Nikon A1r Laser Scanning Confocal & 2-Photon Microscope

pathology.ucsf.edu

Nikon A1r Laser Scanning Confocal & 2-Photon Microscope

Nikon A1r

Laser Scanning Confocal

&

2-Photon Microscope

User Guide

Hardware …………………………………………………………...…………….2

Shut Down …………………………………………………..…………….4

Software ………………………………………………………..………………...4

Multi-Dimensional Acquisition …………………………………………...……..7

Trouble Shooting ……………………………………………………………..….9


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Hardware

1) Sign up for time at the BIDC microscope scheduler:

http://microscopeschedule.ucsf.edu/

a) This can be done up to 4 weeks in advance for owners’ consortium

b) For non-owners reservations may be made beginning Friday for the following

week

2) Turn on the arc lamp for visualization

a) The arc lamp MUST BE on for at least 10 minutes before being turned off

3) The confocal lasers below the wire rack (405, 488, 561, 647) should be powered on

already, turn the keys from “Standby” to “ON” only turn on the lasers you will be

using

a) If the power to a laser has been turned off turn on the power switch and wait

5minutes before turning the key on

4) Check the Mai Tai laser

a) The power supply should always be on, DO NOT turn it off

b) The key should always be turned to “ON”

c) If the screen on the lower box indicates coolant needs to be added, add a small

volume


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5) Turn on the Prior XY stage (1) and the Piezo (2)

6) Turn on the microscope power supply (3)

7) Turn on the microscope control box (4)

***After steps 6 & 7 there will be clicking and optics moving in the scan head, this is

normal***

8) The display on the front of the

microscope can be changed to

display desired information.

9) The display brightness can be

adjusted using the “Brightness”

button

10) The Z-Reset button will set the

current Z position to “0”. All Z

readings are relative.

11) The bottom row does not change

the light path, it must be changed

manually using the knob on the

side of the microscope near the binoculars.

12) On the left side of the microscope is a course/fine

focus knob and the Z-escape

a) The Z-escape brings the objective to the top of

its range to get it out of the way


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) Once it is at the top of its range, if it is pressed again it will return to the previous

Z location. Be careful that the stage and your sample are below the previous Z

so you do not crash the objective into them

13) Place the hydraulic Z stage at its lowest position and load your sample

a) DO NOT raise the manual part of the Z stage until after you have started the

software

14) Shut Down

a) In the OC panel select “Shut Down”

b) Check the scheduler and leave the microscope on if there is someone scheduled

within two hours (close only the software)

c) If no one is scheduled within two hours, turn everything off in opposite order of

start up

i) Software, computer, microscope, devices, illumination

Software

15) Turn on the computer and launch NIS-Elements

16) Adjust the hydraulic Z stage to the appropriate height

17) OC Panel (at the top of the right panel)

a) Select your objective

b) Select how you would like to visualize your sample through the eye pieces (ie:

GFP Eyes, DAPI Eyes etc)

i) Turn the knob on the side of the scope by the eyepieces to “Bino”

c) Once you have found your sample with the eye pieces turn the knob to send the

light to the detectors (F)

d) Select either Default MP or Default Confocal Settings

i) Default MP and Default Confocal will activate the relevant windows, set safe

starting values, and put hardware in the correct place

**NOTE: once you have set your imaging parameters if you go back to the

eyepieces, select “Imaging” rather than either of the defaults, the defaults will

reset any values (gain, laser power, etc) that you optimized in the software**


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18) A1 Plus Compact GUI (in the left panel)

a) If you switch from the detectors to the

eyepieces the Interlock will engage to

prevent laser damage to the eyes, click

the red button to remove it when you

switch to the detectors

This is where you can select:

b) Resonant (faster acquisition) or galvano

(cleaner images)

c) Bidirectional (faster acquisition) or

unidirectional (cleaner images)

d) Frame size

e) Line averaging and summation

f) “Scan” opens a live window

i) To navigate in XY in the live window

with the mouse click the navigation

button

19) Ch. Setup To optimize a single channel at a

time select

a) HV—adjusts detector gain

b) Offset—a number that is added or

subtracted from all the detector’s pixels

values

c) [colored circle + wavelength]—Adjusts

confocal laser’s power (this slider will

disappear in MP mode)

d) AG – the auto gain button will optimize the gain for a given laser power

i) You can adjust the parameters for AG by clicking the arrow next to it

ii) This allows you to change the target intensity and tolerance for maximum

intensity

e) “Ch Series” in confocal mode allows you to adjust which channels are acquired at

the same time to minimize bleed through


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20) After you have set up your acquisition parameters, if you need to switch back to the

eyepieces make sure the DU4 detector is selected and then select any of the

visualization optical configurations

**You will not be able to see anything through the eye pieces if the NDDs are

selected, this is to prevent them from being damaged by the arc lamp**

a) Once you are done with the eyepieces to acquire, DO NOT select Default MP or

Default Confocal, these will wipe out any parameter optimization you have done

for your acquisition. Select “Imaging” this will set the microscope hardware to the

correct places without changing any of your settings in the software.

21) A1Plus MP GUI (above the “A1Plus GUI”)

Controls the Mai Tai MP laser

a) Turn the laser on, when there is

emission a green bar will appear

b) Select your wavelength

Although you can enter numbers

below 740 the laser WILL NOT emit

c) Open the shutter

d) Auto align the beam at the beginning of

each session

i) If this alignment is not optimal place

a uniformly fluorescent slide on the

stage and adjust it manually using

the arrow buttons.

22) LUT (in the bottom panel)

Controls the scaling of your image

a) Auto scale will scale the contrast of your image one time

b) The saturation indicator will color all saturated pixels


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23) A1Plus Scan Area (in the left panel)

Allows you to select an area of interest and zoom by dragging the box or changing

the zoom on the slider

a) In Galvano mode you may also rotate the zoom area to best fit your sample

b) After you have selected your zoom size and area you must right-click on the box

to activate it

Multi-Dimensional Acquisition

24) ND Acquisition (in the left panel)

Allows you to set up time lapse, multi-position, large image, and Z-stack experiments

a) Z-stack

i) You may select absolute, relative, or asymmetrical

(1) For absolute scroll through Z while scanning and define the top and

bottom of your sample

(2) For relative set the center of your sample to “Home” and enter the

distance you want to acquire above and below this point

(3) For asymmetrical select a point and enter the distance you would like to

capture above and the distance below

ii) Select the Z-device you would like to use

(1) Piezo will be fastest, Piezo step-by-step will pause the Piezo at every Z-

plane, or the Ni-E microscope Z motor

(2) If you select the Piezo double click on the center point of your Z-stack in

the illustration, then click the Piezo button with an up and a down arrow on

it, this will home the Piezo to that location to ensure it is scanning the

proper Z range

b) Time Lapse – clock tab (specify

2 of the following three)

i) Interval is how often time

points are acquired

ii) Duration is how long the

experiment should run for

iii) Loops is the total number of

time points

c) Multi-point – XY tab

i) As you navigate around your

sample, mark areas of

interest to be acquired

d) Large image

i) Allows you to acquire a grid

of images of any size with

the current field as the

center

ii) “Stitch” will attempt to

identify common features

between tiles and use them

to stitch the final image


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iii) “Do Not Stitch” simply places tiles next to each other without processing them

***This type of acquisition WILL NOT give individual images for

each tile***

e) Channels – lambda tab

i) This allows you to switch between optical configurations that require a change

in dichroic, light source, IR laser wavelength, or detectors

f) Order of Experiment

i) The drop down menu is written multiplicatively, the experiment will be

executed in the order of options RIGHT to LEFT

25) Image Window

After the acquisition has finished there are several choices for viewing your data

a) Split channel will show each channel as an individual frame

b) Volume view gives an interactive 3D rendering of your data

c) Tile view displays your Zs as a series of thumbnails

d) Maximum intensity projection takes the brightest pixel at each pixel location in

your Z-stack and displays a single image


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Trouble shooting

Let us know when you run into software or hardware problems so that we can add

common problems and solutions here!

• The software does not open with all the usual windows open

o Click the “reload layout button” on the far left panel

• You cannot get light to the eye pieces

o Check if the arc lamp is on and the knob is set to “BINO”

o Open the “filters and shutters” menu in the software

§ Check if the shutter is open

§ Check if the correct filter cube is selected

• No light is getting to the detector

o Make sure the on the side of the scope next to the binocs is on “F” (not “R”

or “binoc”)

o Make sure you have removed the interlock in the A1 Plus Compact GUI


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