Prepare to Image

Producing an Image On Screen

Collecting an Image Stack

SECTION 2 Zooming In

Sequential Collection

Viewing Collected Data

Logging Out and Shutting Down

SECTION 3
Software to open BioRad files on you computer

Parts of the microscope system.

1.      Turn on mercury light. (rocker switch-ON, press and hold IGNITE button until the “click” is heard).

2.      Start computer. Log into Windows NT. Do not start Lasersharp yet.

3.   Turn on BioRad control unit under microscope table (power switch on), wait for self-check to finish (upper green "ready-light" on scan head comes on).   Control Box
4.    Turn on laser: A.     turn key clockwise B.     press lower laser “start” button. Laser “beeps” for ~1 minute. 5.      Start Lasersharp. 6.      Log in to Lasersharp with your username and password   Left side of microscope
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Preparing to Image the Specimen

1.      Choose an objective lens. 2.      Put light-path selector knob in the “A” position. 3.      Using either white light or the mercury light, focus on the specimen and choose the area of interest. Be careful not to bleach the specimen with excessive mercury light viewing! 4.      Once specimen is in focus and oriented, close UV light shutter or turn off white light. A.      Change detector slider to “internal detectors” position (vertical arrow). B.       Rotate light path selector knob to the “C” position.

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Right side of microscope.

Producing an Image On-screen

1.    Select method from upper pull-down menu. Choose protocol  most applicable.Ie: for FITC – Texas Red double stained cells,
use the “red/green labeling” or “red/green/trans” protocols. If you need a Nomarski channel you should choose either the “red/green labeling”, or “Fluorescence/Transmission” protocol.

2.  The instrument control panel toolbox is divided into three panes: scanner control, detector control and focus motor control. 3.       Set the appropriate objective in objective box in scanner control pane (ie. 60x Oil PlanApo). 4.       Set box size = 512 x 512 5.       Check that zoom = 1 and speed  = 500 lps 6.     In the detector control pane, increase sensitivity of instrument by: A.      Opening iris to about 5 or 6mm. B.       Setting gain to about 40-50%. 7.       Set laser power to ~50%.

8.    Open a new “experiment” (on the drop-down file menu, on upper menu bar). Then, create a new subdirectory under "Experiments" in which to save new image files.

9.       Start laser with blue “star” button near top of the scanner control pane.

10.   Optimize Brightness and Contrast

A.      Adjust laser, gain and iris until brightness is optimized and the iris is set as close to optimum as possible (click iris icon on right side of screen to set optimally).

·        Do not close down iris unless increasing the laser power or gain will compensate for decreased brightness!

·        If more brightness is needed, try increasing laser power (this is a very low power laser and will NOT bleach the specimen rapidly).

·        Try to keep gain set below 33%. At 33 and above noise increases exponentially.

B.       Adjust brightness settings (with setcol loaded) until most of background is green (brightness level of zero) and structures in focus are mostly gray, with a few pixels of red (brightness = saturated (level of 256)).

Upper left pane has the setcol LUT loaded, upper right shows the green LUT, lower right shows an RGB merge of the two upper panes.

11.   Determine level of signal averaging appropriate to sample.

A.      Set Kalman in “collection filter”

B.       Leave n = stop (Ignore the Factor setting, it has no function for this filter)

C.       Start laser and watch the image, count passes as the screen updates.

D.      Stop laser when the image has smoothed sufficiently.

E.       Note the value found in step C, it will be needed when collecting a stack (below).

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Collecting  Images

1.      Set stack start and stop positions.

A.      Set collection filter to direct  (scanner control pane).

B.       Start laser.

C.       Find start position:

·        In the position box, click on the down arrow until desired location is reached.

·        Mark position by clicking on start (in limits section).

D.      Find stop position:

·        Click on up arrow until at desired location.

·        Mark position by clicking on stop.

E.       Stop laser.

F.   Set Kalman = n, where n = the value found in step 11.c in the above section.

2.       Click on the blue stack icon.

3.       In the pane that opens, select enable z series (!)

4.       Click on “start” and your stack will be collected.

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Zooming In

Note: Limit zooms to central regions of the field. If an object you want is not centered, use the stage controls to center it.

1.       Drag the zoom slider to an appropriate value (refer to paper chart on monitor). 2.       Turn on laser. 3.       Use the pan controls to center and rotate the image within the filed of view (FOV).

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Sequential Collection

A technique that prevents "bleed-through" from G to R:

1.      Adjust brightness for each channel individually in channels box.

A.      Choose one of the individual channel panes you want to collect.

B.       Adjust iris, gain, black level and laser power

C.       Choose next channel, adjust, etc.

2.      Begin collection by clicking on the stack icon.

3.      Click on the channels tab.

4.      Select sequential.

5.       Click “start”.

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Viewing collected data

1.      If not already opened, open the image file.

2.      The collected stack can be viewed manually or automatically.

- To view automatically, click on the “back & forth” icon at top of image box.

3.      Examine the data (make projections, rotations, add scale bars, etc).

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Logging Out and Shutting Down

1.      Exit LaserSharp by selecting file, then logout.

2.      Turn off laser power supply (turn key CCW)

3.      Turn off Hg-vapor power supply.

4.      Turn off control box (under table)

5.      Data is accessible through two methods: via FTP, or by burning a CD-ROM.

6.      When done copying files, logout from NT and turn off monitor. Please leave the computer running.

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Software that opens BioRad “PIC” files:

            Note: A BioRad PIC file is not a Macintosh PIC file!

• For the PC (Win 95, 98 and NT) use Confocal Assistant, free from BioRad, or from our file server. Confocal assistant duplicates most of the functions Lasersharp contains  with the exception of the morphometric functions. Shareware from Irfanview (www.irfanview.com) also works well.

• For the Mac: use NIH Image ( ftp://zippy.nimh.nih.gov/pub/nih-image) and associated macros (from ftp://rsbweb.nih.gov/pub/nih-image/documents/confocals.hqx). Graphic Converter is a shareware program from Lemke Software (www.lemkesoft.com) that some people prefer.

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