AnalySIS Usernotes for the Fluorescence AX70 Microscope
March 2007
Note: The AnalySIS software is the same on both the Fluorescence BX50 and AX70
microscopes, however there are some camera specific differences in the format of the Image
Preview and Camera Control panels. The rest of the program is identical on both systems.
Turn on the camera, then open Analysis software.
A Hamamatsu DCAM message will appear (The version of DCAMAPI.DLL that is installed
has not been tested ……..)
Click “OK”
The software will appear as shown below, with the “Image Manager” and blank “Image 1”
showing. (These viewing options are selected in the “Window” menu).
Acquire Snapshot Camera Control
(preview) (Capture) (exposure time)
Image
Manager
The current camera configuration will be displayed in the bar at the bottom of the screen as
shown below;
Camera Configuration
Three options;
• Focus
• 16-bit
• 8-bit Æ RGB
1. CAMERA CONFIGURATION
Choose your desired “Camera Configuration” by right-clicking (button on the right-hand-side) the mouse
on the camera configuration icon in the bar at the bottom of the screen. Use the mouse to select your
desired configuration.
Camera Configuration
Three options;
• Focus mode
• 16 bit monochrome
• 8 bit monochrome (Æ RGB)
There are three configuration formats to choose from, your choice will depend on your sample and what
you want to do with your resulting images. (The same three configuration formats are in operation in the
software on both the fluorescence AX70 and the fluorescence BX50 microscopes). If you are unsure of
what to use then always use the 16-bit camera configuration.
The “Focus” camera configuration should only be used as a focus aid, and is not recommended for
taking images (it is set to a lower resolution). If your samples are quite bright, you may not even need to
use this focus configuration. This camera configuration is useful for focusing dim specimens, as it avoids
the delay of otherwise waiting for a longer exposure time when trying to set the focus of your specimen
(by applied a process called “binning” which reduces the image resolution but increases the brightness,
and by applying an “Autogain” to the image). You will need change to the 8-bit or 16-bit configuration to
capture a full resolution image. (On the AX70 you can close out of the preview image, on the BX50 you
will need to capture a (dud) image in order to then change camera configurations). You can determine
the approximate exposure time that will be required whilst you are in the “Focus” configuration by using
the live histogram in the preview mode. As a guide you will need approximately 4x this exposure time for
image capture in the “16-bit” or “8-bit” camera configuration.
The “16-bit” camera configuration is the recommended configuration for acquiring images
(although for some purposes people may prefer the 8-bit configuration). Images captured with this
configuration can be subsequently converted to 8-bit when saving by ensuring the “Convert 16-bit to 8bit” option is selected in the “Save” options. (Alternatively you can deselect this option if you wish to
keep your image in 16-bit format). This conversion process is uniform across all images, and therefore
images taken with the same exposure time will be directly comparable. If you want to subsequently do
any image analysis, or obtain any quantitative data from your images, or be able to make direct
comparisons between images (for example, negative controls and labelled specimens) then you MUST
use this configuration. (If you want to colourise or make merged images in the AnalySIS program, this
needs to be done with 8-bit images. You can still do this with images taken in this format, provided you
have saved them with the convert 16-bit to 8-bit option, but you will need to reload the saved 8-bit image,
as the image in the image manager will still appear as a 16-bit image).
The “8-bit (ÆRGB)” camera configuration is convenient for subsequently performing quick onscreen
merged images. However, you should know that images taken with this format are no longer
suitable for quantitation or direct comparison, even if you have used the same exposure time.
This is because the conversion process which is applied to convert your image from 16-bit to 8-bit during
the image capture in this configuration is not uniform. It converts to 8-bit according to the intensity range
of your image, and therefore the process is different from one image to the next, hence images captured
in this way are not comparable. Because of the different way that this conversion is done, this
configuration may be useful for enhancing otherwise dim images, so long as you only need a pretty
picture and do not need to subsequently use the image intensity information for quantification or direct
comparisons between images.
Confused?
If you are unsure then always use the 16-bit camera configuration.
If you cant remember what camera configuration you used to capture a given image, you can look this
up in the “Image Properties” associated with that image. Right-click the mouse on the image in the
image manager to open the Image Properties, and the Camera Configuration is shown under “Channel”.
2. IMAGE PREVIEW (Specific to Hamamatsu Orca Camera on Fluoresence AX70 Microscope)
Click on the so-called “Acquire” icon to acquire a live preview image, enabling you to adjust the focus of
the specimen and the exposure time. A live “Histogram” will also appear and a “Sharpness Monitor”
may appear (if selected in the “Camera Control” dialogue).
Click on the “Camera Control” icon to also display the exposure time dialogue to adjust the exposure:
Sharpness
Monitor
display on/off
Calculate Exposure Time:
NOTE: This is not useful for
fluorescence images.
Works in conjunction with the
“Target Intensity”.
Adjust exposure time here
according to the intensity of
your image as displayed in
the histrogram.
The intensity of your image
should fall within the range of
0 to 4095. If it exceeds 4095
it is saturated, and you should
reduce the exposure time.
Sharpness Monitor
(may be a useful aid
for adjusting the focus
of your specimen)
Adjust the exposure time using the “+” and “-” buttons (changes exposure time by 2- or 5-fold, do not
go beyond 10 seconds) or by adjusting the actual exposure time displayed.
The live Histogram will help you determine the appropriate exposure time. The intensity range shown in
the histogram is based on a 12-bit image, and shows a range of intensity values from 0 to 4095.
If you are in the “Focus Mode” camera configuration then there is a gain display on, so you will need to
either use the histogram to determine an appropriate exposure time (adjust the exposure so that the Min
and Max intensity value for your image falls within this range of 0 to 4095), or determine the exposure
time empirically when you change to another mode for image capture. As a guide, you will need 4x the
exposure time in the 16-bit or 8-bit configuration compared to the time determined in the focus
configuration.
If you are in either the “8-bit” or “16-bit” camera configuration then this histogram wont show the
maximum pixel value, but you can still use the mean value and the histogram itself as a guide.
If you image is difficult to focus because it requires a long exposure time, try using the “Focus mode
camera configuration.
3. IMAGE CAPTURE AND SAVE (using the 16-bit and 8-bit camera configurations)
To acquire your image, click on the “Snapshot” icon
This will capture your image as a 1280x1024 pixel greyscale image, and a series of autoprompts
will appear;
Save Image?
Yes/No
Set Magnification
Choose the magnification of the objective lens you used and click OK
(or click cancel to ignore this prompt)
Image Information
You can rename your image here or below (leave file name blank).
Renaming your image here renames the image name as it appears in
the “Image Manager” and the blue title bar above open images in
AnalySIS.
You can enter comments about your image, these comments are only
seen.
Save Image As
Rename as desired and save as a tif file, either directly to a USB
memory stick, or to your own folder within your lab folder in the
AnalySIS Images Folder on the desktop.
For the first save you do, click on “Options” to check what is selected;
If you have captured your images using the “16-bit” camera
configuration, you will most likely want to ensure the option “convert
16-bit to 8-bit” is selected. (This is a uniform conversion and maintains
the quantitative integrity of your images, see Section 1. Camera
Configuration).
If you have captured your images using the “8-bit” camera
configuration, then the “convert 16-bit to 8-bit” option is irrelevant.
You will most likely want to ensure that “burn overlay into image” is not
selected (unless you want overlays or the scalebar permanently burnt
into your image – see notes on “Measurements” and “Scalebar”).
Save Options
As well as being saved, the image will default to being displayed in first position of the “Image
Manager” list of images in the left hand side. Subsequent images and any image processing will
be allocated to subsequent positions in the Image Manager unless other positions are selected.
--------------------------------------------If you don’t save your images straight away using the above autoprompts (ie if you click “No”), your
images will still be temporarily stored in the “Image Manager”.
To save later, simply go to “Save as” in the “File” menu (in the “Options” check that “burn overlay
into image” is not selected unless this is what you want).
To set the magnification later, click on the “Set Magnification” icon in the Main Tool Bar
To view / add / edit “Image Information”, right click on the image in the “Image Manager”
--------------------------------------------Important Reminders: Do NOT save images to anywhere else on the computer other than the
“AnalySIS Images” folder. Images saved to here should be deleted as soon as possible once you
have backup up your images to your own computer. This computer is not to be used for any
purpose other than image acquisition and image processing.
4. ADDITIONAL FEATURES OF ANALYSIS
The following pages describe in brief how to work around in the AnalySIS program and how to
perform some basic tasks, including image viewing options, adding a scale bar, obtaining pixel
intensity values and histograms, creating coloured images and image overlays, making
measurements. For further information, refer to the AnalySIS instruction book available in Room
4E421 or consult Jen Clarke, ex66454 [email protected] .
Measurement Tool Bar Display
Pixel Map
RGB Colouriser
Intensity Adjustment
Set Magnification
Fullscreen View
Scale Bar Display
Main Tool Bar
Viewport
Manager
(select/deselect
in Windows menu)
Image Display Options
(Image Tool Bar)
Image Buffer
Displays up to 25 images
In List or Gallery Mode.
Displays measurements
and statistics
In measurement mode
(see below)
Image Buffer
Display Options
Measurement Tool Bar
4.1 IMAGE MANAGER
The Image Manager is displayed on the left-hand side of the screen (assuming that in the
“Window” menu “Image Manager” is selected) and shows the images (as a list or gallery) or
measurements as they are collected. If you choose not to save your images as you go this acts as
a temporary save. Each image captured will be allocated to a sequential position in the Image
Manager. The Image Manager holds up to 25 images, and once full will rewrite over pre-existing
images (so make sure you have saved your images before exceeding 25 images).
4.2 IMAGE DISPLAY OPTIONS
You can view you images (preview and captured images) at full screen size via the “Full Screen”
icon in the main tool bar.
The Image Toolbar operates as follows;
Toggle the display between the selected image and a panel of images.
Choose the layout of multiple images on the screen.
Choose the size your image is displayed at.
The scalebar is “smart” and adjusts accordingly.
Multiple images can be linked for zooming in and panning around, a useful feature for comparing
more than one fluorescence label in the same field of view. To link multiple images, display the
desired images in the viewport and select the ones to be linked by holding the shift key and clicking
on the blue bar at the top of the image.
4.3 SCALEBAR
You can select/deselect the display of a scalebar on your image via the icon in the main toolbar.
The scale bar is only correct if you have told the program which objective lens you are using (“Set
Magnification”). This comes up as one of the autoprompts upon capturing an image. To “Set
Magnification” on an unsaved or preview image select the magnification corresponding to the
objective lens you are using in “Set Magnification” in the “Image” menu.
In the current configuration of the software the scalebar is only visible in the AnalySIS program. It
does not display when you open your image in another program. If you want the scalebar
permanently burnt into your image, select “Draw into overlay” in “Scalebar” in the “Image” menu
(do not select the “burn into image” option here), and, when saving your image, in the “Options”
select “burn overlay into image”.
4.4 PIXEL MAP AND HISTOGRAM
Selecting the display of the pixel map (via the icon in the main toolbar), and clicking on “Move”
enables you to see the pixel intensity at the position of the cursor in image and the intensity of
surrounding pixels (move the cursor around the image with the mouse).
A histogram of the pixel intensity is available in the “Measure” menu, as well as the live histogram
that appears during the image preview
The Pixel Map and the Histogram enable you to check that your image is not oversaturated.
4.5 COLOURING AND OVERLAYING IMAGES
To colourise a single image, or to create a coloured overlay of up to 3 images, click on the RGB
Colouriser icon in the main tool bar to display “Combine Color Separations”.
Allocate the desired colour to the image source number (the source
number is the numbering system of the images in the Image
Manager), and tick to apply the colour. Configure for a single image
(only one colour ticked), or for an overlay of up to 3 colours, and
click ok to generate the colour image.
The intensity of the image or the intensity of each colour within an overlay image can be adjusted
using the Intensity Adjustment icon in the main tool bar.
4.6 VIEWPORT MANAGER
The Viewport Manager (selectable in the Windows menu) can be used in “Navigator” mode or
“Magnifier” mode (respective icons on the left of the Viewport Manager mini screen.
In “Navigator” mode, when you zoom in and pan around an image, the Viewport Manager displays
the location of the zoomed in region with respect to the whole field of the image.
In “Magnifier” mode, the Viewport Manager displays a zoomed in view of wherever the cursor is in
your image. The degree of zoom can be controlled by the zoom in/out icons on the left of the
Viewport Manager mini screen. The Magnifier mode is particularly useful as an aid to position the
cursor accurately at the edges of objects when making measurements.
4.7 MEASUREMENT FUNCTIONS
This version of the AnalySIS software includes a range of measurement functions, including
counting of objects/ groups of objects and measuring lengths, angles, perimeters and areas. The
area of an image which falls within a given pixel intensity range can also be determined (see
“Phase Analysis”, below).
A higher level version of AnalySIS software is available within the Electron Microscope suite of
Flinders Microscopy, which offers additional, more advanced measurement functions such as
configuration of macros for automated counting.
These measurement functions are not exclusive to images captured with the AnalySIS program, so
any tif image can be opened within AnalySIS and measurements done.
Measurements are only correct provided you have set the appropriate magnification for your
image. If you are using tif files that were not captured using AnalySIS, then you will need to
calibrate the pixel size via “Calibrate Image” in the “Image” menu.
To make a measurement, display the Measurement Tool Bar, click on the measurement type, and
perform the measurement in the image using the mouse.
To complete a measurement, or end a group of measurements, right-click on the mouse.
To move the cursor out of the image, right-click on the mouse or press the Escape key on the
keyboard (or the centre button on the mouse).
Tip: to improve the accuracy of your measurements, use the Viewport Manager in Magnifier mode.
To delete a measurement, click on the “Delete Measurement” icon which is the 3rd icon from the
bottom of the Measurement Tool Bar
Changing the Image Manager display to Measurements allows you to view your measurements
and associated statistics.
Measurements and Statistics can be exported directly into Excel using the “Create
Measurement Sheet” icon which is the 2nd icon from the bottom of the Measurement Tool Bar.
To save the measurement annotations on the image, click on the “Export Measurement to
Overlay” icon which is the second icon from the bottom of the Measurement Tool Bar, and save the
annotated image via “Save As” with the “burn overlay into image” dialogue in the Save Options
selected.
Thresholding and Phase Analysis (selecting a pixel intensity range).
“Set Threshold”, the 2nd icon from the top of the Measurement Tool Bar, enables you to set
thresholds to categorise the pixels into a user-defined range according to intensity. For example,
you can select labelled objects based on their intensity. Phase Analysis (3rd icon down) then
provides a excel report of the area represented by that intensity range. This may be used to
compare labelled populations of cells between different treatment groups. If you have determined
the average size of your thresholded labelled cell/organelle/particle, a simple division of area/size
could be used to estimate the number of labelled particles. More complex image analysis,
including automated counting, is available on a higher level version of AnalySIS software in the
Electron Microscope suite, but note that setting up automated counting is a complex procedure in
itself. For further information see Jen Clarke, Kerry Gascoigne or Ian Gibbins.