2D Time lapse Core - National University of Singapore...Single point Time lapse plus ZDC 1) After...

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CBIS Confocal Core Short Manual on FV3000, of 18

Time lapse, 3D Imaging and Stage Scan

2D Time lapse

1) Make sure both [Time] and [Z] on [Series] Tool Window are set to “OFF” position.

2) Press the [Live] button on the Live Window. Pressing the [Livex2] button or the [Livex4] buttonwill display the live image whose repeat interval is reduced by substractive scan. This function isuseful when searching the cells or observing with moving the focus.

3) Rotate the fine knob until you get a bright and clear image of your specimen on the computerscreen.

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4) Once you have found the focus of your image, click “Register” under Origin. You should find that the current position becomes 0.

5) Next click “Stop” to stop scanning.

6) You may finalise all parameters such as scan size, averaging, scan time. Check the Total Scanning Time to know how long you need to complete a single time point.

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7) Select “ON” in [Time] on [Series] Tool Window.

Set the interval to acquire the image in [Interval] on [Time Lapse], the unit for [interval] is in seconds. For example if you want to run the scan for 5 minutes, you will have to input a value of 300 under [Interval]. Also, if you attempt to set the time shorter than the time displayed in [Scan] (time taken for acquiring 1 cycle image) in [Interval], ‘FreeRun’ appears. In this case the interval to acquire the image is the time displayed in [Scan]. Next, set the number of image acquisitions in [Cycle].

8) Create folder and file name, then press “LSM Start” in [Normal] tab in [Acquire] Tool Window to start acquisition. Click “Series Done” when the scan is completed.

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Z stack (3D imaging)

1) Select “ON” in [Z] on [Series] Tool Window.

2A) Select ‘Start/End’ in [Motor] on [Z section]. Next go to Live 2/Live 4.

2A.1)Set Z stacks series with changing the Z position by rotating the focus knob of U-MCZ to register the following positions.

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2A.2) Press the Register button in [Origin] at the Z position when the image is at it’s best focus. You should find the current position becomes 0.

2A.3) Press the Register button in [Start] at the Z position to define the bottom end of the specimen volume.

2A.4) Then go on to press the Register button [End] at the Z position to define the top end of specimen volume.

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2A.5) Always ensure that it is a negative value at the Start position and a positive value at the End position. So that 3D scan will be carried out by moving the objective upwards.

Otherwise press the Start<-> End button to reverse the start position and the end position.

2A.6) You can either select “Optimize” or set the [Slices] and [Step Size].

2B.1) Other than selecting ‘Start/End’ in [Motor] on [Z section], you can select ‘Range’ to do a 3D volume scan. Input a value under “Range”.

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2B.2) Make sure it’s a negative value at the Start position and a positive value at the End position. Otherwise press the Start<-> End button to reverse the start position and the end position.

3) Next click “Stop” to stop preview.


Refer to the above steps to do time lapse plus Z stack

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Single point Time lapse plus ZDC

1) After placing your sample on the microscope stage, press “START” button under Focus search on the touch panel controller. It will bring the objective close to the top surface of the coverslip. If successful, you will hear 2 beep sound for silicone oil lens, and hear 1 beep sound for the other lens.

If unsuccessful, you will hear 3 beeps.

Reasons for 3 beeps and their solutions:

- DIC prism has been engage in the light path, remove it from the light path. - The coverslip position may not be in the search zone. The optimum Z limit setting is usually set

at 2500, so specify the Origin coordinate of Z below and closer to the Z limit setting.

- The coverslip’s thickness is not supported (thickness should be 0.17mm).

2) Select ‘ON’ in [Time] on [Series] Tool window.

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3) Press the [Live] button on the Live Window. Pressing the [Livex2] button or the [Livex4] button will display the live image whose repeat interval is reduced by substractive scan. This function is useful when searching the cells or observing with moving the focus.

4) Rotate the fine knob until you get a bright and clear image of your specimen on the computer screen.

5) Once you have found the focus of your image, click “Register” under Origin.

Fine Knob

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6) Next click “Stop” to stop scanning.

7) Set [ZDC DM] in [Z Drift compensator Control] on [Microscope] Tool Window to “In”.

8) Press [Start continuous ZDC] button in [Z Drift compensator Control] in [Microscope] Tool window. The Z position is adjusted automatically so that it is focused continuously at the focused Z position when this button was pressed.

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You may need to widen the search zone if an error message appears.

9) Set the interval to acquire the image in [Interval] on [Time Lapse]. If you attempt to set the time shorter than the time displayed in [Scan] (time taken for acquiring 1 cycle image) in [Interval], ‘FreeRun’ appears. In this case the interval to acquire the image is the time displayed in [Scan]. Next set the number of image acquisitions in [Cycle].


Note: If you accidentally adjusted the focus during the acquisition, do note that ZDC will pick up the latest defined focal plane and complete the whole time lapse

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Time image plus Z stack using MATL

1) Select “ON” in [Z] on [Series] Tool Window.

2) Select ‘Start/End’ in [Motor] on [Z section]. Next go to Live 2/Live 4.

2A.1)Set Z stacks series with changing the Z position by rotating the focus knob of U-MCZ to register the following positions.

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2A.2) Press the Register button in [Origin] at the Z position when the image is at it’s best focus. You should find the current position becomes 0.

2A.3) Press the Register button in [Start] at the Z position to define the bottom end of the specimen volume.

2A.4) Then go on to press the Register button [End] at the Z position to define the top end of specimen volume.

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2A.5) Always ensure that it is a negative value at the Start position and a positive value at the End position. So that 3D scan will be carried out by moving the objective upwards.

Otherwise press the Start<-> End button to reverse the start position and the end position.

2A.6) You can either select “Optimize” or set the [Slices] and [Step Size].

2B.1) Other than selecting ‘Start/End’ in [Motor] on [Z section], you can select ‘Range’ to do a 3D volume scan. Input a value under “Range”.

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2B.2) Next click “Stop” to stop preview.

3) Set [ZDC DM] in [Z Drift compensator Control] on [Microscope] Tool Window to “In”.

4) Click on this arrow, select ‘Map’.

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5) Register the lst single point by clicking on the icon below. The description line for this lst point will be added to the bottom of the ‘Map’ tab as shown. Any setting changes you made to this point can be updated by clicking on ‘update configuration’.

6) Next move to a new point on the specimen, find the focus for this point, and click ‘register’ origin. Then repeat step 6.

7) Repeat Step 7 for a few more points.

8) Once you have define all the points, you will the list of points you have registered.

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9) If you want a stitching scan to cover all the points, select the icon below to draw an ROI over the

points that you have registered previously.

10) After you have drawn an ROI, delete all the individual points that you have previously registered by clicking line description for these individual points and click on the delete icon below. Leaving behind only a description line for the ROI as shown below.

11) Once done, set cycle interval and no. of cycles below.

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12) Under Acquire tab, select MATL, create a folder and file name, then click ‘Start’ to start the acquisition.

2D Time lapse Core - National University of Singapore...Single point Time lapse plus ZDC 1) After...

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