Introduction to the Nikon A1 microscope hardware
The Nikon A1 system is a state of the art confocal microscope.
1.
The system can scan very rapidly, producing 230 frames per second in a 512
x 64 pixel image and 30 fps (video rate) at 512 x 512.
2.
Every A1 contains two scanning systems: there is a conventional sawtooth
scanning system – galvano, as well as the fast resonant scanner. This
means that as well as the high speed scanner there is the more flexible
slower scan system.
The A1’s two scanning systems can be operated in rapid succession or
simultaneously.
4.
There is a spectral detector, based on a conventional 32 cathode PMT but
equipped with a unique light economiser, using Fresnel rhombs to rescue
signal light, which would otherwise be lost because of inefficient diffraction off
the grating
5.
In the scan head, reflections at a lower angle rather than the conventional
45 degrees have allowed the development of more efficient chromatic
separations.
The system will be fitted with CLEM and VAAS in the future.
6
How to switch on the A1 Confocal
1
Switch on all the main sockets on the back wall.
2
Turn on the keyswitch on the laser power rack.
(The LED will show green)Laser rack in room 125/126
3
Turn on the HBO Mercury Bulb and press the ignition button
Switch on the microscope halogen lamp power unit.
Switch on the X/Y motorized stage control box.
4
Switch on the Ti Microscope power switch located at the back right hand side of the
microscope. If FL light seen at the objectives close off shutter at the right side of the
microscope. If light from the microscope halogen bulb is seen close the shutter from the
microscope controls.
5
Turn on the main A1 Control box using the push switch on the left hand side. After 10s
initialisation the detector units lights will stop flashing be green and the controller unit light will
be amber. Do not switch on the PC until the lights stop flashing.
4- Detector Unit
Spectral Detector Unit
A1 controler Power switch
on left hand side.
6
Switch on the PC. Log on using your own log in.
7
From the desk top select NIS Elements AR 4.1
The NIS Elements logo screen will appear and will stay on the screen while program files are
loaded and checked between PC and confocal controller. This process can take up to 1 minute.
Ti pad microscope controls
controls
Image View window
Confocal
Camera Settings.
If any of the devices do not appear on the screen please see a member of the BAIR staff.
From the bottom tab different screen layouts can be selected depending on what you wish to do. Eg:
bleaching settings etc.
Setting up sample on the Ti for Convensional epi-Fluorescence.
1
From the Light path short cut tool bar select Microscope and filter set you require.( This will be
set up for you on your first session. Everyone has there own desktop layout. This will change the
light path to allow you to view your sample on the microscope with epi- fluorescence.
2
From the Ti Pad controls select the objective and filter you require.( This can also be selected
from the microscope control pad).
Objectives.
X10 dry
X20 dry
X40 oil
X60 oil
X60 water
Ti Control pad.
Light paths
E100 to view cells
on the microscope.
Filters.
Dapi
GFP
TRITC
3
We do not have a motorized shutter in front of the mercury lamp. To open and close off the light
you must manually move the shutter that is located on the right side of the microscope.
Close off the light when you have focused on your sample to avoid damaging the filters.
4
Focus on your sample. Use the Joystick to move in X/Y over your slide.
4
Once you are ready to start scanning select confocal from the light path settings.
5
The ‘ laser interlock’ button will flash
and you cannot activate the lasers for
scanning until you make this message
disappear by clicking on it.
The NIS Elements has a software safety mechanism that locks the laser operation unless the light path is
correctly sent to the scanhead port. If the light path setting is set to other ports, the red “Laser Interlock”
button will flash as shown on the previous page. The laser interlock button will not disappear if the light path
is still set for E100.
Confocal Control Window
Confocal acquisition
controls Live/
freeze,single
image,XY,XYZ,XY
T,XYZT,
Photocativation.
Confocal scanning
mode.
Confocal channels.
Use settings button
to make changes.
Select leases etc
from here.
Chanels,laserpower,
PMT,gain,black
level,offset and
pinhole controls
Home good starting
point. Check this is
set on 1 A.U
Channel series for
more than one dye
should be on “custom”
not “none.”
Scan settings control
or the selected scan
mode Direction,
size, speed and
magnification
Resonant and Galvano Scanning Modes.
With this version of the AIR confocal you have a choice of using the fast resonant scanner or the standard
slower galvano scanner. The Galvano is used for most normal imaging and the Resonant for very fast
scanning. When changing the mode you will hear the mirrors moving in the scanhead.
The Galvano scanning enables high resolution imaging up to 4k x 4k pixels. It offers more scan rotations and
flexible zooming and panning.
The Resonant scanning is recommended for detecting fast biological processes. It has a maximum resolution
of 512 x 512 pixels scanning at 30fps and up to 230fps at 512 x 64 pixels resolution.
Setting up the software for an XY scan
1
Select Galvano from the Confocal control window for normal imaging. Resonant for fast imaging.
2
From the filter and dye window select the channels you require. Open the setting tab if they need to be
changed. Please check with Margaret ,Tom or David on how to set the channels and lasers up. Open the
filter dye window from here.
Use DU4 and
AUTO.( see mop)
Settings Tab Window
Select and deselect
lasers from here
This window will allow
you to select the different
detectors, lasers and
scanning mode. For two
or more channel imaging
we recommend CH series
custom.
Select transmitted light
detector from here if
required.
The Nikon system default is that all selected lasers and detectors remain on throughout scanning so the scanning is
simulataneous. This makes for fast data collection, but creates problems when one dye’s emission spreads
spectrally into more than one detector. Sequential scanning should be used to reduce this problem. Set Ch series on
line.
For example, the emission from DAPI may spread into all channels, but DAPI is excited only by the 405nm laser.
If the 405nm laser is used first, on its own, with only one of the detectors, the other detectors will not register a
DAPI signal, since the 405nm laser will be turned off and other lasers will be used when those channels are active.
Settings
tab
Ch Series set to custom. Will show here as line instead of
none.
.
Scanning Settings:
Select uni directional scan and select your scan resolution from here. For set up try 512x512 and for imaging try
1024x1024. If you need high resolution see a member of the BAIR staff and we will help you set up for an
optimal image.
On the Acquisition/photo activation window make sure Acquisition is selected.
Set the pinhole width.1 A.U is a good starting point.
Select Live to continuously scan the
sample.This button allows you to toggle
between Live and Freeze.
XY Captures a single image.
XYZ opens the Z series set up window.
XY Time opens the time series set up window.
XYZ time opens the Z and time acquisition
dialogue box.
Photo Activation Opens the bleaching set up
window.
1
2
Select Live to continuously scan with the lasers and see a live view.
If no image appears in the image window increase the HV, Laser Power for each channel.
3
Focus on your sample using the microscope focus Knob. ( select fine or extra fine focus)
4
Use the colour look up table to optimize your image.( see next page for more details)
This will show you over saturated and under saturated pixels.
Scan area, cropping and zooming
The scan area can be changed by using the square or band scan tools.
Show scan area
toggle button
Select square
or band scan
Crop tool
Reset scan
area
The image can be displayed in a gallery view or in an overlay view by toggling this tool.
Zoom In
Select show scan
area. The Square
scan area window
will be in green –
grab sides to
resize, centre to
position and top
point to rotate.
Right click to
apply the change,
frame colour new
changes from red
(modified) to
green again.
Cropping area tool
window
By selecting a smaller scan setting you can zoom into the image without losing the resolution. If the scan size is set to
512x512, the scan area will retain its size thus giving an enlarged image scanned at the same speed.
Crop function will reduce the pixel numbers according to the area being cropped but will increase the scan speed.
Use the colour LUT to check for oversaturated pixels By selecting the LUT tab. Over saturated pixels will be red by
default unless changed by users. Use the HV and laser power to reduce the number of saturated pixels. Switch off
LUT when optimized.
Each pixel has a gray scale intensity
value. The LUT will make sure that
the values are not over or under
saturated. This is important if you
want to quantify the intensity of your
image.
Scan resolution and pixel size is
important. To large a pixel size
will mean loss of resolution.
You can adjust the laser power and HV for each channel from the software. You can also set the offset value but be
careful when using this function!!!!!!!. Please see Margaret or Tom for instructions on how to use this correctly.
What happens in the PMT when I adjust the HV? The signal is amplified and the digital image on the screen will look
brighter.
You can also adjust the laser power and HV from the control hub.
To capture a single image, click the XY button. The new image will need to be saved.
File > Save As. The best file formats for confocal images are Tiff or Jpeg 2000 as they preserve the acquisition
control information. They will open in imageJ ( Please see Margaret, Tom of David)
Z stacks will be saved as nd2 files.( These should open in volocity and the Nikon NIS elements offline
software) See Margaret or Tom.
JPEG2000 Format (JP2) An advanced format with optional compression rates. Image calibration, text
descriptions, and other meta-data can be saved together with the image.
Standard JPEG files are used in many image processing applications.
Tagged Image File Format (TIFF) This format can save the same amount of meta-data as JPEG2000.
TIFF files are larger than JPEG2000 files but are loaded faster. TIFF files have several ways to store
image data therefore there are many versions of TIFF. NIS-Elements support the most common TIFF
modalities.
Windows Bitmap (BMP) This is the standard Windows file format. This format does not include
additional image description information such as author, sample, subject or calibration.
LIM Format (LIM) Developed for the needs of laboratory image analysis package, nowadays all its
features (and more) are provided by the JPEG 2000 format.
The British Medical Research Council advice on good record-keeping. The ORI states that data should
be stored in such a way that it permits a complete retrospective audit, and that it is monitored regularly
to ensure completeness and accuracy.

Raw data should be recorded and retained. The original data should not be
altered in any way. All manipulations should be carried out on a copy of the
original data. Modifications should be clearly identified and dated.

The original and all other important date should be stored on an area that is
backed up by IT.

All raw data should be included. Be honest.

Data interpretation should be carefully written.

Electronic records need to be carefully monitored.

Electronic data should be backed up on a disk with a hard copy; relevant
software must be retained to ensure future access, and security of data is an
issue.
Nikon A1R Confocal Microscope
XYZ Acquisition
Z-stack acquisition will allow you to capture a sequence of images from different focal planes of the specimen.
The Z-series capture can be done through Acquire menu by selecting XYZ. This will open the set up window.
Select Live and scan sample as normal. Set top and Bottom value and step size. Select run now to start XYZ acquisition.
Save as nd2.
Tick to save to HD otherwise images utilise
system RAM.
Range define methode:
1 Top/Bottom
2 Home position symmetric range
3 Home position Asymmetric range.
Select Top and Bottom.
Select the required Z step
Select Run Now to capture the Z stack
The Z can be controlled in two different ways. Select Ti Z Drive for normal microscope Z control. Select Nikon A1
Piezo Z Drive for the accurate nano drive. MCL nano drive controller on the shelf to right of the microscope must
be switched on to use this.
Z Stack Gallery View
Save stack as nd file if not already
saved.
Nikon A1R Confocal Microscope
Select 3D volume view
The image stack can now be rotated and viewed form all angles. (For avi movies please see M.O’Prey)
Nikon A1R Confocal Microscope
Image improvements for better quality if under sampling.
Movie maker:
Nikon A1R Confocal Microscope
TURNING OFF
1.
Stop scan
2.
Close software.
3.
Use Windows shutdown procedure.
4.
Turn devices off by reversing actions in steps for start up.