Zeiss AxioObserver Manuale utente

Zeiss AxioObserver with ApoTome

Quick Start User Guide (AxioVision4.8)

LSU Health Sciences Center-Shreveport

Research Core Facility (RCF)

Microscopy

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 2of 19

Table of Contents

Start up the system……………………………………………………………………………………………………..

Page

Touch screen controller (TFT DISPLAY)

Page

Mount and view the sample through the microscope ……………………………………………….

Page

Start up the AxioVision software….……………………………………………………………………..........

Page

4.1

Experiment settings……………………………………………………………………………………………….

Page

4.2

Apotome Settings………………………………………………………………………………………………………

Page

4.3

Channel Creation……………………………………………………………………………………………………….

Page

4.4

Setting Exposure

Page

Acquiring and saving an experiment …………………………………………………………………………..

Page

5.1

Acquire………………………………………………………………………………………………………………………..

Page

5.2

Save and Export……………………………………………………………………………………………………….

Page

Z-stack ………………………………………………………………………………………………………………………

Page

6.1

Adding a Z-Stack to Your Experiment…………………………………………………………………………..

Page

6.2

Setting the Limits of the Z-Stack………………………….…………………………………………………….

Page

6.3

Z-Stack Settings……………………………..…………………………………………………………………………..

Page

6.4

Z-stack Cut View…………………………………………………………………………………………………………

Page

Shutdown……………………………………………………………………………………………………………………

Page

Technical Issues and Errors….……………………………………………………………………………………..

Page

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 3of 19

If the room lights are not on when you arrive to use the Axioobserver, DO NOT TURN THEM ON. Please be courteous of

those using the other microscopes, since they may have light-sensitive slides, dishes, or plates on the microscope stages,

which could be damaged by the room lights. This is particularly the case with the Leica confocal, since some Z stacks can

take over half an hour to complete. Use a torch if needed.

Check whether the floating stage is functional. If not, please contact the Research Associate. Make sure the

table is clean. No food or water is allowed in the microscope room. Carefully take off the microscope cover

and put it in the white storage cabinet. Check whether the holder is properly in place.

Please check the objective turret has the 10x objective in place. This is a safety precaution. If the 10x objective

is not in place, please clean the current objective and manually switch it to the 10x objective. To manually

switch objectives, push on the teeth of the objective turret in either a clockwise or counter clockwise fashion

as shown adjacently in the boxed region.

1. START UP THE SYSTEM

Please sign in on the login sheet. The format for the date is MM/DD/YYYY, eg. 05/25/2015. Write legibly for

this is how the staff knows who has been using the microscope. Check in and Check out time is written in 24

hour format, e.g. 1pm is 13. The format for writing the bulb time is ###h ## m, eg. 100h 25m. (You will not be

able to write the bulb time until you turn on the power supply.)

Our system has the startup labeled

numerically with yellow stickers.

First, turn on the mercury lamp power source.

The green power light and the green lamp

light will come on. (This is where you record

the bulb time.) If the lamp light indicator does

not light up, please contact Core facility staff.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 4of 19

Once turned on, the lamp should remain on

for a minimum of 30 minutes. Do not turn the

lamp off if another user is scheduled within 2

hours after your session.

Secondly, turn on the Power Supply 2 by

flipping the switch which will have a green

light turn on.

Third, you will need to go in between the

microscope and the power supplies to press

the button below the yellow sticker with the

number 3. See image to the right. This will

turn on the TFT Display, shown adjacently, and

please wait for this display to fully load before

proceeding.

Lastly, turn on the Apotome power supply

labeled with a yellow 4 sticker.

The computer should be on and logged in with your LSUHSC ID and password. Make sure the domain is set to

LSUMC-MASTER, selected from the drop-down menu. If you only wish to access documents/files, then login

and do not turn on the mercury lamp power supply or the other buttons. Please indicate this on the sign in

sheet.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 5of 19

2. TOUCH SCREEN CONTROLLER (TFT DISPLAY)

To change the position of the microscope stage, and

thus your sample, you will need to use the adjustment

knob and joystick attached to the TFT display. The

adjustment knob, on the right side of the TFT display,

has two “wheels” for different adjustments and controls

the Z-direction of movement. The larger wheel is used

for course adjustment while the smaller one is used for

fine adjustment. The joystick behind the TFT display will

control the X and Y direction of the stage. The degree of

which the stage will change will depend on which

objective is in the light path.

The image to the right displays the Home screen and we

will go over useful information, in the center, that can

assist you in imaging.

Objective: Description of the current objective in the

light path along with N.A.

Resolution: How fine of detail the current image will

appear

Optovar: A tool that is part of the system that can be

used like a magnifying glass. This should always read 1x

and should be changed to this in compliance with

Apotome use.

Total Magnification: The number of times the image has

been “expanded” compared to the actual size of the

image

HAL: The current voltage of the halogen bulb, this is only

a concern for brightfield imaging.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 6of 19

To select an objective, you can either move the

objective manually, or using the TFT display. We

recommend using the TFT display for it is easier to

select objectives. On the home screen displayed, push

the icon labeled Microscope which will pull up a new

screen.

The adjacent picture shows the screen for the

Microscope control panel on the TFT display. Make

sure that the Objectives tab is selected as it will be a

light blue colored compared to the rest. Also, ensure

that you are on the Control display by checking to see

if the Control icon on the sidebar is white and not

blue. An icon is selected if it is white and not selected

icons will appear blue, as shown to the right. The

Objectives displayed tell you which one is an air

objective versus an oil objective. If you need to switch

between different objectives that use different

immersion, the software and TFT display will inform

you and confirm that you are ready for the next

objective.

Reflector: Displays which position of the reflector turret

is in the light path and what type of cube is in that

position.

Condenser: Describes which type of brightfield imaging

is in place along with the N.A.

VIS: Displays how much of the light from the sample is

being sent to the eyepiece

Sideport: Displays how much of the light from the

sample is sent to the camera/computer

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 7of 19

To illuminate your sample with fluorescent light,

press the header icon labeled Reflector to pull up the

fluorescent filters. On the right sidebar, make sure

that the Transmitted light (TL) Illumination is off and

that the Reflected Light (RL) Illumination is on.

Filter choices are labeled as, on the TFT display:

34 BFP (EX 390/20, EM 460/60) –for DAPI, Hoerchst, AMCA

38 HE GFP (EX 470/40, EM 525/50) –for GFP, FITC, and

prevents red emission bleed through

31 AF 568 (EX 565/30, EM 620/60) –for Cy 3, Texas Red

17 AF 488 (EX 485/20, EM 540/25) –for GFP, FITC, and

prevents red emission bleed through

50 Cy 5 (EX 640/30, EM 690/50) –for Cy 5 and far red dyes

3. MOUNT AND VIEW THE SAMPLE THROUGH THE MICROSCOPE

If you are using an immersion objective, place a VERY small drop of the appropriate immersion fluid on the

objective face, or the coverslip. Be sure to only use the immersion fluids located next to the microscope. If you

accidentally use the wrong fluid, please contact staff right away. If you are using a slide, insert it coverslip

down into the specimen stage template. Below is a table of our current objectives, imaging medium, and

brightfield capabilities.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 8of 19

Obj

Objective

Medium

Grid

10x

EC Plan-Neofluar 10x/0.3 DIC I

Air

20x

Plan-Apochromat 20x/0.8 DIC II

Air

40x

LD Plan-Neofluar 40x/0.6 Ph2 DIC II

Air

NA*

40x

Plan-Apochromat 40x/1.3 DIC (UV) VIR-IR

Oil

63x

Plan-Apochromat 63x/1.4 DIC III

Oil

100x

Plan-Apochromat 100x/1.4

Oil

The 4 buttons on the template move the support brackets along the track. The template will hold a variety of

shapes. If you need a multi-well plate template, please ask staff.

Start with DAPI to find and focus on your sample. Be aware that fluorescence light is damaging to the eyes.

Avoid looking directly at the lasers. Block the light infront of you if the orange light shield is not strong enough

to block the light.

4. START UP THE AxioVision SOFTWARE

Double-click on the AxioVision icon on the desktop to

start the program.

Make sure your Apotome power supply (labeled with

number 4) is turned on before starting up the

software, otherwise apotome may not work properly.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 9of 19

4.1 Experiment Settings

The default page for the AxioVision

software is shown on the right. A

majority of commands can be found

in the left side bar labeled

Workarea. You can control filter

cube selection, light path direction,

objective selection, camera settings,

etc.

The image on the right shows a

more detailed view of the options in

the Workarea. The Microscope

selection allows you to adjust parts

of the microscope similar to the

options in the TFT display. All

options with a plus sign to the left

of the icon indicate that a pull down

menu will appear when the plus

sign is clicked on. In order to adjust

the Camera, Apotome, and to work

with Multidimensional Acquisition,

you must select the plus sign next to

the designated icon. The toolbars at

the top of the screen give you short

cuts to features such as light path

selection, filter selection, and

illumination.

RCF Microscopy Manual 5/2/2017

Authors Cory Nook and Chaowei Shang

Page 10 of 19

4.2 Aptome Settings

About the Aptome

The ApoTome uses a projected grid along with a

mathematical algorithm to remove out-of-focus

light, similar to the function of the pinhole on a

confocal microscope. When not in use, it should be

pulled out slightly from the microscope. There are

three small grids (L, H, D) that can be used in the

ApoTome; each objective requires a particular grid.

The software will prompt you to change the grid if it

senses that the grid and the chosen objective do not

match. Sometimes, because objectives are retired

and new ones created, the match between

objective and grid may not be reflected in the

software. A Zeiss engineer helped me come up with

the best pairing for objectives and grids that we

have (see below).

When the ApoTome is pushed in all the way, it is

engaged. The grid moves through 3 positions as it

captures one image. When you need to disengage

the ApoTome from the microscope, turn off its

power supply first (Labeled #4), which allows the

grid to return to its original position, then turn in

back ON before you pull it part or all of the way out.

Failure to do so may cause damage to both the grid

and the Apotome. Should the grid become loose

from its niche in the Apotome, let me know, and I

will retrieve it.

From the menu, choose the ApoTome button. A dialogue box

labelled ApoTome will pop up. Choose the second tab, Settings,

and choose the following:

ApoTome: Live Mode –Grid visible

ApoTome: Acquisition Mode –Optional Sectioning

ApoTome Grid –Choose the correct grid for your

objective from the drop down menu

ApoTome Filter –Medium

ApoTome: Averaging (Noise Reduction) –2

ApoTome: Status –this should say Calibrated in a green

field (if not, come get me!)

Obj

Objective

Medium

Grid

10x

EC Plan-Neofluar 10x/0.3 DIC I

Air

20x

Plan-Apochromat 20x/0.8 DIC II

Air

40x

LD Plan-Neofluar 40x/0.6 Ph2 DIC II

Air

NA*

40x

Plan-Apochromat 40x/1.3 DIC (UV) VIR-IR

Oil

63x

Plan-Apochromat 63x/1.4 DIC III

Oil

100x

Plan-Apochromat 100x/1.4

Oil

*The 40X Air Objective is a Phase Objective and shouldn’t be used with the Apotome.

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