a particular slide(s) in the batch, select the slides in the list and click on Exclude slides from the batch scan button 16) If ready to proceed, click on Scan Now. to exclude from batch scan. Note: If you need to scan a single slide NOT loaded in the cassettes, click on Priority Scan to interrupt the batch process. Page 10

11 Troubleshooting Sample not recognized For weakly stained samples with poor contrast (for example, brightfield slides with weakly staining dyes, very sparse stained areas OR brightfield overviews of fluorescently stained samples). During the editing scans dialog you will see the following settings in the left column: Figure 1. To increase the threshold of sample detection, modify the Sample detection sensitivity slider. In the example below (Figure 2) with the slider at 0, you see only a small percentage of the spinal cord tissue sections detected by the system as indicated by the white, highlighted background. With the slider set to +10 (Figure 3), almost all the sections are highlighted. Sample detection is also important when creating focus maps on scan areas. Under Detailed Scan Settings, if the Scan and focus sample only or Scan whole area and focus sample only (first and second button from left) is selected. Focus maps will only be placed on samples that have been detected (as described above). If your sample is still too faint to be detected, you will have to choose the 3 rd button Scan and focus whole area after manually drawing the scan area and/or manually add focus points. There are various options in the Sample Detection Settings and Advanced Sample Detection Settings menu to assist in fine tuning detection of your samples. When your samples are successfully highlighted, you can now click the Create Scan Area button which will automatically put a scan area around the detected sections (Figure 4). This is especially useful if you have many sections on the one slide to avoid manually drawing scan areas. Page 11

12 Figure 2. Brightfield overview image of fluorescently stained mouse spinal cord cross sections, sample detection sensitivity set to Zero. Figure 3. As above with sample detection sensitivity set to +10. Figure 4. As above after clicking on Create Sample Area button. Page 12

13 If you choose Brightfield overview scan for your fluorescent sample, there are various options in the drop down arrow menu. Choosing BF (brightfield) you have a choice of Normal, Dim and Faint samples. Depending on the choice, the program will manipulate the brightness and gamma to improve the contrast of the samples on your slide, giving you a greater chance of sample detection (mentioned above). BF Normal Samples BF Dim Samples BF Faint Samples Page 13

14 Focusing Issues After setting focus maps on your samples and clicking Scan Now, the system will go to each focus point to set the focus. If the focus is found, the focus point square will change colour to green. If the focus could not be found, the focus point square will change to grey. The scan will proceed if there is at least ONE green focus point square. Various focusing issues and possible solutions: 1. Samples are weakly stained (either brightfield or fluorescent). o In the case of weakly stained brightfield samples, go into Live mode before proceeding with the scan to manually set exposure times. o In the case of weak fluorescent signals, increase the exposure time of the focusing channel (channel on the top of the list) so the mean intensity on the histogram is at least 1500 counts (Page 5). Always choose the strongest and most widespread fluorescent channel to be the focusing channel. 2. Samples are too thick or too thin. o Thin samples <5 microns will have a smaller volume of fluorescent signal. Ensure that the stage is close to the focus by going into Live mode before proceeding with the scan. o Thick samples >50 microns will have many points of focus. The algorithm will usually choose the top of the sample. 3. Stage is too far away from sample or focusing algorithm gets lost. o Pertaining to both points 1 and 2, double check in Live mode to make sure the stage is at least close to the focus of your sample. o Avoid putting focus maps on empty areas where there is no sample. Focus maps on a glass slide can often bring the stage far from the focus. Page 14