Andor Dragonfly Spinning Disk Confocal Microscope (User's Manual)

1. Power strip (#1)
2. Spinning disk module (#2)
3. (Optional) Switch on power for 'Mosaic3' photostimulation (power strip is at the back of the system)
4. Computer (#3); Login as 'lmcfuser' 
5. (Optional) Power strip (#4) for heater and CO2 supplying
6. Open Fusion. It is strongly suggested that you refresh the 'Fusion User Data' folder every single time before you start Fusion. The LMCF 'Fusion User Data' folder is in BackupDisk drive. That will clean up the settings and correct possible errors from the previous user. This is done in two steps: (1) delete the 'Fusion User Data' folder in 'Documents' folder; (2) Copy/paste back the same-titled folder from the 'BackupDisk' drive. 

(1)  Choose an objective lens:  On the touchscreen display panel of the Leica DMi8 microscope, push ‘Magnification’ icon (1 in Figure 1) to open the control panel. 

• Push the ‘eye’ icon (2 in Figure 1) to open the port for eyepieces 

• Choose the objective (3 in Figure 1) you want to use; Be aware, when you switch between the dry and oil/water objectives, you need an extra push to the objective icon to confirm the choice. Otherwise the objective does not change.

(2)  Loading a sample:  Put a sample on the sample adapter. If the objective position is high, lower it down first to avoid scratching of it when loading or moving the sample. Remember, imaging through a #1.5 glass coverglass will ensure the best image quality.

(3)  Moving the stage:  Using the joystick on the ASI stage controller to move the stage and position the sample above the objective. Toggle the button on top of the joystick to choose between faster (‘f’ shows on the LCD panel) and slower speeds (‘s’ shows on the LCD panel) (1 in Figure 2).

(4)  Lights on the microscope: two LED light controllers are attached to the microscope: one (pE-100) is for viewing samples with bright-field light; the other (pE-300) is for fluorescent samples (three LED lights are available for blue, green, and red fluorescent channels). The two controllers have push-button switches to turn the lights ON or OFF. The light intensity is adjustable. 

(5)  Bright field or fluorescence:  Push the ‘Contrast Methods’ icon (1 in Figure 3) on the touchscreen to open the page. BF, Ph, and DIC are for bright-field illumination; For best bright-field contrast, you also need to manually select a filter in the condenser to match the objective used;

Selections for the DAPI, GFP, and TxRed filter cubes are for blue, green, and red fluorescent channels separately.

(6)  Focusing:  The focus knobs on the microscope move the objective in faster (bigger knob) or slower (smaller knob) speed. On the Leica touchscreen open ‘stage and focus controls’ panel (1 in Figure 4) where you can further set up the focus knobs in ‘coarse’ or ‘fine’ modes (2 in Figure 4). A Leica SmartMove is connected to the microscope for focusing as well. The ‘coarse’ or ‘fine’ modes can also be activated by buttons on the Leica SmartMove (2 in Figure 4).

Be alert that most of the objectives on the microscope have very short working distances. It is important to avoid crashing of the objective with the sample. In this touchscreen panel, the red triangle icon indicates the current position of the lens. The gray bar on top marks the lens position where the sample should be in focus (3 in Figure 4).

(1) Fusion saves a single image or serial of multidimensional images automatically. Therefore, you need first to set up a folder on the local hard drives, which is preferred, and a file name for your image.

A basic image name should be given (1 in Figure 5). Click on the icon ‘…’ (2 in Figure 5) to open the ‘File Manager’ where to set up the folder (1 in Figure 6) and to complete the file name by optionally adding imaging date, time, channel name, or protocol label (2 in Figure 6) that are appendices to the basic image name. Then return to imaging window.

Warning: LMCF may delete users' data at anytime without prior notification. Users are responsible for the safety of their data.

(2) Open the triangle on the right side of Fusion window (3 of Figure 5) to expand the work area.

‘Channel Manager’ tab is displayed by default. However, users are not recommended to make changes in here before they know how the system works. Changes on many items in ‘Channel Manager’ will be remembered by the system and are carried on to later experiments. LMCF provides basic channel settings that are included in the 'Fusion User Data' folder for users to use (see 'Power-up and Open Fusion' section above).

(3)  Instead, open ‘Protocol Manager’ tab (step 1 in Figure 7) to set up a protocol: for image acquisition in combinations of single/multiple channels, time lapse, z-stacks, multiple positions, montage acquisition (tile scanning and stitching), and hardware autofocusing

(4) Acquired images in Fusion may be opened in Imaris, ImageJ, or Fiji for further processing.

Set up channels and single point image acquisition:

(1)   Open ‘Protocol Manager’, click ‘New Protocol’, and name it (2 in Figure 7)

(2)   LMCF channel settings are grouped in ‘CF25’, ‘CF40’, ‘TIRF’, ‘DIC’, ‘Dual Channel Acquisition’, DIC for CF25, DIC for CF40, and etc. 

In general, for the 10x and 20x dry objective lenses, the settings under ‘CF25' (confocal with 25um pinhole size) should be used while settings under ‘CF40' (confocal with 40um pinhole size) should be used for the 40x oil, 63x oil, 100x oil and the 63x water immersion objective lenses. Channels under 'CF40' can also be used with 10x and 20x lenses for brighter images.

(3)   Select channels from the preset channel pool and add them to the protocol (3 in Figure 7). It is suggested that the same camera is used for channels in one protocol.

(4)  Activate ‘Auto Map’(1 in Figure 8) to allow image displyed in disregard of image quality

(5)   Finding good exposure for each channel: Use ‘Active Channel’, start ‘Live’ mode (step 1 in Figure 9), adjust exposure time, laser intensity, and EM gain if the EMCCD camera is used (EM Gain at 200 is suggested);

It is not appropriate to set up those exposure parameters based on the display of the images; Rather, you need to read the fluorescent intensities from the structure of interest and from the background by hovering the mouse cursor over them. In general, at least 3 times (the higher the better) difference in intensity between them is desired for publication quality images. Increasing the exposure (by exposure time or laser intensity) usually increases the signal to background contrast.

(6) When a good exposure is determined for every channel, tick ‘Protocol Channels’ 

(7) Name the image to be captured and choose a location on the data drive where those images will be saved

(8) ‘Snap’ (step 2 of Figure 9): images in the protocol are then acquired and automatically saved to the designed hard drive space.

(9) In the 'Camera' section: (i) the cameras can be set to take multiple images ('Frame Averaging') and the images are averaged to generate a cleaner image. 2 or 4 times averageing is usually good. For the iXon EMCCD camera; (ii) 'Symmetrical Binning' can increase the camera sensitivity and image contrast, but at the cost of lower resolution of the images.

Multi-dimensional acquisition setup (you may only need some of the following functions)

Time Repeats:

(1) Time interval: The time between the starts of two consecutive acquisitions

(2) Total time points or total time period of experiment: These two parameters are linked to each other. Changing one also changes the other. For single point acquisition, the time point is ‘1’.

Focus Stabilization (hardware autofocusing): 

Start the function by ticking ‘Draft Stabilization Enabled’ (better to choose stabilization for ‘All Fields’) and by ticking ‘Draft Stabilization Mirror Inserted’. Both items have to be activated or inactivated together. Otherwise the system will report error.

Multi-Field Positions: 

That is to add multiple imaging positions on the sample. In a time-lapse acquisition, those positions will be re-visited at each time point. Adding (‘+’) a position in the list allows the system to memorize its coordinates, focus (x,y,z), as well as the autofocus offset ('Focus Stabilization' (see above) needs to be set up first). The saved positions can be deleted, refreshed, or re-ordered.

Z Scan Settings:

(1) Z stack range: 

• For multi-field position acquisition (see above), use ‘Center/Range’ method in ‘Scan Mode’ (1 in Figure 10); Add a ‘Scan Size’ in micrometers (3 in Figure 10); The system will mark the current focus as the central point of the entire z-stack.
• For single-field acquisition, you may use ‘Start/End’ method in the ‘Scan Mode’; Use the ASI piezo Z (2 in Figure 2) to find one end of the future z-stack, set ‘Scan Start’ (2 in Figure 10); Then find another end of the z-stack and set ’Scan End’;

(2) Step size: Choose  ‘Auto Step Size’  (4 in Figure 10) if the future z-stacks will be used for 3-dimensional reconstruction or deconvolution; if you plan to do maximum intensity projection of the z-stacks, a step size that is around 5x of the ‘Auto Step Size’ may be entered.

(3) If image from a fixed sample, it is recommended to choose ‘For each Channel, acquire all Z points’ to avoid constant switching of the channels at each Z position, hence faster image acquisiton.

(4) Start 'Acquire' 

Montage: 

This is an image tile-scanning procedure. It involves: (1) marking a region and (2) (optional) setting up a focus correction map. Enable the 'Montage function' (figure 11). Also, activate ‘Apply Stitching’ under the ‘Image Procession Options’ section near the bottom of the window to have the image tiles stitched right after all the tiles are acquired.

(1) Setup of the scanning area. There are two ways to do it: 

(i) 'Tiles': adding numbers of tiles in x and y axis

(ii) 'Perimeter': this is the preferred method (figure 11 and figure 12).

• First, generate an overview of the tissue after adding tiles in width and height. Clicking ‘WxH’ icon to start acquring images in a spiral manner. After the overview is created, clicking the ‘WxH’ icon again will add one additional row and lane of tiles on each side of the overview image.
• In Montage setup, ‘+' to turn on 'Draw Region for Montage’ (figure 11). Left-clicking the mouse to mark the border of a region you wish to image on the overview image; Right-clicking the mouse to complete the job. The program then lays minimal number of tiles that cover the desired area on the overview image. The size of the region can be adjusted when necessary (figure 13).

• Please note: before adding a new region, an existing region and its associated focus map should be deleted (figure 11 and figure 14).

(2) 'Focus Map': This allows focus correction while acquiring tiles from an uneven or slanted tissue section.

Important: Only use microscope focusing devices to set up focus map because ASI piezo Z is not supported for this job.

(3) Turn on ‘Enable Focus Map’ and use ‘Global’ mode (Figure 14).

(4) ‘Enable Image Click’: On the overview image, adding focus map points (FM) in the montage region (figure 13). It is best that the focus map points are evenly distributed and are not missed in the perimeter of the region.

(5) Double-click on one focus map point in ‘Focus Map’ section to move the stage to that specific position. Start ‘Live’ and adjust focus. When best focus is found, click ‘Confirm Positions’ to update the focus. The stage will then automatically move to the next position. Repeat this process until all focus map points are confirmed.

(6) (Optional) If Z-stacks are acquired around the image tiles, go to 'Z Scan Setting' section, use ‘Center/Range’ method in ‘Scan Mode’ (1 in Figure 10); add a ‘Scan Size’ in micrometers (3 in Figure 10) and 'step size'; Piezo Z can (and is preferred to) be used for this operation.

(7) Name the images to be captured and choose a folder on data drive to automatically save those images; Click ‘Acquire’ to start the multi-dimensional acquisition, which is followed by stitching of the tiles.

(8) The overview image, individual image tiles, and stitched image tiles are all separately saved to the folder.

Super-Resolution Radial Fluctuations (SRRF, pronounced as ‘surf’) :

(1).  Use the 63x oil or 100x oil objective to achieve the best resolution

(2).  Set up a protocol that uses the iXon EMCCD camera

(3).  Set image acquisition at 2x magnification for the EMCCD camera

(4).  Choose a small region (ROI: region of interest, see 2 in Figure 8) for fast imaging and smaller file size

(5).  Use a short exposure time (for example less than 50 ms). Loss in fluorescent intensity due to the short exposure time can be compensated by increasing the laser power. But make sure the structure of interest is not oversaturated. As a matter of fact, relatively lower fluorescent intensity generates better SRRF images with less artifacts in Fusion.

(6).  Turn on ‘SRRF-Stream’ (in Channel Manager and under 'iXon EMCCD camera')

(7).  Set ‘SRRF Frame Count’ between 50-150;  ‘SRRF Radiality Magnification’ to 4x;  ‘SRRF Ring Radius’ to around 1 (a smaller ring radius produces better resolution, but is prone to more artifacts); Use ‘mean’ for ’SRRF temporal Analysis’

(8).  Click ‘Snap’ for single SRRF image acquisition; Or click ‘Acquire’ for multidimensional image acquisition with SRRF.

(1).   Lower down the objective and very gently wipe off the oil or water if an oil or water objective has been used;

(2).   Check the online booking calendar and

A.  If next user comes to use the microscope in one hour, close Fusion, and leave the system on;

B.  If nobody will use it in the next hour, turn off the heater if it has been used (switch #4), close Fusion and shut down the computer from Windows Start Menu, switch off #2 and #1.

C. After the Fusion closes, the 'Fusion User Data' folder is updated with a user's specific setups. The folder can be saved in the user's space and re-used for future experiments (see 'Power-up and Open Fusion' section above).