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Cornell University - Biotechnology Resource Center – Imaging Facility

Zeiss 710

Confocal Microscope

User Guide

B46 Weill Hall

BRC Imaging Facility

8-6-2014

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Table of Contents

Startup – General Procedure

Microscope General Info

To see your sample thru the microscope

Zen software – Locate (Eyepieces)

Zen software – Acquisition

Configurations

Smart Setup

Initial Settings for Live Scan

Confocal Scanning

Saving Image Files

Z-Stack

Saturated Pixels

Scale Bar

Optimizing Image Quality Chart

Multi-tracking (Sequential Imaging)

Time Series

Tile Scan

Reflected Light Confocal

Transmitted Light (Bright Field)

Positions or Multi X-Y-Z

PhotoBleaching (FRAP) or Photoactivation

Definite Focus

Zen Light 2012 Browser

Adjusting the Brightness and Contrast

DIC

Specifications

Common Errors and Problems

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Startup – General Procedure



See instructions posted



Start Zen software



Start System



Choose objective, add immersion if needed



Place sample on stage



Focus on sample with bright field or fluorescence



Switch to Confocal Acquisition



Load configuration



Start Live Scan

Microscope General Info

Objectives

Right focus knob, far buttons will change objectives.

Objectives are covered with spill protectors but are in

this order:



10x is dry –has large top lens and yellow ring



25x is multi-immersion, has red and green top rings

Remove protective cover

Set black line on knurled ring for immersion



Oil use red tape setting



Water use blue tape setting



40x is water immersion, has blue ring, set knurled ring

for cover slip thickness (black line to 0.17 = #1.5)



63x is oil immersion, has black ring and paper collar

Note: See Stats at end for more info about objectives

40x H2O

cover slip setting

63x Oil

40x Dipping

10x Dry

25xOil/H20

H2O setting Oil setting

Ending your Session:

Check schedule

Save your images

Exit Zen software

Transfer your files to

Imaging Lab FileShare

Logoff Windows

Lower objective, remove sample

Wipe oil or water off objective

STOP HERE

Total Shutdown:

Start as above

See instructions posted

Also:

Shut down Windows

Cover microscope

NOTE: after hours, if the next

person is not there,

Do Total Shutdown.

When in doubt, do total shutdown.

Change Objectives

with buttons on

Right focus knob

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Stage control



X-Y joystick - Top button toggles between very slow and

normal speed (beeps)



Focus

Manual coarse and fine knobs on each side

Away is up (see arrows above left knob)

You must manual focus the first time



Quick Focus

Auto up and down with buttons on right focus knob,

see arrows on scope

Always lower objective with down button before

placing or removing samples.

Up button returns to previous position



Do not use up button until you have found the focus manually.

Sample loading onto microscope



Put oil or water on objective (not 10x)



Put sample on stage, cover slip down, center with joy stick



Manually focus up (away) until immersion liquid contacts sample, lower slightly but don’t

lose contact with immersion liquid



Find focus through eyepieces.

To see your sample thru the microscope

Without using Zen

Brightfield

Touchscreen



Press ‘Set Work Position’ to get rid of this screen



Home-Make it Visible for viewing bright field



Look thru eyepieces and focus up



Wiggle the stage as you go, look for movement

Fluorescence

You must Start Zen software first, see next page

Once you set up for fluorescence, you can change the filters on the

microscope:

Fluorescence Filter Cubes:



Use 2 back buttons on left focus knob to change filters



Last button

Blue->green->red (towards red)



Second last button:

Red->green->blue (towards blue)



If spinning, hit same button twice

Note: Fluorescence at the microscope uses the Mercury-

Halide lamp, not the lasers. The Blue and Green filters

are Long Pass (LP) meaning you will see all colors through them.

These filters are not used for confocal imaging.

Make it Visible

for bright field at

the eyepieces

Right Focus Knob

Quick focus

Left Focus Knob

blue>green>red

red>green>blue

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Zen software – Locate (Eyepieces)



Choose Bright Field or Fluorescence



If no buttons or buttons don’t work:



Turn on desired lamp

(12% for fluorescence)



Open shutter



Close other shutter



Choose filter (for fluorescence)



Find sample with eyepieces, focus



When ready for confocal:

Hit Light Off avoid photobleaching

Acquisition—to do confocal

Bright Field

Fluorescence

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Zen software – Acquisition

Configurations



To use an existing configuration you must

open the folder and choose the config



Or, open a previous image and Reuse



To make a new configuration or change an

existing one, use Smart Setup



Show all tools should always be checked

Note: You have to load a configuration each time you

start Zen. It does not load automatically.

If the software asks about the 561 laser and you have a red dye, say yes to turn it on.

Smart Setup



Use this rather than making your own or adding to an existing configuration. Adding a dye to

an existing configuration often will not work.



Choose dyes and colors, starting with lowest wavelength



Choose Current (default)



Choose Smartest (Line)



Apply

Smart Setup Options

Name

Autoscan

Frame size

Scan Speed Bit Depth

Scan direction

Pinhole

Current

512 x 512

single

1 AU

Quality

Yes

1700 X 1700

single

1 AU

Speed

Yes

400 x 400

bi-directional

1 AU

Standard

Yes

512 x 512

single

1 AU

Widefield Like

Yes

512 x 512

single

Max

Note: numbers may vary. Recommended to use Current (no autoscan) and set these parameters yourself.

See Optimizing Image Quality for guidance.

Lasers



For Green dyes the 488nm laser is turned on with the key on the laser control box



For Red dyes (rhodamine, dsRed) use 561nm laser.



405nm (DAPI) and 633nm (AF647) lasers automatically go on as needed

Note: 488, 514 and 458 nm lines are all from the same laser

Light Path



Adjust emission (spectral) regions if desired



MBS (Main Beam Splitter) Filters

must match laser lines in use



For a Bright field/DIC image click on T-PMT

(transmitted light detector)

Note: bright field uses whatever lasers are on as the light

source, not a separate lamp

Detector ChS1

This channel can be divided into many colors,

designated S1, S2, etc. ChS only has one master

Gain. To adjust the brightness of the individual S

channels, use the digital gain. See next page.

Notes on layout and windows with blue bars:



Can be open or closed – click on blue bar



Can be expanded (Show all on blue bar) or not



Can be moved, drag blue bar (arrow in upper right corner

returns to home)



To make a new column, click in grey area and drag over



You can save your arrangement of windows using

Workspace config in the upper right corner of the

software

Zen2012 is the default

Once loaded or saved, it will load automatically

every time

Initial Settings for Live Scan

Note: see Optimizing Image Quality for more information.

Acquisition Mode window



Show All



Check that correct objective is listed



Pixel format: Click on X*Y to change



Use 512 x 512 or 1024 x 1024



Bit Depth: use 12 bits for measuring

fluorescence, else 8 bits



Use Bi-directional scanning unless using

very high resolution

Channels window



Laser Power (2% is a good starting point)



Pinhole: 1 AU for optimal section thickness



Master Gain (~500-750)



Digital Offset (keep near 0)



Digital Gain leave at 1.0



For 4+ colors, use the Digital Gain to

adjust the S1, S2 channels separately

Confocal Scanning

Live for a fast continuous scan

Continuous for a high quality continuous scan

(slow)

Snap to take an image

Save Immediately or open a new window

See Saving Images (p8).

Split to see individual color channels

Zoom-Crop Box

You must stop scanning to use



Position box on object of interest



Resize, move, rotate

Live to see zoomed image

Live Zoom

Active while live scanning



Adjust zoom value



Move, resize, rotate with

Graphic

Values

Sliders

Little arrows (most accurate)



Reset All to return to zoom 1, center

You can zoom out to 0.6 but the corners and

edges may be less bright

Notes on Zoom:



Real Optical zoom



Increases Resolution



Decreases Image Area

Resolution



Collect 2-3 pixels over the smallest object you

want to resolve



Do not collect smaller than the minimum pixel

size



Use higher mag/NA lens if needed

Minimum pixel size

10x pixel size ~ 0.30 um (300 nm)

25x pixel size ~ 0.10 um (100 nm)

40x pixel size ~ 0.08 um (80 nm)

63x pixel size ~ 0.07 um (70 nm)

Note: To get small pixels but a larger field you can collect 1024 x 1024 pixels or use tiling.

Can be adjusted while Live Imaging

Stop First, then Crop

Saving Image Files



Single images - Save Immediately



or open a new document window.



Z-stack, Time series, Tile, etc are not

overwritten. You can save later.



Save files to



D:\ALL USER IMAGES HERE



Save files as .czi.



stores all the hardware settings



Reuse the settings from any

image



Information to see settings



Copy Images to Z:Imaging Lab

FileShare



Retrieve from CU campus only



Retrieve and backup in a timely

manner

Opening .czi files later



Zen Lite 2012 Blue or Black



Free (see below)



Find in the Imaging Fileshare

Zeiss 710 folder



Zen 2012 Full version

On our workstation



FIJI (is Image J)



Free software for PC or Mac

Autosave



Saves your file automatically



Breaks up your .czi files



Separate files for each channel, z-plane, time point or position (not tiles)



Save as .czi

If you save as .tif, the colors will be wrong



Name is a base name and will be followed by date, time and other identifiers (ch, z, t)

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