Nikon A1+ User Manual
1
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-imaginghelpdesk@igmm.ed.ac.uk
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
2
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
3
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
4
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
6
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.
7
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 “ConfocalChannel-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
8
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.
9
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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