Using ImageJ

Introduction

This is only a terse note on what is needed to read the PIC files acquired on the Bio-Rad MRC 600 and Radiance 2000MP LSCM. There are excellent manuals and tutorials available on the NIH ImageJ website and the ImageJ Documentation Portal.

Components needed

ImageJ (download from ImageJ website)

You will need the Java runtime library to use ImageJ. Java is often installed as a standard add-on for some operating systems but not all. Some packages of ImageJ are bundled with certain versions of Java. Please read carefully the description provided in the download page to determine what you are getting, and the installation instructions on how to configure the software. Assigning an appropriate amount of RAM to ImageJ is a sensible thing to do. In general, it should not be more than 75 % of the installed amount of RAM.
basic plugins (download from ImageJ website, look under the same category on the ImageJ website as listed below for the plugins)
Input/output
- Open Biorad Z-series: install also "Handle Extra File Types" will allow using either File | open or dropping the file on the ImageJ window or icon to open Bio-Rad PICs
useful plugins
Analysis
- Sync Measure 3D: allow measurements across all selected windows
Color
- LUT Panel: load and unload LUT
- Biorad_Aligner from the WCIF plugin collection: splitting the dual channel images from MRC 600
Stack
- Biorad Zproj: do Z projections for the stack at each time point and then create a stack from the projections to represent the time series (this plugin is not found on the ImageJ website and the source is unknown; it is available here only for facility users)
More Sites with Plugins
- VolumeJ: create 3D images and movies from stacks (manual and a useful tutorial on indexing)

Useful information

the Bio-Rad PICT file used by LaserSharp stores calibration information and acquisition parameters viewable in ImageJ which also applies the calibration information to the displayed image

from Image | Show info… (notes at the end of the file, annotations added are in [ ])

SCALE_FACTOR = 20.000000 [system specific scaling factor]

LENS_MAGNIFICATION = 60.000000

PIXEL_BIT_DEPTH = 8

Z_CORRECT_FACTOR = 1.000000 -10.198988 [mountant:immersion medium refractive index ratio, corrected aspect ratio; objective specific]

PIC_FF_VERSION = 4.5

RAMP1_MIN = 0

RAMP1_MAX = 255

RAMP_GAMMA1 = 1.000000e+000

RAMP2_MIN = 0

RAMP2_MAX = 255

RAMP_GAMMA2 = 1.000000e+000

RAMP3_MIN = 0

RAMP3_MAX = 255

RAMP_GAMMA3 = 1.000000e+000

AXIS_0 004 0.000000e+000 1.000000e+000 Intensity

AXIS_2 001 0.000000e+000 9.883333e-002 Microns [?, pixel size]

AXIS_3 001 0.000000e+000 9.883333e-002 Microns [?, pixel size]

AXIS_4 001 2.025000e+001 -1.008000e+000 Microns [Z start position, step size]

AXIS_9 011 0.000000e+000 3.000000e+000 RGB channel [number of channels: 0/1, 2, 3]

METHOD_NAME = Green/Red/FaRed (pang)

GUID = {E2071B9E-847A-4231-BF74-CE1BFE7B6805}

SETTINGS_COUNT = 5 [many lines omitted, parameters of the channel settings e.g., power level, iris, gain, etc…]

…

INFO_IS_RADIANCE = 1

LAST_SETTING_IS_LINE_BY_LINE = 0

LAST_SETTING_IS_SIMULTANEOUS = 0

INFO_OBJECTIVE_NAME = 60x Oil 1.40 NA [lens name]

INFO_OBJECTIVE_MAGNIFICATION = 60

INFO_OBJECTIVE_ZOOM = 2

INFO_PAN_COORDS_X = -0.05 [pan x, corrected for zoom]

INFO_PAN_COORDS_Y = -0.025 [pan y, corrected for zoom]

INFO_SCAN_ROTATE = 20

INFO_RASTER_RATE = 500 [line frequency]

INFO_FRAME_RATE = 0.0440733

INFO_PIXEL_DWELL = 1.30208e-006 [pixel dwell time]

INFO_FILTER_NAME = Direct [acquisition filter]

------------------------

[summary information]

Title: 20050527grfp60x2-0z01_raw01.pic [filename]

Width: 101.21 Microns (1024)

Height: 101.21 Microns (1024)

Depth: -20.16 Microns (20) [height of stack, number of slices]

Voxel size: 0.10x0.10x-1.01 [x, y, z dimensions in µm]

Resolution: 10.118 pixels per Microns [0.099 µm per pixel]

Bits per pixel: 8 (color LUT)

Frame: 1/20 [current slice position of the image displayed: slice 1 of 20]

Frame interval: 22.68947 seconds [during acquisition]

No Threshold

Magnification: 0.75 [current display magnification]

Uncalibrated

No Selection
the Bio-Rad PICT file used by CoMOS stores calibration information and acquisition parameters viewable in ImageJ which also applies the calibration information to the displayed image (the appropriate unit e.g., micron, can be added by doing Analyze | Set Scale…)

from Image | Show info… (notes at the end of the file, annotations added are in [ ])

Number 13 of 13

TS60XB10.PIC 27-Oct-104 8:24:53 PM Zoom 10.0 Kalman 2, 4.0 m

SCALE_FACTOR = 16.500000

LENS_MAGNIFICATION = 60.000000

AXIS_0 4 0.000000e+00 1.000000e+00

AXIS_2 257 -1.056000e+01 2.750000e-02 [?, pixel size in µm]

AXIS_3 257 -7.040000e+00 2.750000e-02 [?, pixel size in µm]

AXIS_4 1 4.000000e+00 -5.000000e-01 [Z start position, step size]

TS60XB10.PIC 27-Oct-104 8:24:57 PM Zoom 10.0 Kalman 2, 3.5 m

TS60XB10.PIC 27-Oct-104 8:25:01 PM Zoom 10.0 Kalman 2, 3.0 m

TS60XB10.PIC 27-Oct-104 8:25:05 PM Zoom 10.0 Kalman 2, 2.5 m

TS60XB10.PIC 27-Oct-104 8:25:09 PM Zoom 10.0 Kalman 2, 2.0 m

TS60XB10.PIC 27-Oct-104 8:25:12 PM Zoom 10.0 Kalman 2, 1.5 m

TS60XB10.PIC 27-Oct-104 8:25:16 PM Zoom 10.0 Kalman 2, 1.0 m

TS60XB10.PIC 27-Oct-104 8:25:20 PM Zoom 10.0 Kalman 2, 0.5 m

TS60XB10.PIC 27-Oct-104 8:25:24 PM Zoom 10.0 Kalman 2, 0.0 m

TS60XB10.PIC 27-Oct-104 8:25:28 PM Zoom 10.0 Kalman 2, -0.5 m

TS60XB10.PIC 27-Oct-104 8:25:32 PM Zoom 10.0 Kalman 2, -1.0 m

TS60XB10.PIC 27-Oct-104 8:25:36 PM Zoom 10.0 Kalman 2, -1.5 m

TS60XB10.PIC 27-Oct-104 8:25:40 PM Zoom 10.0 Kalman 2, -2.0 m

------------------------

[summary info]

Title: TS60XB10.PIC [filename]

Width: 21.12 (768)

Height: 14.08 (512)

Depth: -6.50 (13) [height of stack, number of slices]

Voxel size: 0.03x0.03x-0.50 [x, y, z dimensions in µm]

Resolution: 36.364 pixels per [CoMOS omitted the unit, it should be micron]

Bits per pixel: 8 (grayscale LUT)

Slice: 1/13

No Threshold

Uncalibrated

No Selection

Examples

Cell counter plugin

This is a simple block diagram for some usage of this plugin.

Things to watch out:

do not initialize more than once, you will have problem reloading your saved markers; e.g., if the title bar reads counter window-counter window-filename, you have initialized it twice, and that may be the filename recorded in the counter xml file
clicking reset will erase all the marks thus setting all counters to zero