1 Setting Laser Power and PMT Gain Begin with laser power at 20% Set PMT gain to 6.00.

2 Acquire 2D Images Basic Select objective Select lasers (Color is on) Select detectors Select pinhole (based on objective) Set PMT gain Live for continuous scanning (on/off) Single for a single scan pass Basic-Intermediate Set pixel dwell (EX/EM Exposure) Set scan steps (resolution) (changes # of pixels in FOV and pixel size) Set field zoom (FOV changes, # of pixels is constant, pixel size changes) Define a region (FOV Crop) Average (noise reduction) Frame Lambda (Multi-Channel) Line Lambda (Multi-Channel - LU-4 only)

3 Image Display - Color Tab The Color tab sets the channel display parameters for the active image window. Color Mode selects color multichannel or single channel (grey scale) Color assignment is arbitrary, but is usually set to coincide with the fluorescence colors seen in the eyepieces or pseudo color.

4 Acquisition Menu Screen Select lasers Select detectors Select pinhole Set gain & offset for each channel Live for continuous image update Single for a single pass Set pixel dwell Set scan resolution 160 x 160 through 2048 x 2048 Set Scan zoom Define a region (FOV) Select Mode

5 Setting Pixel Size & Resolution Select XY tab, Zoom button You can increase resolution to 1024 x 1024, or even to 2048 x Field size remains constant so pixel size changes. Changing the field zoom changes scan area but keeps the number of pixels constant by changing their size Changing pixel size keeps zoom & scanned area constant while changing X & Y steps (resolution)

6 Color Tab The Color tab sets the channel display parameters for the active image window. Color Mode selects color multichannel or single channel (grey scale) Color assignment is arbitrary, but is usually set to coincide with the fluorescence colors seen in the eyepieces. Select saturation and black level indicators Basic post processing (Intensity scaling) of display images Best color fit (select if image is black) Set dark level, saturation, and gamma Reset views Channel color selects active color channel or custom colors (Also on Tool bar) Can view ROI spectral graph. Intensity vs. wavelength

7 View Tab The View Tab sets the display mode for the active window. The available options will vary with the acquisition mode View selects the display screen type from 1D graphs, 2D & 3D displays Axis displays different XYZ combinations Use Z slider to scroll through Z stacks. Select animate options to play, change speed, and playback direction Checking Force Window height to data Document automatically fixes Live window to image size & disables Zoom slider

8 Scan Head Pinhole Alignment Setup live screen in Pseudo color for the laser being used for alignment. Use Chroma plastic slide & 20x objective. Green slide is best for 488nm Preliminary settings for Gain at about 6.0 & Laser power 10-20% Turn on Live (512x512) and focus just below the surface of the slide and adjust Gain and Power until the screen is light Blue/Green. Adjust pinhole centering screws on scan head until brightest intensity is realized. The screen should change to yellow/pink indicating maximum intensity. Reduce gain if image turns white. If the pink area is centered no further alignment is required. Pinhole alignment should be done every time the scan head dichroic is changed.

9 Scan Head Pinhole Alignment To verify system is aligned check the field using a 1D graph. Click on the 2D image and create a copy of the window in a new screen. Create an image using Averaging 4 frames. On new image select 1D graph X-Y from View Tab and check for uniformity. Intensity should not very more than 10% in X or Y and 20% diagonally If pink area is not centered coupler alignment is required A simple check of scan uniformity can be done by placing a piece of paper on stage with no objective, zoom to max, pixel dwell to longest and check spot. It should be uniform in center 3/4

10 Zoom Images The resolution changes, but the frame size does not Can be done with live scan on or off. Click on XYZ button to open Navigation window. Verify you are in Zoom. If Navigation window is black click on Live and select “Copy” to add image to Navigation window Size, rotate, and position boundary box in navigation window Select Live or Single Do not switch to crop from zoom while in live scan Reset Zoom prior to closing Close navigation window prior to switching to crop

11 Crop Images The frame size changes, but the resolution does not Can be done with live scan on or off. Click on XYZ button to open Navigation window. Verify you are in Crop. If Navigation window is black click on Live and select “Copy” to add image to Navigation window Size, rotate, and position boundary box in navigation window Select Live or Single Do not switch to Zoom from Crop while in live scan Reset Crop zoom prior to closing Close navigation window prior to switching to zoom

12 EZC1 Contains Great Averaging Algorithms Improvement in signal to noise through simple frame averaging is an exponential function. Averaging 4 frames doubles the signal to noise. To double it again, you must average 16 frames. Stop at quality allows you to measure, not guess at your signal to noise. For applications such as deconvolution 8 db to 12 db is a good starting point Stop when Quality decreases stops adding frames at the point where quality, measured in dB, decreases.

13 Check the PREVIEW PASS block for setup, turn on LIVE Start usually with highest wavelength in Pass 1 (Red laser) and progress to lowest wavelength (Blue laser). Highlight each Pass line and set individual Gains, Offsets, Pinholes, Lasers, Shutters, Detector etc. exactly the way you want to acquire that wavelength. Highlighting the next Pass line will auto save the settings for the previous pass. After setting the last Pass turn off LIVE and uncheck PREVIEW after programming. Select Clear Image> Acquire SINGLE Channel Series-Frame Lambda

14 Acquiring Z-Stack Select your objective lens. EZC1 will automatically select a predefined step size but see chart on pg. 65 & 66 for recommended step sizes based on Nyquist. Optimal Z stacks are acquired when nosepiece or stage travel against gravity. – If using RFA make sure specimen “Top” is highest positive number – Run a trial Z stack to verify if Z stack is acquired with stage or nosepiece moving against gravity. If not check settings in Configure > Microscope > or RFA – You want to set the order of acquiring the Top and Bottom slices of your Z stack so the stack starts where you finished the Z setup. (Normally this would be bottom to top) Get a good image of a representative slice within the stack while in the LIVE Mode; usually in the middle Click the two red buttons in the Z-stack tab. This will set your reference position

15 Acquiring Z-Stack Click Top and focus through the specimen just past the highest plane you wish to include in your stack. Watch for oversaturation and optimize the gain. Click Bottom (jumps to ref plane) and focus to the bottom. It is very important to optimize gain as you approach the bottom. Turn off Live. Verify correct Step Size & Step Count. (See chart on page 65 & 66) Click on the green Z-stack button. Click on the green Average button if necessary. Select View in 3D Orthogonal and click SINGLE Upon completion SAVE DATA

16 Z stacks can also be acquired by setting a reference section and assigning a specific range above and below the reference slice Turn on LIVE and focus on the reference plane and click on the two red buttons in the Z stack tab and then turn off LIVE Select the Range & Step Size required. (See following chart) The actual range above and below the reference plane and the total number of slices will be displayed Click on the green Z-stack button. Click on the green Average button if necessary. Select View in 3D Orthogonal and click SINGLE Upon completion SAVE DATA Acquiring Z-Stack - Reference

17 Viewing a Stack in 3D Select View 3D Orthagonal Place cursor (hold left mouse button down) in any of the three panes and you can scroll in XY, XZ, YX, YZ, ZX and ZY axis Adjust X, Y, and Z sliders to view through individual axis

18 Viewing a Z Stack in 2D After acquiring the Z stack select the View Tab Select 2D Image Use Z slider to scroll through stack Select animate options to play, change speed, and playback direction Checking Force Window Height to Data Document automatically fixes Live window to image size & disables Zoom slider

19 Viewing a Stack in Tiled View