Zeiss 710 Confocal Microscope User Guide

Cornell University - Biotechnology Resource Center – Imaging Facility

Zeiss 710

Confocal Microscope

User Guide

B46 Weill Hall

BRC Imaging Facility

1

Table of Contents

Startup – General Procedure 2

Microscope General Info ₂

To see your sample thru the microscope ₃

Zen software – Locate (Eyepieces) ₄

Zen software – Acquisition ₅

Configurations ₅

Smart Setup ₅

Initial Settings for Live Scan ₇

Confocal Scanning ₈

Saving Image Files 9

Z-Stack 10

Saturated Pixels 11

Scale Bar 11

Optimizing Image Quality Chart 12

Multi-tracking (Sequential Imaging) ₁₃

Time Series ₁₄

Tile Scan ₁₄

Reflected Light Confocal ₁₅

Transmitted Light (Bright Field) ₁₅

Positions or Multi X-Y-Z ₁₆

PhotoBleaching (FRAP) or Photoactivation ₁₆

Definite Focus ₁₇

Zen Light 2012 Browser ₁₈

Adjusting the Brightness and Contrast 19

DIC ₂₀

Specifications ₂₁

2

Startup – General Procedure



See instructions posted

 Login to computer with net id and netid password  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 o Remove protective cover

o 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:

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:

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

3

Stage control

 X-Y joystick - Top button toggles between very slow and normal speed (beeps)

 Focus

o Manual coarse and fine knobs on each side o Away is up (see arrows above left knob) o You must manual focus the first time  Quick Focus

o Auto up and down with buttons on right focus knob, see arrows on scope

o Always lower objective with down button before placing or removing samples.

o 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

o Blue->green->red (towards red)  Second last button:

o 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

Left Focus Knob

blue>green>red red>green>blue

4

Zen software – Locate (Eyepieces)

 Choose Bright Field or Fluorescence  If no buttons or buttons don’t work:  Turn on desired lamp

o (12% for fluorescence)  Open shutter

 Close other shutter

 Choose filter (for fluorescence)  Find sample with eyepieces, focus  When ready for confocal:

o Hit Light Off avoid photobleaching o Acquisition—to do confocal

Bright Field

Fluorescence

5

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

6

Smart Setup Options

Name

Autoscan

Frame size

Scan Speed

Bit Depth

Scan direction

Pinhole

Current

No

512 x 512

9

8

single

1 AU

Quality

Yes

1700 X 1700

6

12

single

1 AU

Speed

Yes

400 x 400

10

12

bi-directional

1 AU

Standard

Yes

512 x 512

9

8

single

1 AU

Widefield Like

Yes

512 x 512

9

8

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

o 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

o Zen2012 is the default

o Once loaded or saved, it will load automatically every time

7

Initial Settings for Live Scan

Note: see Optimizing Image Quality for more information.

Acquisition Mode window

 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

8

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

o Graphic o Values o Sliders

o 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

9

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

 Free (see below)

 Find in the Imaging Fileshare Zeiss 710 folder

 Zen 2012 Full version o 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

o 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)

Saving .tif images

There is a macro that will export a list of files to .tif all at once. File- Export to .tif

10

Z-Stack

 Check Z-Stack box and open window  Focus (down) to desired starting point  Set First

 Focus (up) to desired ending point  Set Last

 Stop scanning

 Choose optimal step size  Start Experiment

Note: always focus down to set First. Zen only collects against gravity. Set Zero

 Only if you want, not required  Go to Focus window

 Show all

 Manually set zero position

Optimize Sectioning and Step

o Best for 3D imaging

 Match Pinhole: no overlap, no gaps  X:Y:Z=1 gives cubic pixels

Imaging your Z-Series

 Ortho- gives you a cross section at any location, cover slip (start point) is to the outside

 3D – to do a live rotation. o Use Maximum Projection o House icon to return o Create image to save  Cut—slices in another direction

Note: Volocity is another software option for doing 3D visualization and analysis

11

Saturated pixels

Range Indicator

 Saturated pixels are red,

 Black (zero value) pixels are blue.  Use Split view and adjust each channel  Aim for a few red and a few blue pixels,

max

 For quantification, avoid all red and blue pixels

 collect 12 bit images See Optimizing Image Quality

Note: You can always make your images brighter

and the background blacker, with image processing, but you cannot unsaturate pixels after acquisition

Merged Image-Fewer channels

To remove a channel from the Merged Image, Click on the name of the channel above the color bar, eg Ch1 or T-PMT. This only changes what you view, the image is still acquired and saved.

Scale Bar

Overlay  Click on ruler

 Click on image and drag to set length.  Change color, etc

To save scale bar on image:

 Change to single image (2D vs Split)  Use 100% for screen zoom

 File-Export

 Choose High resolution contents of image window

 Choose channels  Save as tif

Note: scale bar is saved as a screen shot.

Saving contents of image window will save exactly what you see in the Image Window.

12

Optimizing Image Quality

Option Optimal Range What it Does Pros Cons

Increasing Brightness

Master gain - increase 450-750+ Amplifies signal, makes image brighter No effect on photobleaching High gains amplify noise Laser Power - increase 2% - 20% More excitation light No increase in noise Can increase photobleaching Pinhole - open wider 1AU - > Collects thicker slice, more emission light

No effect on

photobleaching _{Decreases Z resolution} Adjust Display Best Fit or min/max Only affects view, not data See low signal better Use with 12 bit images

Increasing Signal to Noise Ratio

Master Gain - decrease < 700 Lower brightness of image Image less noisy Need to increase laser power Line Average - increase 2-8 Repeats scans to collect more excitation light Image less noisy Slow, increases photobleaching Scan Speed - decrease 7-9

Slower speed collects more

excitation light Image less noisy Slow, increases photobleaching

Increasing Speed

Scan Speed >9 Scans faster Fast, less damage Collects less light

Bidirectional Scanning On or Off Scans in both directions Cuts time in half, no loss of light May produce artifacts at high resolution

Increasing Resolution (decreased pixel size)

Zoom up to minimum pixel size Scans smaller area Pixel size is reduced Image area reduced, increases photobleaching # pixels 1024 x 1024 Divides image into more pixels Image area not reduced Less light per pixel

Zoom + tiling up to minimim pixel size Scans a larger area Pixel size is reduced Takes time, increases photobleaching, poor stitching High NA objective 1.2-1.4 Can resolve smaller

13

Multi-tracking (Sequential Imaging)

Use Smartest (Line) in Smart Setup

Check  Channels  Laser Lines  Colors  Pinhole

 Main Beam Splitter

Note: Set Pinhole to 1 AU on track with longest laser wavelength

Start Live scanning Adjust

 Gain (use Digital Gain for ChS1 and ChS2)  Laser Powers, etc

Save Configuration in upper left corner of Zen

Note: With line method, only the channels (on/off) and lasers (on/off) can change between tracks

Line method is preferred unless you absolutely need to change something else between tracks, like pinhole, or beam splitter. Then you need the frame method. This is often the case with reflected light.

14

Time Series

 Click on time series at top

 Set interval and total number of scans  Start Experiment

o If you also have Z-Stack checked, you will get an xyzt series Definite Focus will keep sample in focus over time.

See more notes below

Tile Scan

Use to get large field at high resolution  Set number of tiles (eg)

 3 x 3 (odd numbers) puts current image in the center

 2 x 2 (even numbers) cuts up the current image

 2 x 8- can be non-square  Rotate image to fit tile grid

 Scan overview image for quick view  Set# tiles

 Set objective  Start Experiment

o You can do a z-stack at each tile, set up as usual

Note: Tile Scan creates one large image and the stitching is not perfect. To get a perfect, stitched image, or to get separate tiles, you can use the Multitime Macro. It tends to be much slower and does not rotate. We will be getting a better stitching algorithm.

15

Reflected Light Confocal

Light Path

 Set Main Beam Splitter to T80/R20  Check box for Reflection

 Create small region under desired laser line  Can do fluorescence and reflected light Note: For brighter fluorescence, use frame sequential Will not work with 405 line.

Transmitted Light (Bright Field)

 Click on T-PMT.  Adjust gain and offset  Gain = brightness

 Offset = blackness or contrast

o Can be negative to increase contrast  Uses whatever lasers are active

o Changing any laser power affects this image

Note: This image is not an optical section For a high quality image see DIC

Reflected Light

16

Positions or Multi X-Y-Z

 Click on positions in upper left  Got to desired X-Y-Z location  Add position, repeat

 Return to first position  Start Experiment

For Z-stack at several positions

 Use the center option to define the z-stack at the first position  Then just add each location when in the center of the object.

o You must do the same range and step size for each position

o X-Y-Z coordinate is recorded

o Software sets an offset to relate to center of the first position

Center Option for Z-series

 Manually focus to the center of sample  Hit Center

 Choose Interval, then calculate the # of steps needed to get the distance you want and set steps

o Use the focus window to set the center to zero, if desired

Range Select

Will do x-z scan, you can change center or top/bottom on this image

PhotoBleaching (FRAP) or Photoactivation

 Choose Bleach at the top

o Time series and Regions should also activate  Choose # of before scans

 Iterations is the number of scans during the bleaching o More iterations will bleach longer

 Repeat bleach-don’t use this, it repeats the before scans, too.  Set laser and laser power

o 405nm at high percent will give fastest bleaching

o Slower scan speed will give better bleaching, use fewer iterations o Zoom bleach can be faster but is less spatially accurate

 Define region(s)

o Define control regions, too

o Choose which to bleach and analyze  Set up time series

o Time series does not include the bleach step, just pauses  Start Experiment

17

Definite Focus

 Uses a very far red laser (750nm) to find the coverslip and maintain the same distance from the objective to the coverslip.

 It does not follow your moving cells

 Works with a time series +/- Z series, positions, tiling.  Helps to have >10 sec between acquisitions (end to begin)

o Other things are possible with the Multitime Macro

Touch Pad:

 Home, Settings

o Set how often to check focus (this is disabled during the time course and Zen uses its own interval so you don’t really need to do this)

 Home, Microscope, XYZ, Definite Focus

o ON or Once – watch on Touch Pad or on Power supply o Will be ‘Waiting’ between focus checks on control box  Watch it on the Touchpad. Let it check once or twice.  Hit OFF

Focus Devices and Strategy (Zen Blue Bar)  Need to have Time checked

o Choose Definite focus o Choose how often to check  Start Experiment

 You will see it ‘Setting Focus’ on the control box  Or watch the Touchpad to see it check focus.

 This is usually fast but sometimes can take up to 10 seconds

Batch Export

There is a macro that will export a list of files to .tif all at once. To Load the macro the first time

Macro-macro

Assign macro

Menu entry - choose 1 or whatever

….Browse, find C:\zen\macros\Batch export

Text, type in Batch Export (this will appear in the dropdown menu) Apply, wait

Close window

To Use, Macro – Batch Export Find directory (folder) – Open

Highlight files – choose only single image or series files, do not mix Single image with raw data or Series with raw data

For individual channel tifs, check the box for Monochrome For merged tifs, leave unchecked and choose channels

You cannot mix these, you have to do them separately You can only use Red, Green, Blue, no other colors No greyscale for brightfield images

Click box for Long file names (?) Do not click box for overwrite files

Name will be the original file name plus ch1, etc Choose .tif

18

Zen Light 2012 Browser

Get this free browser from the fileshare where you retrieve your image files. Look under Zeiss710. Install on any PC anywhere. They now have a Blue and a Black edition. Both have 3D capabilities, but the Black has more confocal features and the Blue has better export and image processing features. If you have a 32bit computer, you can only use the Blue edition. Zen Installation Instructions for a 64-bit PC: (Note: this takes a while.)

1) Find the folder Zeiss 710 on the fileshare where you download your files from 2) copy this file to your computer: ZEN_2012SP1_black_SP2_blue.exe

3) double click to extract

4) follow instructions to install but X-out AxioCam, CanCheck, System Maintenance Tool and MTB (see pic below)

5) Install drivers (there are many)

6) This will install both the black and the blue versions a. The black has confocal features-recommended

b. The blue has more image processing/analysis features c. Both will do 3D rotations

Zen Installation Instructions for a 32-bit PC:

1) Download and install the 32bit.exe version. This is only the blue version but you do get the 3D rotation feature. (I think)

Sorry -- there are no Zen options for a Mac

Mac and PC users can open .czi files with the FIJI version of ImageJ available at

19

Using the Zen 2012 Browser

This looks just like the acquisition software, so you should be able to do basic things. Here are some extras:

Export to .tif

Use File-Export- Export Raw Data

Choose single plane or series, RGB image or pallet for single color Chose tif, 12 bit tif, 16 bit tif

Choose Full resolution image window makes the pixel size of the image match the pixel size of the monitor. This is the best way to capture a screen shot.

Use this to:

Save a scale bar or other overlay items (numbers, arrows, regions, etc) Save a greyscale image or an overlay including greyscale (use single, not tile) Save any colors other than Red, Green Blue.

Contents of image window gives a screen shot of whatever is in the image window. If you zoom up (make image pixelated) you will get only the area visible on the screen.

Processing-Copy-Subset: to remove frames from a Z-stack or Time series or X-Y crop Select current image, Adjust, Apply

3D

You can do lots of 3D stuff with ZenLight 2012. Rotations

Surface shadowing Make AVI movies

Etc. You should just play around.

For better 3D try Volocity (see Carol or Johanna)

Adjusting the Brightness and Contrast of your image

Photoshop (is the BEST)

Image--Adjustments--Levels Choose color(s)

Adjust histogram with little triangles on right or left of histogram

Center triangle is gamma, this effects linearity of brightness, use with care Gamma can brighten dim areas without saturating already bright areas. Save with new name as this is permanent and changes the image data.

Adobe Elements does the same thing, just look here: Enhance—Adjust Lighting—Levels (Ctrl-L) ImageJ (Fiji)

Image-Adjust-Brightness/Contrast or Color Balance

This gives you both the histogram adjustment and, brightness and contrast adjustment. No gamma

Note: Save with a new name. Always save your raw image or .czi file. These procedures change the pixel values of your image!

20

DIC (Differential Interference Contrast or Nomarski)

Note: Get help with this the first time, at least

At microscope (Ocular)

Set Kohler Illumination First:

 Close Field Stop near top of microscope

 Adjust condenser height to see and focus on small spot of light (polygon) o Should have sharply focused edges

 Center with centering screws  Open field stop to fill field

Note: you should set Kohler every time you collect a bright field image Condenser Settings

 On left is aperture -- Open fully for DIC

o Close partly for Bright Field (to increase contrast)

 Closing too far lowers resolution, better to increase contrast by lowering offset in software

 On right are settings—must look on Touch Screen to see current setting o Options are PH2, PH3, BF, DICII, DICIII

o Use DICII for 25x, DICIII for 40x or 63x

Note: you must have this setting on the Touch Screen even when in confocal acquisition mode. You may have to resave your config so it stays.

Filter Cube Setting (right focus knob)

 Use POL for DIC. See setting on Touchscreen.

o This is only for eyepieces, it will change when acquiring an image.

Upper Polarizer

 This must be rotated to the left when using the eyepieces  When switching to confocal, rotate to the right

 Clicks into correct position Wollaston Prism

 This is a small filter with an adjustable screw just below the objective. Each objective has one (except 10x) but they are not kept on the microscope.

 Must be inserted (and removed) each time and this is best to do before moving objective into position.

 Adjust the screw as desired (try to get your hand in there while looking thru the eyepieces)

o The center of the adjustment should be darkest, as you move away in either direction the DIC effect maximizes and then goes away.

o Light / dark shadows flip from one side to the other as you cross the center. You may get nice colors in the eyepieces.

Confocal Acquisition of DIC

 Make sure condenser setting is still on DICII or DICIII  Click on T-PMT

 Feel free to lower offset to a very negative value, increase gain as needed. This increases contrast.

21

Specifications

Lasers

Objectives

Mag/ NA Imm Name Coverslip WD(mm) Ring Color

10x/ 0.3 dry EC Plan-NEOFLUAR - 5.2 Yellow

25x/ 0.8 Oil/W* LCI Plan-APOCHROMAT +/- 0.55 Red/Green

40x/ 1.2 Water C-APOCHROMAT 0.14-0.19** 0.28 Blue

63x/ 1.4 Oil Plan-APOCHROMAT +0.17 0.19 Black

*25x: Set to Red mark for oil, Green mark for water

**set for cover slip thickness. #1 cover slip is 0.13 μm, #1.5 cover slip is 0.17 μm. #1.5 cover slip is recommended, esp for oil immersion

WD = Working distance: how far the objective can focus past the coverslip

Focus

Coarse 1 revolution = 2mm

Fine 1 revolution = 0.2 mm (200μm)

Filters in Microscope – Axio Observer .Z1

Position Set# Ex BS Em

1 empty

2 02 G365 FT 395 LP 420 (Long Pass)

3 09 BP 470/40 FT 510 LP 515

4 15 BP 546/12 FT 580 LP 590

5 POL Polarizer for DIC

6 Mirror for LSM

Stage Speed Focus Speed

10x 7 4

25x 5 3

40x 5 2

63x 3 1

Resolution

Collect 2-3 pixels over the smallest object you want to resolve Maximum zoom (at 512x512) and minimum pixel size

Objective Max Zoom Minimum pixel size

10x 5.0 0.30 um (300 nm)

25x 6. 0.10 um (100 nm)

40x 6.3 0.08 um (80 nm)

63x 4.0 0.07 um (70 nm)

22

Common Errors and Problems

Inkubator

When starting, small box in lower right will say “Error…Inkubator…”



No light at microscope in Ocular mode

 Is the lamp on in the screen graphic?  For fluorescence is the power >0% ?  Is the shutter open?

 If yes to all of these, hit Offline, then Online again. This usually works.

Zen not loading

Blue bar is stops moving after ~1/4 inch:  Stop and exit

 Find large black box on the table in the corner, it looks like a computer and is called the Real Time Controller (RTC).  Open front (like a door—far side opens).  Hit reset button (yellow triangle).

 Watch for red flashes, takes a minute  Restart Zen.

Blue bar stops moving near the end:  You will also get more error messages in

the lower left box.

o You will need to restart everything.  Stop Zen and restart Windows

 Turn off all buttons on white control box.  Then quickly turn Main back on

 Wait 5 sec and turn on 2 small buttons  Turn the laser key off and on again.