Nikon A1+ Manuel

LBIC

Nikon confocal A1+

Standard operating procedure

Version 1.2 June 2019

(1)

Motorized stage

power

Brightfield lamp power

A1 controller

Laser

on/off

key

Laser

buttons

(2)

Turning on/off the system

1. Turn on computer

2. Power on A1 controller and laser

power supply.

3. Turn the key and select the

needed lasers by pressing the

buttons.

4. Turn on stage power supply and

DIA lamp if bright field is needed

5. Power on microscope, wait until

startup sequence is finished.

6. Log in with your LBIC account

and start NIS-Elements AR

7. Turn off system in reverse order

(3)

Finding your sample

1. Put your sample in the appropriate holder, if you have slides make

sure the coverslip is facing down.

2. Start with either 10x or 20x objective

3. Navigate to a suitable place in your sample with the joystick.

4. Press “Eye port” to change the light path to the eye piece, if

needed press the Sola button (on Sola pad) to activate the LED

lights.

5. Use the coarse focusing mode and find focus (for slides around Z=

2100-2200, for 30mm petri dishes around Z=2500-2700)

6. Press “Eye port” again to switch to confocal mode (the first time

the system is started do this twice, this is a bug in the software).

(4)

Confocal Acquisition: Quick Start

1. Setup your channel requirements

2. HV (Gain), start at 100 for 405/640 and 25 for 488/561 PMTs

3. Offset adjustment, leave at 0

4. Laser Power, start at 2%

5. Start with a scan size of 512x512 and scan speed of 2.2 µsec.

6. Select “Normal” and “Ch Series” if using more than one laser

7. Select 1.2 AU for Pinhole

8. Start “SCAN”

9. Adjust detector and lasers settings if needed

10. Change pixel size by manually changing zoom factor in A1+ Scan Area” dialog

(View>Acquisition Controls>A1 Scan Area) or pressing Nyquist for optimal pixel

size for the selected objective and pinhole size.

11. Quickly Capture an image with the current settings. Image must be saved

manually afterwards. Save as .nd2.

*If you need to go back to the eyepieces to check your slides, click “Eye Port”,

click it again to return to the confocal!

2-4

(5)

Confocal Acquisition: ND Acquisition

Very useful for setting up advanced imaging protocols

1. Enter file save path (O:\is recommended)

2. Combine function tabs as needed by clicking the boxes

•Time is for time lapse videos

•XY saves coordinates (Z can be included with the include Z box)

•XY points carry over with objective change (as long as the sample is not

moved in the holder

•Large image captures multiple fields of view and stitches them to one image

•Very useful for creating maps of your sample with lower resolution (10x or

20x) that can be used to find points of interest and adding to XY (right click

on the point and select ”add this point to ND acquisition”.

•needs at least one XY point to work (this point will be the center point of

the large image).

•Z is used for capturing Z-stacks

•Can be setup by either manually selecting top and bottom Z-values or in

relative mode where the center Z-value is taken from XY (include Z needs

to be selected). The latter is used when you want to capture Z-stacks in

more than 1 XY point.

3. Contextual GUI for each function tab

*XY points can be added also when viewing in the eye port. Useful for quickly

adding points of interest before starting scanning.

(6)

(7)

(8)

(9)

Z-focus adjustment

Joystick

Changing sample holder

(10)

The confocal sample holders:

1. Multi-holder (slides and small petri dishes)

2. Multiwell plate holder (for all standard multiwell plates)

3. Oko-labs stage top incubator (see next page) with fittings for two 35mm petri dishes and

one 75mm x 25mm slide (standard slides and chamber slides)

Exchanging between them is simple

1. Remove plastic screws with

screwdriver located on top of

shelf.

2. Remove holder and put on shelf.

3. Change to the desired sample

holder (they only fit one way)

4. Fasten plastic screws gently.

DO NOT OVER TIGHTEN! The

screws break quite easily.

5. Put back screwdriver on shelf.

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