Introduction to Live Data Mode - Capturing images for SRRF

I was asked to help set up an acquisition for SRRF on the SP5 the other day. It turned out that the method of image acquisition was one that was unusual for the SP5 (or any confocal). We had to acquire 100 time points in as short at time as possible and repeat that process for the slices of a stack and three fluorescent channels. So the acquisition needed to be done in the order XYTZC when the normal procedure would be to acquire images in the order XYCZT. To acquire the images in that order I needed to use a module in the SP5 software called Live Data Mode (LDM). LDM allows you to program different acquisition phases as separate Jobs. Each Job can have different acquisition parameters; so for example, one job could use the 488 nm laser to take stacks of the whole field while another job only illuminates an ROI at a single plane. Sequences of Jobs can be looped so patterns can be repeated again and again. I’m going to use this particular case to introduce and give a flavour of LDM.

You can either start setting up your imaging parameters (laser power, channels, etc.) before you open LDM or after. It doesn’t seem to matter that much. If you’ve created some parameters before opening LDM then these will be copied into Job 1.

Click on the banner in the top left of the LASAF user interface and select Live Data Mode.

In the LDM interface you will see that the current imaging parameters have been copied to Job 1. In my case I had not set any imaging parameters so I needed to do that. To image 100 frames before changing Z slice I needed to use the XYTZ mode. I needed speed so I set the scan format to 512 x 64, selected bidirectional mode and changed the scan speed to 1000 Hz. This gives me a minimum frame rate of 35 ms using the conventional scanner. Note that the resonant scanner can also be used for this kind of acquisition would certainly be better if you want to scan a larger field of view than me.

I set up the Z series at this stage because it makes things easier later on.

To set up multiple channels I would normally use Sequential mode but in this case that won’t work because I need to capture 100 time frames before switching channels, and Sequential always switches channels before time. The solution is to create different Jobs in LDM for different channels.

Click Add and select New Job. Job 1 will be duplicated and called Job 2. You can change the parameters of Job 2 without affecting those of Job 1, so a different laser and detector can be used for a different channel.

Note that all the parameters of Job 1 will be copied, including the absolute Begin and End position of any Z series. If I set up the Z series in Job 1 after creating Job 2 I have to manually enter the Begin and End in the second Job to match the first, so in this particular case it was more sensible to set the Z series up before creating Job 2. This is also true of any additional Jobs created. I have not discovered a workaround to this yet but that doesn’t mean there isn’t one.

Clicking Live will repeatedly scan the specimen with the settings of whichever Job is currently highlighted whereas clicking Start Record will acquire a one-off image using the highlighted Job. Clicking Start Pattern will execute all the Jobs in the sequence (called a Pattern in LASAF).

In my case I only needed the Pattern to be executed once but in other cases it may be necessary to loop the patterns a number of times. To create a loop, hold down Shift and select the Jobs that you want to include in the loop. Then click the loop icon to create a new loop. You will have to define the number of times you want the Pattern to loop.

It may also be necessary to insert a Pause into the Pattern. For example, you may want to execute a loop repeatedly as part of a time-lapse but have a delay between each execution. To insert a Pause, select the Job immediately after where you want the Pause to be, click the Ins button and select the Pause item.

You can delete a Job or remove it from the Pattern by right-clicking the Job and selecting what you want to do from the menu. You can also do this with a loop by clicking on the loop itself and right-clicking.
Clicking on different Jobs usually causes the hardware to immediately change configuration but you may only want to view and edit a Job without changing hardware settings. The button below disconnects the software from the hardware so you can edit a Job without making any immediate changes to hardware.

Jobs and Patterns can be saved and recalled

You must be very careful when recalling saved Jobs and Patterns because the absolute Begin and End of the Z position is stored, so reloading Jobs or Patterns may cause the objective lens to move to an entirely incorrect focus position, potentially damaging specimen and objective lens