Basics

Normal operation of the StedyCon microscope through a normal confocal scan with the Pollen slide.  Additional books will cover other types of scans, e.g., Z-Stacks, Tiled scans, STED.

These documents are to reinforce, not replace, hands on training on the microscope.  All users MUST recieve training from Cell Imaging Staff prior to use of the instrument.

Stop: This is a first draft in progress. The information is not/may not be accurate.

Power On

One Photo of the bottom of the laser box and the bottom rack - all lit up with numbers 1-6

One photo of the microscope power switch with 7

1. Laser Box:  
   Turn
2. the Laser Key to ON.  The laser box is normally in standby (There aretop two lights on:Power Power(Red) and Standby/Ready and(Orange) twoare lightson, off: Emission and Scanning). (Color of lights….)

Turn on the Laser Key.  Vertical is Standby, Horizontal is on.  The lightsbut do not change.  The bottom

Istwo therelights, aEmission powerand switch or is that just the power strip?

ThereScanning are 4 boxes under the table: 

off. Piezoconcept: Turn on switch on front left.  A green LED on the front will turn on. Tango Desktop: Turn on switch in back top left.  A green LED on the front will turn on. Nikon Brightfield Lamp Power Supply: Turn on switch on front right.  A green LED on the front will turn on. Fluorescent Lamp Power Supply:

Turn on the power switch, lower left.  The green Power LED will turn on. Fluorescent Lamp Power Supply: Press and release the Ignition buttonButton above the power switch.  The Orangeorange Lamp Ready LED will turn on. Microscope:  Turn on the microscope with the switch on the lower right side near the back.  The microscope will make noise and the front display should be on. Computer and software: 

1. If the computer is off, press the button on the front of the box.
2. If the monitor is off, there is a small power button on right bottom.
3. Double click the StedyCon icon on the left side of the screen.  This should bring up the Welcome Screen

The Microscope

Your first task will be to get your sample in focus and in the correct position.  This is done with the microscope and through the controls on the microscope itself.

1. Apply a drop of oil to the lens if needed (60X, 100X).  (The lenses are parafocal,parfocal, so you can start at lower magnifications first.)first and work up to the 60X, 100X.
2. Place your slide with the coverslip down.  Tilt the transmitted light arm back using the white housing at the top.
3. Use the X-Y controlledcontroller to get close to position.position (below).
4. Set up the appropriate light path (below).
5. Use the microscope to adjust focus and position (below).

BrightfieldXY Stage Control

Joystick with labels for fast and slow

The XY stage controller is the joystick on the air table.  Press the top right button to set the mode to fast / coarse motion.  Press the bottom right button to set the mode to slow / fine motion.

Bright-field Light Path and Controls

3/4 view of microscope with numbers for all the steps and arrows for the light path

Light path set to Eye and Epi shutter set to ---

Epi Control switch

Bright-field controls

1. Verify that the lamp is on by putting your hand in the air path between the lamp and the condenser.  If it is not, it may be switched off on the left panel or the intensity set to off.

Verify that the condenser turret is not blocking the beam. Bring the Transmitted Light arm forward. Set the light path (on front panel) set to Eye CondenserUse dialthe EPI Shutter button on the right panel to set to not block the upper beam. Transmitted Light arm should be forward. EPI Shutter--- (buttonsSecond online right panel,will read on front) set to PSF Out ---). (or Otherwise you will get a combination of Brightbright and Fluorescent)fluorescent. LightUse controlthe islight controls on the left side panel onto set the microscope.intensity of the light.  On/Off is for the brightfieldbright-field light.  The knob is for intensity.

If you cannot see anything in the eyepieces, check your light path again.

Fluorescent Light Path and Controls

3/4

Light path (on front panel) set to Eye Condenser dial set to block the upper beam, or turn the brightfield off on the left panel. Lever on the right sideview of the filtermicroscope turretwith mustnumbers befor back.all the steps and arrows for the light path

Fluorescent filters

Beam block

Epi shutter button

Eyes and channel

Block the bright-field light with the condenser turret.  This is a beam block. Just to the left of the lamp, there are a number of filters.  These do get moved to the wrong spots.  Left to right, they are:

1. Iris: Should be pulled out.
2. A.S. Two knobs
3. ND4: Neutral Density Filter: should be pulled out for maximum intensity
4. ND8: Neutral Density Filter: should be pulled out for maximum intensity
5. Filter: Should be pushed in.  If out, it will block the path.  Can be used as a beam block.

Lever on the right side of the filter turret must be back.  This is a beam block. EPI Shutter: set to one of the epifluorescent channels: 

1. DAPI
2. FITC
3. TxRed
4. D-F-T

Light path (on front panel) set to Eye

XYZ ControlsFocus

The1 stageFocus XYknob control- isUp the/ joystick.Down, 2 Coarse, Medium, Fine.

The Z control is the focus knob.  Top towards you is up.   There are coarse, medium and fine settings.

Data Acquisition

 StedyCon Welcome screen

 

Acquisition

On the welcomeStedyCon Welcome screen, press Start Up.  The process will take a few minutes.  Wait for the status to change from Starting Up... to Ready for Imaging.

Click on Startup.  The start process will take a few minutes.  The laser box Standby/Ready light will turn green.  Eventually, the status will be Ready for Imaging.

Status and Interlocks

Interlocks tab with 5 green and 2 red

Click on the Status Info button and a Status window will pop up in a new tab.

Double click on the Interlock line in the STEDYCON State section.  This will pop up a new tab with the Interlocks status.

There are 7 interlocks.  The first 5 shouldmust be Green before starting a scan.

1. Key: Is the key on the laser box on?
2. Head:
3. Interlock 1:
4. TL arm & Condenser:  There are two interlocks:
   1. TL arm: is the transmitted light arm (tower) forward?
   2. Condenser: is the dial set so that the brightfield path is blocked
5. Beam path: On the front of the microscope, switch the path to L100
6. Software: will still be red.
7. Combined: will still be red.

The fluorescence source is not part of the interlocks.  You still need to block its path to get a good image.  
- Use the lever under the filter wheel to block the fluorscencefluorescence source.  
- Set the fluorescence filter to ----.

Start New Session

Click on Start New Session.  This will bring up the main window and the Define New Session dialog.

New Session Dialog

• Session Name:
• Sample Name:
• Objective Magnification:
• Objective NA:
• Choose Your Dyes:
  • Dye Preset: pick from drop down
  • Display Name
  • Colormap
  • Colormap in Overlay
• Load Settings: pull up settings from ?
• Let's Go

Scan Controls Overview

YouMain canwindow getwith numbers for areas

1- Scan Control

Scan Control section

Session Settings: Goes back to this with the Session Settings buttondialog. topOverview: left.

Overview button will doDo a quick scan.  LoopThe loop will do continuous quick scans.

 This is good for focus and placement.

Gallery: opensOpens a new tab with thea gallery of images from this session.

Acquire

Acquire: will doDo a normal scan.  LoopThe loop will do continuous normal scans.

Stop: Stop the scancurrent

scan.

Auto: I think that this is autoAutomatically save new images to the gallery.

Finish Session:

finish this session, with the option to begin a new session or quit.

Acquisition Mode: Cancan turnset onto Confocal and/or STED

Confocal: Excitation Powerpower and Acquisitionspeed. Speed Set

so that the photon counts are high enough (>70?) but do not saturate.

STED: Excitation Power,power, ResolutionResolution, andSpeed. Acquisition Speed

2- Image Viewer

Image Viewer

Z:  YouThis can controlcontrols the focus or Z position of the scanscan. either with If the focusmouse knobis onover the sliderZ line, the mouse wheel can be used to themove leftup ofand down.  If you hover over the image.Dot,

the Z-position will pop up.

Channel display:/  Overview: At the top is a list of the channels.  The channel being displayed is underlined.  IfAs you are usingsetting moreup thanlaser one channel,power, make sure the correctright onechannel is underlined.selected.

3- Scan Parameters

Scan Parameters - with important ones opened up

Z-Stack:

Acquire z-slice and select z-stack: The z-slice can be useful in selecting the z-stack and in setting the laser power based on all the depths. 

Lenses

Magnification	Immersion	Numerical Aperature	Working Distance
4X	Air
10X	Air
20X	Air
40X - Not There
60X	Oil
100X	Oil

Save Your Data

Gallery View

In the gallery, you can edit what data you want to save.

Select images by clicking on the check box beside them - or use the Select all button.

Selected images can be downloaded, or you can download all.

Alternately, when you click Finish Session, you will have the option to download or delete the session images.

 

Copy

Whereyour dodata theto imagesyour go?own  

drive

Your data ends up in two places:

• The C drive downloads folder as a .zip file named:  yyyy-mm-dd session name.zip
• The S drive as a folder: yyyy-mm-dd session name

To copy your data, copy the S drive folder to your own hard drive.

What happens to the C drive folder?

What is our data clean up policy for this microscope?

Clean up

Take your sample off the microscope.

If you used oil on the objective, please use lens paper to remove the oil.  The process should be taught during the hands on training.

Power Off

TheOne power off procedure is basically the reversephoto of the microscope power on.switch with number 1

One Photo of the bottom of the laser box and the bottom rack - all off up with numbers 2-6

1. FinishMicroscope: Session.

Click Shut Down the system in the StedyCon software.  Wait for it to shut down.  Status will now be Standby. Close the software. Reverse order on the switches.

 

Turn off the lasermicroscope key.with the switch on the lower right side near the back.  The display will be off. Normally,Fluorescent weLamp doPower notSupply: turnTurn off the laserswitch box.on the front right.  Green and Orange LEDs will be off. Nikon Brightfield Lamp Power Supply: Turn off the switch on the front right.  Green LED will be off. Tango Desktop: Turn off switch on back top left.  Green LED will be off. Piezoconcept: Turn off switch of from left.  Green LED will be off. Laser Box: Turn the Laser Key to OFF.  The top two lights Power (Red) and Standby/Ready (Orange) remain on.

Normally, the computer is left on or put to sleep.