Zeiss Axiovert 200 Manual de usuario

18 Feb 2011,<qsg-wf1> p. 1/8

QUICKSTART GUIDE:

WIDEFIELD 1

ZEISS AXIOVERT 200 /VOLOCITY

(SAFB 409)

Startup procedure................................................................................................................2

Adjusting brightfield Köhler illumination ...............................................................................2

Observing fluorescence through the eyepiece.....................................................................3

Acquiring images with the Improvision Volocity software.....................................................3

General informations................................................................................................................................. 3

Acquiring the image ...................................................................................................................... 3

refocussing/ re-centring: ........................................................................................................................... 3

adjusting channel presets:........................................................................................................................ 4

multidimensional acquisition setup: .......................................................................................................... 4

Shutdown procedure............................................................................................................5 

Important additional information...........................................................................................7

Volocity Video Toolbar.................................................................................................................. 7

Auto Exposure .............................................................................................................................. 8

Auto Contrast................................................................................................................................ 8

Keypad controls: ........................................................................................................................... 8

Control buttons on the lefthand side of the microscope:............................................................... 8

File formats ................................................................................................................................... 8

merge colours: .............................................................................................................................. 8

Facility for Imaging

by Light Microscopy

Observing Life As It Happens

Imperial College London

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Startup procedure

Turn on (in this order):

•if required: heater unit and heater, 4 hours before live imaging to allow focus to stabilise:

•cplate holder

•dchamber

•if required: CO2supply

•switch on the mercury fluorescence lamp

•MAIN switch on the wall, on the right side of the microscope

•computer

Adjusting brightfield Köhler illumination

•make sure the LIGHT PATH (button on the left / bottom) is switched to 100%

BINOCULAR: press button until displayed on the microscope top display

•switch to 10x objective with the objective buttons on the right side of the

microscope

•you can move your specimen in xy-direction with the joystick, holding the

button on top of the joystick makes the stage moving faster

•focus on your sample

•if focus beeps, override the top limit by pushing the top focus button on the

right side of the microscope while turning the focus wheel (when you hear double beeps, the

mechanical limit of the focus is reached which cannot be overrun)

•open brightfield shutter with S1 (labelled ‘BF’ on the filter wheel pad)

•adjust brightness with large black button on front / bottom of

microscope)

•make sure condenser iris is not completely closed (two buttons on

top of illumination filter wheel)

•completely close field iris (top silver lever) (if the image turns

completely black, reopen until you see some light, proceed to next

step and close again after you have focussed the iris)

•focus iris (black condenser focus wheel)

•centre iris (2 silver Köhler screws)

•reopen field iris until the edge is just not visible anymore

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Observing fluorescence through the eyepiece

There are 2 options to observe fluorescence: the common way would be to use the filter cubes

in the cube revolver; for some applications the use of a dichroic mirror in the filter cube position

together with the filters in the filter wheels is necessary. The latter setup will only work with the

camera, not with the eyepiece.

•make sure the excitation / emission filter wheels are in the open position,

unless needed:

•on the filter wheel pad, select FW1 and then HOME

•repeat for FW2

•with the REFLECTOR buttons choose the illumination filter (microscope,

right / bottom behind focus wheel)

•switch between brightfield and fluorescence

with FL ON / OFF (left / front / top button on

microscope)

•Æturn fluorescence immediately off if not

needed, to avoid bleaching!

•find your sample

Acquiring images with the

Improvision Volocity software

General informations

All microscope parts can be controlled by the software (shutters, filters, focus, …), except the light

path (BINOCULAR/ FRONT PORT / BASE PORT) and the objectives

Acquiring the image

•start the VOLOCITY software (make sure the dongle is attached to a USB port)

•click on the camera icon to activate the live view and microscope controls(for more information

about the Video Toolbar in the upcoming window see Additional Information section)

•on the microscope stand, change the light path to BASEPORT

•refocus/ re-centre (if needed, see "refoccussing/ re-centering")

•adjust the channel presets of the channels you need (see "adjusting channel presets")

•adjust other acquisition parameters (see "multidimensional acquisition setup")

•with a click on the camera icon on the video toolbar you capture a single frame with the

settings actually displayed in the video toolbar

•with a click on the red button icon on the video toolbar you start your recording as defined in

"Acquisition Setup"

Useful: by clicking the "Freeze" button in the software you can keep your actual live image

freezed on the screen; the acquisition stops and the shutters will close, preventing your

sample from bleaching.

refocussing/ re-centring:

ochoose the brightfield preset (preset button 1)

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orefocus/ re-centre

ochoose a flourescent preset

oif there is only a faint signal switch "Auto Contrast" on

orefocus/ re-centre

oswitch off "Auto Contrast"

adjusting channel presets:

ochoose a preset button for your channel settings by clicking on one

ostandard presets for filter cubes are

1 = Brightfield

2 = DAPI/ Hoechst

3 = rhodamine / TRITC/ Alexa543

4 = GFP / FITC/ Alexa488

oadjust

oexposure time, binning, gain and offset (make sure "Auto Contrast" is off!!!)

obit depth

oright click on the preset button > Overwrite

multidimensional acquisition setup:

oset multiple channels

oopen "Acquisition Setup" dialogue

otick "Change channels"

oadd as many channels as you need with the "+" button

ofrom the appearing drop down menus choose the desired channel presets

oif you don`t want to do a stack, time series or multiple positions set timepoint to 1,

choose no focus and no xy

oset stack

obefore any other adjustments: click on Z position button and set "Top" and "Bottom"

to the mechanical limits

ousing defined focus positions

move focus to mid focus plane

click on Z position button, choose "Set Zero"

move focus to top position

click on Z position button, choose "Set Top"

move focus to bottom position

click on Z position button, choose "Set Bottom"

in "Acquisition Setup" dialogue

•set "Change focus using" to "Zeiss Focus Drive"

•choose and set Z-spacing or number of slices

ousing Autofocus

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in "Acquisition Setup" dialogue

•set "Change focus using" to "Zeiss Focus Drive"

•choose and set Z-spacing or number of slices

•set "Auto-focus using" to "Zeiss Focus Drive"

•choose the lightpath where the Autofocus should take its cues from

oset time series

oin "Acquisition Setup" dialogue: set duration

oin video toolbar: set intervals

oset multiple positions

ostage needs to be calibrated for this

remove sample!!!

switch to shortest objective (10x, 2,5x or empty position)!!!

move objective turret to the lowest position!!!

choose "Calibrate Stage" from the "Stage" menu

in the "Acquisition Setup" dialogue set "Change XY using" to "Ludl XY

Stage"

o"handmade" positioning

"Video" menu > "Live Video"

"Stage" menu > "Clear All Points"

move to desired point

"Stage" menu > "Add Point"

with "Stage" menu > "Review Points" you can adjust the focus for each of

your points separately

oautomated positioning

"Video" > "XY Stage

"Stage" > "Make Well Overlay"

"Stage" > "Create Points from Well Overlay"

follow the instructions

Shutdown procedure

•check if anyone is booked after you within 2 hours

If nobody is booked within two hours:

•remove specimen

•clean oil objective lenses with lens tissue

•Important: close incubation chamber completely (Æprotects from dust)

•update time in Sharepoint

•secure your data (e.g. copy them to the server)

•shut down hardware in reverse order from startup

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If someone’s booked within two hours:

•update usage in Sharepoint

•remove your samples

•clean objective lenses with fresh lens tissue and close incubation chamber

•clear up the desk

•save files onto the server or on a mobile harddrive

•log off

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Important additional information

Volocity Video Toolbar

18 Feb 2011,<qsg-wf1> p. 8/8

Auto Exposure

•sets max pixels to 80% (not 100%) (can also be applied to a selected area)

Auto Contrast

•unlike Gain only affects appearance of the image on the screen(!!!)

Keypad controls:

•top level button 1 = transmitted light on/off (#2-4 = empty)

•FW button switches between control of:

•FW1 = excitation filter wheel (orbit excitation filters, only used for FRET or CFP+YFP)

•FW2 = emission filter wheel in front of camera, underneath microscope

the orbit excitation filters must be in HOME position for use of the emission filters; if

they aren’t, press the HOME button on the external keyboard

Control buttons on the lefthand side of the microscope:

•lowest button:

•switches between frontport (empty) <> eyepiece <> baseport (camera)

•middle button:

•switches between sideport 100% (empty) <> 50/50 <> eye/ front/ base 100%

•top: Optovar in / out

File formats

•The original Volocity libraries are stored as "mvd2" files together with a folder containing the

image data in "aisf" files

•TIFF:looses all enhancements but keeps original ("raw") data

•all stacks/ channels are stored in 1 multipage TIFF with the original bit-depth (8 or 12 bit)

•TIFF for publication

•screen version of image, including colours, merge, enhancements, on-screen image

resolution, it is always 8-bit

loss of original ("raw") data!!!

merge colours:

•CREATE NEW IMAGE SEQUENCE > MULTIPLE CHANNELS

•drag single channel (or z / timepoint) images onto new image sequence window

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