Leica Sp5 ii User manual

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Instructions for the Leica SP5 II laser

scanning confocal microscope

Content: Check-in and Start up • Set up acquistion parameters • Optimize acquistion parameters • Acquire

a z-stack • Sequential scan • Check out • Appendix

Check-in and Start up

1. Main Switch Board: switch on PC Microscope, log on and wait until Windows XP is fully loaded

2. Main Switch Board: switch on Scanner Power, wait 5 S

3. launch LAS AF (desktop shortcut),

select the desirable Configuration

and scanner mode, then click ok

Note: Usually, Configuration is

Machine and Activate Resonant

Scanner is unchecked. However,

if the previous user has selected

simulated SP5 or used resonant

scanner, these will become the

default when start up, therefore please check carefully before clicking OK.

4. make sure the condenser is in the "up" position, click Yes to initialize the stage when prompted

this enables Mark and Find, and Tile scan

stage is configured to return to the position before initialization

5. Main Switch Board: when the software finish loading, switch on Laser Power and turn the key clockwise to

On-1 to enable Laser Emission

6. in LAS AF, click on the Configuration Operating step on top left to bring up Hardware Configuration

Laser: check to activate lasers that will be used for the session

Note: If you need the Ar laser, set the power to 20%. You can use higher power which will

shorten the life span of the laser; but one should never go above 80% into the red region.

(optional) Settings > Resolution > Bit Depth: select 12 Bit for 4096 grey levels, the maximum this

system can offer

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Set up acquistion parameters and acquire an image

1. Beam Path Settings in the central Work area: there are 3 ways to

configure excitation and emission parameters

A. use a preset

i. click on Load/Save single setting

ii. select a Leica or User Settings (for multichannels, the default

is simultaneous scan, see below for sequential scan)

B. reuse a setting from a previous scan

i. open a previous experiment

ii. in the Experiments tab, click to select the desirable scan

iii. right-click to choose Apply Setting or hit Apply button at the bottom

C. assemble from scratch

i. set laser power

activate the shutter

set power level of the desirable laser

line, 5% is a good starting point

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ii. select the beam splitter (dichroic)

selections for different laser lines

beamsplitter

laserline(nm)

RT 30/70 for

backscatter or

reflection

Substrate TD

488/561/633

458/514

RSP

500

488/561

spectra or details for the beam splitters are in the Appendix

405 ✓ ✓

458 ✓ ✓ ✓

476 ✓ ✓ ✓ ✓

488 ✓ ✓ ✓ ✓

514 ✓ ✓

561 ✓ ✓ ✓

633 ✓ ✓

iii. setup the detector

select the emission band: drag the lower and upper

bounds or double click the slider to enter the begin

and end wavelengths

Caution: Drag the sliders slowly or else they will

jam!

click on PMT n to adjust PMT Gain* and Offset*;

good starting values are 800 and 0, respectively

select a LUT as default

activate the PMT

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2. select the Acquire Operating step on top left, click the Acquisition tab to set up the data format

A. Acquisition mode panel

defaults to xyz simultaneous scan

seq. button enables sequential scan

Tile scan, and Mark and Find

Best Focus

B. XY panel (click ▶ on upper right corner to expand)

Format: frame resolution defaults to 512x512pixels, max 8192x8192pixels

Speed: line frequency defaults to 400Hz, scan area will be reduced above 700Hz, max 2800Hz

Zoom* and panning

Average and Accumulate

Scan Field Rotation*

Pinhole*: default is 1AU and displays in µm

3. acquire an image (buttons in the lower left pane of LAS AF)

Live: scan, apply accumulate if set

Capture Image: acquire an image at the current z position, apply both average and accumulate if set

Start: acquire all configured scans, e.g., z-stack, stage positions, tile scan, time points, spectral series etc.

*these parameters are also adjustable via the Smart Panel with the default configuration

Optimize acquisition parameters

1. center your prep in the field of view with either BF or FLUO (see Modes of operation in the Appendix)

2. to toggle on scanning, click Live or use the button on the left side of Smart Panel

Caution: if there is no signal in the viewer display Window on the right, DO NOT move the stage

controls (XYZ) or increase the laser power; try the following

i. with BF or FLUO, make sure object of interest is still in focus

ii. open up pinhole to increase the slice thickness

iii. raise PMT gain

iv. lastly, increase laser power

3. bring the object of interest into focus, adjust PMT gain, offset, and laser power to obtain an image

with good dynamic range

Note: the "glow-over, glow-under", Glow (OU) LUT which displays 0 intensity in green and

saturation in blue (255 for 8-bit or 4095 for 12-bit), facilitates this setup

i. top left side of the viewer display Window: activate the Glow (OU) LUT via the Quick

LUT button which cycles through the default , Glow(OU) , and grey LUTs

ii. adjust offset so the pixels in the area you think should be darkest just turn green, usually

around -0.2%; increase the sensitivity of the control panel offset knob may help

iii. vary the PMT gain or laser power so a few pixels in the brightest object of interest just turn

blue

4. with multichannel simultaneous scan, always check for cross-talk between channels by dropping the laser

power of the suspected channel to zero; if cross-talk does present, use sequential scanning

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Z-stack

Z-Stack panel

focus control is selectable: z-Galvo (default) or z-

Wide

focus (z-Position) can be adjusted with the

microscope controls, Smart Panel and also via

software (see z-drives in the Appendix)

z-drive controls

controls

z-drives

microscope

focus knob* Smart Move* Smart Panel range min.

step size

*the objective turret i.e., focus is always adjustable with the microscope focus knob and Smart Move, regardless of the z-drive

selection in the software

z-Galvo ✓±250µm 42 nm

z-Wide ✓ ✓ ✓ >4mm 49 nm

Acquire a z-stack

1. in Live mode, move deeper into the prep (z is more positive) to where the stack should start, click

Begin to register this z-Position (black: unset, red: set)

2. move towards the outside of the prep to where the stack should stop, click End to register this z-

Position

3. stop scanning, click the z-step size button and then enter the appropriate slice thickness (check setting

slice thickness in Appendix for recommendations)

4. select the number of passes to average in the XY panel

5. click Start (lower middle) to collect the stack

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Sequential scanning

1. set up a simultaneous scan for the fluorophores needed with regard to laser power, beam splitter, emission

band, and PMT settings; activate one laser line and detector combination at a time to find out the optimal

acquisition parameters

2. click the button on Acquistion Mode on the upper left to open the Sequential Scan panel

3. select between lines, -frames, or -stacks (see useful tips below)

4. click the + button to add as many scans as needed

5. for each scan

drop the unwanted laser power to 0%

select the appropriate beam splitter

deactivate the unwanted PMTs

6. (optional) save the setting

useful tips:

if you use different beam splitters, have overlapping emission bands, apply different number of passes in

average or accumulate, or need different size pinhole for different scans, you will need to use the slower

"between frames" or "between stacks"

with "between frames" or "between stacks", keep hardware changes to a minimum between scans will ensure

fast and smooth operation e.g., use the same emission band for the same PMT in each of the scans

each scan can have multiple simultaneous channels e.g., adding the transmission detector, or have 2

simultaneous channels that do not show any cross-talk

the function buttons act differently in different sequential scan mode

display behavior of the function buttons in sequential scan

mode

buttons

scan mode

Live Capture Image Start

*to obtain all scans at the current focus (z-position), switch to xyt mode, set frame to 1, and then click Start

between lines all scans all scans acquire all scans

between frames currently selected scan currently selected scan* acquire all scans

between stacks currently selected scan currently selected scan* acquire all scans

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Stage automation

Caution: whenever the Tile Scan or Mark and Find window is launched

i. any tiles or points marked will be processed when you click Start

ii. the grey area represents the stage, a left mouse click will cause the stage to move to that specific

location and can be dangerous for any objective lenses except the 10×

Tile Scan

1. click to toggle displaying the Tile Scan window

2. check that Scan Field Rotation is set correctly (see

r elevant details

below)

3. go Live

4. move the prep/stage to the left boundary of the region of interest, focus

5. click to mark the current position

6. repeat the last 2 steps for the top, right, and bottom boundries, the software will calculate the matrix

needed

Note: Marking any points outside the established coverage area will expand the matrix but

marking any points inside will not change anything. Unfortunately, there is no provision to

save the points here or apply any points already saved from Mark and Find.

7. move about to make sure the coverage is sufficent, especially with multichannel acquistion

8. setup Z-stack, configure Average etc., as needed

9. click Start to acquire data

Mark and Find

1. click to toggle displaying the Mark and Find window

2. if there are existing coordinates, you can save or clear them first

3. go Live

4. move the prep/stage to the desirable area and focus on the object of interest

5. click to mark the current position, which will be listed as positionN

6. repeat the above 2 steps to mark more positions as needed, i.e., positionN+1, N+2... etc.

7. you can go back to any position by selecting it from the drop down list

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1. viewer display window 2. at the microscope

viewer display windows rotated 90 ° right

3. Tile Scan or Mark and Find window

viewer display windows rotated 90 ° left

relevant details

for tile scan, current rotation calibration for stitching is -0.19 ° (-0.18 ° is also OK) with a 10% overlap

tile scan order is row-wise in the viewer display window, starting from the lower left, which

corresponds to column-wise starting from the lower right as displayed in the Tile Scan window

Note: The orientation of the specimen as shown in the viewer display window (1) is rotated

when compared to that as observed through the ocular of the microscope (2) or represented

in the Tile Scan or Mark and Find window (3). Confusing? Indeed!

coordinate system

values (mm) displayed in the window denotes the upper right corner of the image in the viewer

display window

upper left of the stage is 0, 0

lower right of the stage is 126, 82

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Check out

1. lower the turret, remove your prep; if applicable, remove oil/immersion fluid on immersion objectives

2. switch to the 10× objective

3. if there are users signed up within the next 6 hours do the following, otherwise go to the next step

i. save your experiments and quit LAS AF

ii. copy your data to external storage media or network file servers, as needed

iii. submit your usage using the google form and log out of Windows

4. if no one signed up within the next 6 hours, please proceed to shutdown everything

i. save your experiments and quit LAS AF

ii. Main Switch Board: turn the key counterclockwise to Off-0 to disable Laser Emissions

iii. Main Switch Board: wait 10 s, switch off Scanner Power

iv. copy your data to external storage media or network file servers, as needed

v. submit your usage using the google form

vi. shutdown Windows XP

vii. Main Switch Board: after computer has shutdown, switch off PC Microscope

viii. Main Switch Board: when the Argon laser cooling fan stops, switch off Laser Power

ix. turn off FLUO, if applicable

x. put the cover back on the microscope

5. clean up the work area

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Appendix

Content

DMI 6000 inverted microscope: basic controls • modes of operation • using immersion objectives

beam splitter spectra • working with lif files • scanner

software overview and layout

LAS AF user interface

DMI 6000 inverted microscope

Controls

focus knob on either side of microscope (z-Wide)

commonly used functions are controlled by 5 sets of buttons

1. left rear variable function buttons

CHGTL◑: switch to transmitted light, TL

and rotate through different modes TL_BF

(bright field), _DIC, _POL (polarization)

CHANGECS: toggle between confocal SCAN

and the last selected TL mode

COMBI◑: epifluorescent + DIC

Z COARSE Z FINE: toggle z control

2. left front: Illumination Manager

TL/IL: toggle between TL and IL (incident-

light i.e., epifluorescence)

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AP: aperture diaphragm for TL

INT : light intensity, press both together to toggle between Coarse and Fine mode

FD: field diaphragm for IL

3. front top row switches should not be activated, or

else it may block viewing through the

binocular, to rectify

press the leftmost button to select

binocular

press the rightmost button to deselect

the supplemental magnification changer

SHUTTER: for epifluorescent illumination

bottom 6 buttons: for selecting specific cube

(DAPI, GFP, DsRed) for epifluorescence; press

the middle 2 buttons together to display the programs assigned to the variable function buttons

4. right rear variable function buttons

CHGOBJ↻: rotates the objective turret one position

clockwise in the current mode (dry or immersion)

CHGOBJ↺: same as above but counterclockwise

DRY/IMM: toggles between dry and immersion mode

5. right front: Focus buttons

Z↑: moves the Z drive up when a upper focus stop has

been set

Z↓: moves the Z drive down

SET+Z↑: toggles set and unset upper focus stop

SET+Z↓: toggles set and unset lower focus stop

SmartMove remote control for stage movements, as observed in the viewer

display window of the software

1. X

2. Y

3. Z (focus, z-wide)

4. left button set selects Precise or Fast XY movement

5. right button set selects FINE or COARSE focus control

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front LCD panel displays the current status

1. Contrasting method e.g., TL, FLUO, or SCAN;

and IL SHUTTER status

2. Objective lens and Magnification, see front top

buttons above

3. Illumination and Apertures

4. Camera Ports/Eyepieces, see front top buttons

above

5. Focus control

Note:display on the right shows z at 0µm

with upper focus stop set and lower

focus stop unset

if upper focus stop is unset, display will indicate --.--mm and

indicates lower focus stop is set

Modes of operation

1. press CHGTL◑ to rotate

through BF, DIC, and POL

2. adjust intensity (INT) and

aperture (AP) as needed

3. optional, press CHG CS to scan

mode thus shutoff transmitted

light

4. a knurled wheel (arrowhead) on

the left side below the objective

turret adjusts the DIC shear; it's a

rather tight space!

IL/epifluorescence

1. turn on FLUO (fluorescent light source)

2. press the button for the desirable cube, see the front buttons in Controls

3. press Shutter to illuminate specimen

4. adjust the intensity (INT) and field diaphragm (FD) as needed

5. press Shutter to block illumination when done viewing

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Using immersion objective lenses

2 ways to switch from DRY to IMM objectives

semi-auto with Mark and Find (recommended)

1. locate the area of interest with a dry objective and focus

2. click to bring up the Mark and Find interface

3. click to record the current position: positionN

4. press and hold Z↓ to lower the turret completely

5. switch immersion objective into light path

via the objective link in LAS AF (recommended)

or at the microscope: press DRY/IMM to switch mode, press either button CHGOBJ↻

or ↺ to select the desired objective

6. move the stage to expose the objective so you can apply immersion fluid

7. apply an appropriate amount of immersion fluid

8. select positionN from the drop down list and the stage should move back and bring the

objective to focus

9. click to dismiss the Mark and Find interface

manual (not recommended)

1. locate the area of interest with a dry objective and focus

2. make sure upper focus stop is set

3. press and hold Z↓ to lower the turret completely

4. switch immersion objective into light path (see semi-auto procedures above)

5. offset the turret ~15° counterclockwise manually and apply a small drop of immersion fluid on

the objective

6. swing the turret back into position

7. press and hold Z↑ to put the prep back to the previously set focal plane

when finish

1. press and hold Z↓ to lower the turret completely

2. remove slide

3. at the end of the session, use the provided lens paper to remove the excess immersion fluid from the

front lens

4. switch in the 10× objective

Best practice

always set the upper focus stop whenever you put on a prep, press SET+Z↑

switch objective lenses via LAS AF software or the right rear buttons on the microscope

if you want to manually switch the objective, DO NOT grab the objective lens barrel, use the turret

instead

Caution: All our immersion objective lenses have aperture or correction collar, grabbing the

barrel can easily move the collar and also strain the lenses.

select the 10× objective at the end of the session

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20×/0.7 NA multi immersion objective, currently set

to use oil only

OIL: oil, with or without coverglass

GLYC: glycerol, with or without coverglass

0.17: water, with #1.5 coverglass

W: ca. 3% NaCl, without coverglass

0: water, without coverglass

63×/1.2 NA water immersion objective, set at 0.17

T+ ←: turn to this direction for higher

temperature or thinner coverglass

0.14 0.16 0.18: water, with specified

coverglass thickness in mm

→ SW: turn to this direction for higher salt

concentration, lower temperature, or thicker

coverglass

40×/1.25 NA oil immersion objective 63×/1.4 NA oil immersion objective

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