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3.10 Basic Confocal Operation - Time Series |
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Two different approaches to acquiring time lapse series are described.
Firstly 3.10.1 describe the automatic method. One first sets up the image
then sets the confocal to periodically sample the image. At the end of the
run the images are saved as a single multi-paged tiff format image
file. In the second method 3.10.2 one setups up, then scans a single
sliced, saves it as a unique name, and then waits until the sample period has
passed before repeating the process. This last methods allows inspection
of the cells focus, which can be readjusted if necessary before acquiring the
image. In both methods the file(s) have to be assembled into a form that
can be viewed as a movie.
This section is still in preparation. Errors and omissions may be present!
3.10.1.
Acquiring a time lapse series using the build in Leica time lapse acquisition
mode
- Start Confocal System
- Find area to be scanned and set up acquisition parameters (i.e. laser,
pmt, pin hole, zoom, accumulation, sections to 1, etc.)
- From the pull down menu select "settings" ->
"time lapse/photo bleach"
- Define parameters for acquisition. e.g. choose parameter set 1
definitions
- set "number of frames" - set to number of
time points to acquire (set to more than than required and use stop to
finish
- set "accumulation" - number of scan to accumulate (average) per time point.
e.g. 4
- set "frame duration" - time between start of each frame, i.e. the time
period. e.g. 120 seconds
- set "delay interval" to zero
- set "repeat interval" - set to 1.
- press "Finish"
- Press "series" button to open scanner and be ready to start
acquisition
- Press "1" to start time lapse
- When time lapse is finished may have to un-depress
"stop" manually before saving images
- Save image data using "file -> save_as" ("save ->
selected" is not reliable)
- Time each frame was acquired and other information may be read with
PhotoShop (note that PhotoShop will only display 1st image in a series) or extracted
frame
images using tiffsplit4.exe.
- Convert bitmap image series into a single Biorad pic
format image file
- Display pic image series using Confocal Assistant
3.10.2.
Acquiring a time lapse series by manually saving each reading and using a stop
watch
- Start Confocal System
- Find area to be scanned and set up acquisition parameters (i.e. laser,
pmt, pin hole, zoom, accumulation, sections to 1, etc.)
- Press series to acquire the image
- Save the image using file -> save as and a unique
file name, preferable one that denotes the order of the image in the time
lapse series.
- Repeat process until desired time period has elapsed
- Convert each save image from tiff format into bitmap
format using tiffsplit4
- Convert bitmap image series into a single Biorad pic
format image file
- Display pic image series using Confocal Assistant
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