Olympus LEXT OLS4000 Confocal uScope - Quick Start

^{Demis D. John –– 2018-07-19}

Do not use the LEXT unless trained first!

You MUST be officially trained on this tool before you are allowed to use it!  

These instructions are a quick-reference for users who have already received the full training. 

Quick-Start General Procedure

In order to acquire a profile height measurement, you must do the following:

1)  Get the sample surface in focus with the regular “color” optical microscope & lock the focus knob

2)  Switch to “laser” mode & readjust the focus

3)  Specify the bottom and top range for the focus motor to scan over & acquire the scan data (in Fine/Manual mode)

4)  In the “Measure” tab, Specify a region on the image of which to take a profile

5)  Move the cursors to the desired step-height measurement positions

Quick-Start Detailed Procedure

Initial Setup

Make sure microscope is raised and stage is away from the objectives in the “Sample Exchange” position.  Make sure software is started (should show a Login screen). If this is not the case, do the following:

1. Open the software “OLS4000”. 
2. Ensure no sample is inserted, and microscope objectives are lifted away from the stage – use the coarse focus knob to lift the objectives to a safe height & Lock it, if needed. Then press “OK” on the warning & wait for motor initialization.
3. At the login screen, click “OK” to login as Guest.
   1. 
   2. System should automatically display the “Acquisition” tab

Load & focus on sample

1. Place your sample near the center of the stage.
2. Make sure 5x objective is selected and objectives will not hit stage if motors move down.  If unsure, click “Cancel” on the “Move to Z reference position” dialogue, and lift the objectives using the manual focus knob.
3. Click the “Scan Position” button to move the stage to center 
4. System should already be imaging live in Color mode.  If not, click the “Color” imaging button , and ensure the “Live” window-tab has a halo around it by clicking on it, like so:
5. Focus on the sample surface by using the black manual focus knob first, unlocking/re-locking if needed. This prevents the electronic focus motor from hitting it’s limit during imaging.
   1. Make sure to engage the focus “Lock” ring (finger-tight only), otherwise the focus will drift.
6. Fine-Focus (electronic) by using the Mouse-Wheel (Right-click the live image to set wheel to Coarse focus), or by using the “Focus” up/down arrows on the right of the image.
7. Use the joystick to drive to feature of interest.  
   1. Make sure sample's TOP surface is in focus before Stepping up in objectives/magnification, to prevent crashing.
   2. The higher the magnification, the better the height resolution.
8. Click the Laser imaging mode button
9. Readjust electronic focus until the sample surface lights up.
   1. May need to adjust laser Brightness (vertical tab on image pane), for max brightness but not saturating can use “Saturation Highlighting” to show this <<pic of saturation highlighting>>

Acquisition Settings / Scan Range

1. Set the Acquisition Settings to [Fine▽] & [√] Manual, like so:
   1. 
   2. [√] Color mode will scan the in color mode and then in laser mode, and superimpose the true-color image onto the 3D image.
2. Expand the [+] on the right of the image and enable Saturation Highlighting
   1. 
3. Focus ⇓on the lowest surface of your sample. Go a few mouse-wheel clicks below that focus point until lowest surface has reduced in brightness and click the [Bottom] button to save this Z height as the lower point of the scan range.
4. Focus ⇑on the highest surface, and go a few clicks above that until brightness decreases, and click the [Top] scan range button.
   1. If you see any regions of the sample surface reach saturation anywhere in the scan range, lower the image brightness using the Brightness arrows on the right of the imaging panel.  <<pic of brightness buttons>>. 
   2. Higher brightness reduces noise, but saturation reduces accuracy of surface height detection.
   3. 

      Top & Bottom focus range buttons for Manual mode
5. Click [Acquisition] to perform the scan
   1. 200 height steps takes about ~30sec.  Watch out for excessively high scan steps that could take a long time.

Measure Height Profile

1. Switch to the [Measure] tab at the top 
2. Use Profile > 2-point mode
   
3. Click two points on your image to extract profile
4. Optional: level the surface (done automatically, but sometimes can be better)
   1. [Image Correction] > (•) 3-Point > Click 3 points on image on same surface> [Execute]  
      

      [Image Corrections] button
   2. 

      3-point Leveling Screen with relevant options highlighted
5. Place cursors on height profile & observe “Height” value in corner.
   1. Optional: Click ▽under Profile buttons to average around the cursors
      1. 

         Cursor Averaging Panel
   2. Optional: Save  as Screenshot or LEXT file (for later analysis)

Shutdown at End of Session

1. [Imaging] at top > Color <<color icon>> > 5x > Sample Exchange
2. Log Off (NOT the [X] button!)
   1. This saves each user a few minutes of boot-up time
   2. “No to All” to discard unsaved scans.