Nikon A1+ User manual

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Title: Firefly Confocal (Nikon A1+) User Manual
Date of first issue:
23/10/2015
Date of review:
28/08/2017
Version:
Admin
For assistance or to report an issue
Office: CG.07 or CG.05
Email: Igmm-[email protected]
Website: www.igmm-imaging.com
Download a PDF copy of manual:
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Manuals\Confocal\Firefly
Facility Usage Policy
1. You must have the relevant Risk Assessment/COSHH form for the
work you are undertaking before using imaging facility resources
2. Users must be trained before using facility equipment
3. Please leave the microscope clean and tidy for the next user
4. Please report any issue, even if it seems minor, to facility staff
5. Any clinical waste must be placed in the orange bins provided

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Table of Contents
System Startup..................................................................................................................... 3
List of devices ...................................................................................................................... 4
System Shutdown ................................................................................................................ 5
Mounting a sample............................................................................................................... 5
Finding the sample by eye ................................................................................................... 6
Nis-Elements software layouts............................................................................................. 7
Selecting a confocal channel setup ..................................................................................... 7
Adding a brightfield channel................................................................................................. 8
Capture an image ................................................................................................................ 8
Pre-scan acquisition settings ............................................................................................... 9
Optimising scan settings for best image quality................................................................. 10
Image Saturation................................................................................................................ 12
Capture a single XY image ................................................................................................ 13
Image Window Options...................................................................................................... 13
Adding a scale bar ............................................................................................................. 13
Capture images with Optical Zoom.................................................................................... 14
Z Stack Acquisition............................................................................................................. 15
Z stack viewing options...................................................................................................... 16
XYZT Experiments............................................................................................................. 17
Multi-position XYZT Experiments....................................................................................... 18
Tile-Scan Experiments....................................................................................................... 19
FRAP/Bleaching Experiments............................................................................................ 21
Appendix........................................................................................................................... 24
Saving Images ................................................................................................................... 24
Export images in tiff format ................................................................................................ 24
Kohler Illumination ............................................................................................................. 25
Perfect Focus System (PFS) Operation............................................................................. 26
Frequently Asked Questions .......................................................................................... 27
1. My images look noisy, what can I do to improve image quality? .................................. 27
2. I can't see anything down the microscope .................................................................... 27
3. I can't fit all of the tissue into the field of view ............................................................... 28

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System Startup
*See the next page for labelled images of all devices
1. Prior brightfield LED (only if required). Switched on via Prior Optiscan II box
2. Lasers-Turn the key on the front of the laser bed clockwise to the horizontal position
(405/458/488/514/561 excitation lines)
3. The 640nm far-red laser (only if required) is turned on via the black rocker switch.
The LED light will stay orange for 30secs or so and then turns green. Once this
happens turn the key on the box to the horizontal position.
4. A1 controller (button on left side of tower)
5. XY stage controller (on shelf)
6. MCL Piezo controller (on shelf, only if required)
7. PC
Additional devices required for Live Cell Imaging
•Incubation chamber heater (chamber doors must be on while heater is in use)
•CO2 controllers (see labelled diagram)
•CO2 flow valve on wall (see labelled diagram)
8. Log in to Windows using the “User” account
9. If using the 640nm laser launch its control
software from the desktop icon
10. Select the Power mW button and type 30 into the text
box
11. Press the “On” button
12. You can minimise the Fiber laser window but don't close it
13. Launch Nis-Elements from the desktop icon. If you have been trained then you
will have a username to log in to Elements

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List of devices

5
System Shutdown
1. Clean any oil immersion lenses used with the lens tissue
2. Close Nikon Nis-Elements
3. If the 640nm laser is turned on, maximise the 640 control software and select Off
then close the window
4. Shutdown the PC
Incubation chamber heater and CO2 supply if used
5. MCL Piezo (on shelf)
6. XY stage controller (on shelf)
7. A1 Controller
8. Prior brightfield LED, Switched off via Prior Optiscan II box
9. Lasers- Turn the key on the front of the laser bed anticlockwise to the vertical
position
Mounting a sample
1. Ensure the correct stage insert is mounted (see facility
staff if unsure)
2. Select a low magnification (10-20x) lens from the
touchpad objective menu
3. Use the joystick to move the stage if necessary.
Change the speed of the stage by twisting the joystick.
4. Before mounting the slide on the stage, turn the microscope focus
knob, if the Z value on the microscopes front LCD display doesn't
change as you turn, the lens is in the Escape position. Press the
Refocus button on the right side of the microscope body.
5. Remember to invert your slide so that the cover glass is the closest surface to the
lens

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Finding the sample by eye
1. Use the shortcut buttons (OC Panel) in Elements to select a wavelength to view.
The “Eyes” heading indicates the relevant buttons.
2. Set the microscope focus to course movement using the button the
right side of the microscope body.
3. It may not be obvious in which direction you need to move the lens to find the focal
plane. If you are unsure, lower the lens (nosepiece) as far as it will go using the
microscope focus knob (turn away from you to move the lens down). When it will
not lower any more (Z position on microscopes LCD display stays constant) start to
turn the focus knob towards you while looking down the eyepieces.
4. Use the OC Panel to switch filters & check your labelling at each wavelength
5. Press the “Fluorescence Off” OC button to turn off the fluorescence LED
6. You can check brightfield by eye using the Brightfield (Eyes) button
7. At this point you can either change to a higher magnification lens if required or
begin the scanning process
8. Lenses 60x and above always require a drop of oil between the lens and the
coverglass. 40X lenses can be either a dry or oil type. The touchpad used to select
the lenses tells you if the lens requires oil.

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Nis-Elements software layouts
1. Nis-Elements has many toolbars which need to be changed depending on the
experiment you are carrying out. The Layout tabs at the bottom left side of the
screen allow you to change the software layout which essentially changes the
toolbars visible too you.
2. When you start Elements the “Confocal” layout will be selected by default. This
contains all of the toolbars required to capture confocal images. The padlock icon
denotes that the “Confocal” layout cannot be altered.
3. The “User” layout can be selected if you want to customise the layout of the
software. If you change this layout profile, any changes will only be applied within
your Nis-Elements user account.
Selecting a confocal channel setup
1. Some default channel presets are available from the OC Panel under the “Confocal-
Channel-Settings” heading. Selecting one will initialise the relevant lasers/detectors.
Ask facility staff if the defaults are not suitable for your application.
2. The number of detectors and lasers that are active will
display in the A1Plus Compact GUI in the right hand margin
on the screen

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Adding a brightfield channel
1. Press the Channel Settings button in the A1Plus Compact GUI window
2. Press the Transmitted Detector “In” button to add a
Brightfield channel
3. Select Ok to confirm
4. You will see that a TD detector has appeared in the list
of channels and signifies that a brightfield image will be
scanned
5. If the Transmitted Detector is active and you wish to view the sample looking down
the microscope under brightfield illumination, press the Brightfield(Eyes) Optical
Config button
6. Press the Eye Port button at the top of the A1Plus Compact GUI window
Capture an image
1. Before you can scan with the lasers you have to remove the laser
interlock by pressing the red “Remove Interlock button”
2. You can see the live image at any time by pressing the Scan button
3. To acquire an image press the Capture button
4. The captured image is displayed as a merge of all the channels by default. You can
view individual channel images by selecting the channel tabs at the bottom of the
image window.
5. To see a montaged view of all channels, select this icon from the top of the
image window.

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Pre-scan acquisition settings
*Consult the labelled diagram of the A1Plus Compact GUI (page 10) if you're not sure
where any of the buttons mentioned below are
1. Before you can scan with the lasers you have to remove the laser interlock by
pressing the red “Remove Interlock button”.
2. The scan direction buttons indicate unidirectional and bidirectional scanning. When
bidirectional mode is selected the image acquisition frame rate doubles. Only
consider bidirectional mode when carrying out a live cell experiment.
3. Select an image size (e.g. 512x512 pixels in xy). The size affects the xy image
sampling with larger sizes resulting in smaller um/pixel values and thus better
spatial resolution but the scan time is also increased. The most commonly used
sizes are 512 or 1024.
4. Now choose a scan speed, start by selecting the lowest number when the control
by “Pixel dwell” option is enabled. Higher values represent a longer pixel dwell and
hence a slower scan. You may need to use a slower scan speed if your signal is
low but this could
increase the
photobleaching rate.
5. Ensure that the “Normal” and “Ch Series” buttons are enabled (green).
Channel Series enables sequential scanning mode which means only one
laser is scanning at any particular time. If Channel Series is turned off then
all lasers scan simultaneously which can lead to fluorescence crosstalk
where emission from one fluorophore is detected by more than one detector.
6. Press the “Optimal Pinhole” button, this sets the detection pinhole to a diameter that
gives the best compromise between light throughput, noise and resolution. Closing
the pinhole results in a thinner optical
section and better lateral and axial
resolution due to rejection of out of focus
light.

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Optimising scan settings for best image quality
1. Press the Scan button (lasers scan and the live image is constantly
refreshed). Pressing Scan again will stop the scanning process.
2. The Find button can be used to increase the live image refresh rate by
decreasing the size of the scanned area and the image quality. Use
this option if you cannot find the sample by scanning with the
continuous Scan button.
3. Each detector, signified by the coloured band and labelled with the fluorophore
name in the compact GUI, generates its own image. The Live window shows the
merged colour image of all the active detectors.
4. The default detector settings, HV (Gain), Offset and Laser power are set
conservatively. If the live window is black this usually means the detector settings
need to be adjusted.
5. It is recommended that you optimise one channel (detector) at a time. An easy way
to do this is to enable the Channel Setup tick box. When selected the square tick
box next to each detector changes to a radio button (circle), the active detector is
the one with the filled in grey circle. You can toggle between the different channels
by clicking in the circle next to the channel heading.
6. Note that when Channel Setup is enabled you will only see the image generated by
that selected detector.
7. If the image is black you can raise the HV (Gain) of the detector to a higher value
until you can see an image. Alternatively you can use the AutoGain (AG) button at
the top of the Compact GUI to calculate the optimal Gain value based on the
current laser power.
8. Note that increasing gain leads to increased image noise and eventually a lower
signal to noise ratio (SNR). Therefore the best image quality is usually attained by
adjusting several parameters, namely the gain, laser power and the scan speed.
9. Keep laser power as low as possible to minimise bleaching of the sample. Gain
should be set so that image noise is negligible. Offset at zero and scan speed 6.2
pixel dwell or faster.

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Image Saturation
1. As well as the image being too dark, it can also be too bright. The confocal
acquires 12bit images. This means that any pixel in the image can have an
intensity value between 0 (black)-4095 (white). The aim is to set the scan
parameters so that the pixels are just below saturation level (4095).
2. In the right hand margin in the software below the
Compact GUI is the LUTs tab. Select the tab and click
on the Pixel Saturation Indicator button. When active this
will colour any pixels in the image that have reached the
4095 maximum intensity value. The colour given to over
saturated pixels can be selected through the down arrow
next to the button. An image window must be open in
order for the Pixel Saturation Indicator button to appear.
3. Images showing the saturation indicator turned on and
off. Saturated pixels in this case have been coloured
yellow.
4. Continue to scan using the Channel Setup option, switch between the detectors
optimising each image so that it is just below saturation intensity level (no or
minimal pixels at 4095 intensity).
5. If you stop scanning this automatically turns off the Channel Setup option and
pressing Scan again will activate all lasers/detectors.

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Capture a single XY image
1. Once the scan parameters have been optimised, press the Capture
button to acquire a single scan of each active channel.
Image Window Options
1. The captured image is displayed as a merge of all the channels by default. You can
view individual channel images by selecting the channel tabs at the bottom of the
image window.
2. To see a montaged view of all channels, select this icon from the top of the
image window.
Adding a scale bar
1. Every Nis-Elements image window contains a scale bar icon. Press the button to
add a scale bar to the image
2. To adjust the scale bars length, width or colour, select the
down arrow next to the icon and select Scale Properties
3. The scale bar is an image overlay so can be switched on/off by selecting the scale
bar icon in the image window on which the scale appears

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Capture images with Optical Zoom
1. Optical zoom enables capture of a ROI within your image at higher magnification
without the need to change the objective lens. Optical zoom can improve spatial
resolution by changing the pixel size but can also increase photobleaching rates.
2. The easiest way to apply a “useful” level of zoom is to
use the Nyquist XY button found in the A1Plus Scan
Area window.
3. Nyquist xy sets the zoom level based on the optimal
pixel size for the selected lens.
4. A green ROI in the Scan Area window shows the area
of the image that will be included in the new zoomed in
image. A text box displays the actual zoom level
applied, in this case 2.92x.
5. If you move the green ROI zoom box or shrink/enlarge it, it turns red. Right click on
the image or within the A1plus Scan Area window to apply the new zoom settings.

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Z Stack Acquisition
1. To acquire a 3D dataset you need to define a Z stack
acquisition
2. Enable only the Z Stack tick box within the ND
Acquisition window
3. Choose the Z device from the dropdown box either Ti or
MCL Nanodrive Piezo. The piezo can move between z
planes at a faster rate but it only has a total travel range
of 100µm
4. Press the Reset button to clear any pre-existing values
5. Select the Defined by Top-Bottom
button
6. Press Scan
7. Turn the focus knob towards you to move to the top of the sample
then press the Top button
8. Now move the focus away from you to move to the bottom of the
sample then press the Bottom button
9. Press Scan again to halt the scanning process
10.To set the optimal step size between slices press this button. The calculated size
determines what step is required to produce optimal sampling according to the
Nyquist-Shannon theorem
11.To acquire the Z stack press the Run Now button at the bottom of
the ND Acquisition window
12.While the stack is being acquired
a progress window will appear. Pressing Abort will stop the acquisition and close
the z stack window. Pressing Finish will stop the z stack but the stack window will
remain open.

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Z stack viewing options
1. You can view the different slices of the stack by left clicking with the mouse on the
horizontal blue bars running along the bottom of the z stack image window
2. A z stack can be viewed in various ways. Pressing the Slices View button will
display the XY and YZ orthogonal views as well as the standard XY view
3. To display a volume rendering of the stack press the Volume View button
4. To see all slices of the stack displayed as a montage, press the Tiles View button
5. To generate a Maximum Intensity Projection press this button
6. To save an image of any of these views, select Edit>Create new document from
current view. Then Select File>Save As from the top menu, choose either ND2 or
Tiff as the file format.

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XYZT Experiments
1. Set up the scan settings as required
2. Enable both the Z and Time check boxes in the ND Acquisition window
3. Select the Time tab and tick the box under the Phase column. You can add
multiple time phases which will run sequentially.
4. The Interval time sets the delay between image captures
5. Set the total time lapse time under the Duration column
6. Once these parameters are filled in, the number of loops will be calculated
7. Tick the Save to file check box and input a destination for the data
8. Select the Run Now button to start the time lapse

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Multi-position XYZT Experiments
1. Enable the Z and Time options and set up these parameters as described above
2. Enable the XY check box in the ND Acquisition window
3. Here you can add multiple stage positions. Move to the position of interest and
select the check box under the Point Name column
4. Repeat this process to add multiple xy positions to the list
5. If you enable the Move Stage to Selected Point check box, when you select a
position in the list the stage will move to that point
6. To update a positions XYZ coordinates select it from the list, adjust the
focus/position on the microscope and then press the horizontal arrows that point
towards the XY and Z coordinates.

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Tile-Scan Experiments
Basic Tiling
1. Enable the Large Image tick box in the ND
Acquisition panel
2. Choose whether the tiled area is defined by the
number of consecutive fields of view or by
absolute area (mm x mm)
3. Enable the Stitch option if you want the output image to be the montage of all the
tiles joined together
4. Select an overlap of between 10-15%
5. You can also enable any of the other ND Acquisition tabs to combine experiment
types i.e. tiling z stacks or time lapse experiments
6. Select Run Now to start the acquisition
Advanced Tiling
1. Select Acquire>Scan Large Image from the top menu
2. In the Capturing section select the lens to use to capture a low magnification
overview (Macro image) from which you will decide how big an area you will tile at a
high magnification (Scanning).
3. At this stage you can also choose the high magnification lens to be used for the
actual tile scan (Scanning)
4. You can see the live image and choose your position to tile by pressing the preview
button
5. Select Capture in the Stage Overview with Macro Image
section. This will capture a single image at the stage's current
location and display the macro image result.
6. Red squares will appear on the image representing the fields
of view to be scanned at the currently selected Scanning lens.

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7. You can alter the number of fields of view to be scanned in the Area section and the
number of fields of view on the macro image is automatically updated.
8. The Fields Placement option determines whether the tiling
is around the current stage position or if the current
position is the top left corner of entire tile scan.
9. Set a degree of overlap for stitching in the Overlap section
(10-15%)
10.Select whether the final output is the tiled image or the individual tiles
11. You can select whether the tile scan is automatically saved if you set up a file path
12.To acquire a tiled Z stack enable the Z Series
option
13.Select the number of Z planes and the step size
14.The Z stack will be acquired symmetrically around
the current Z plane
15. If the ultimate aim is to generate a maximum intensity projection of the tiled z stack
then you can just enable the Max IP option. Note that the MIP will be generated
automatically and you will lose the individual z stack images.
16. To begin the tiling experiment press the Scan button