Leica TCS SP8 MP Manual de usuario

User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope

This version: 7.24.14.

Introduction

The IBIF confocal microscope is made available on a fee-for-use-hour basis to all users who have been

trained. Unlike our previous instrument, it has a number of hardware features, specifically the

objectives, the stage, and the condensers, that can be added or removed by the user, with the

permission of the Imaging Specialist. To minimize inconvenience to other users, and to reduce the risk

of inadvertent damage to the equipment, we ask that users obey two rules:

1) Leave the confocal as you found it. Replace and/or reset everything that you modified.

2) Be aware that other users may have failed to obey rule 1. Be alert, especially in regard to

mechanicals such as stage translation, condenser height, and objective rotation.

Thank you, and we wish you every success in your imaging. We are here to help.

The Confocal Microscope and Accessories

The instrument is a Leica TCS SP8 MP confocal

microscope, equipped for multi-photon confocal

microscopy. The components are the microscope

body, two automated stages, the scan head, the

electro-optical modulator (EOM), the monitor

and computer, and the Spectra-Physics Mai-Tai

tunable near-IR laser.

The items on the desk (along with the monitor,

keyboard, and mouse) consist of a confocal

controller, a microscope controller, a metal-

halide power source (which performs the

function of the Hg bulb in the previous system),

and a controller for the Dodt contrast

enhancement system (which replaces the

Nomarski contrast enhancement method of the

previous system).

The unlabeled switch is a shutter for the

Mai Tai laser and should only be needed in

emergencies in which the beam is exposed.

Two photomultiplier tube (PMT) detectors and

two hybrid-PMT (HyD) detectors are available

for conventional (single-photon) confocal

microscopy.

Some cell-phone carriers use frequencies that

interfere with the HyD detectors. Switch your

phone off or leave it at the front of the room.

Two high-sensitivity Super HyD detectors are

installed for non-descanned (multi-photon)

confocal microscopy. These detectors are

electrically- and liquid-cooled for decreased

background. They should only be used in a

darkened room and with the microscope

covered by a dark room cloth (available).

The Super HyD detectors are also vulnerable to

cell-phone interference.

Behind the air table at the rear are three boxes of

instrumentation. The lower box is the Compact

Supply Unit, which houses the visible light lasers.

The smaller of the two boxes on top of the

Compact Supply Unit is the power supply and

cooling unit for the Super HyD non-descanned

detectors.

The microscope has no manual focusing

adjustment. Instead, it has two stages and two

computer-controlled focusing mechanisms. The

large X-Y stage performs lateral translation in all

modes. The nosepiece has its own focusing drive,

controlled from the microscope controller, or the

confocal controller, or from the software.

The smaller Super Z Galvo stage rides on

top of the large stage and includes the slide

holder. It is used for stacks. The large translation

stage can be used without the super X Galvo stage,

primarily for whole animal work or specialized

experimental chambers. Contact us for

instructions on how to do this.

System Specification

Lasers

The system has four visible-light lasers: 405 nm, 488 nm, 514 nm, 552 nm, and 633 nm. The 405 nm

should be used to excite DAPI or similar blue-emitting dyes, therefore use of the multi-photon laser will

not be necessary. The 488 nm and 552 nm lasers are for FITC and rhodamine bands respectively. The

514 nm laser can be used to excite YFP, while the 633 nm laser is for longer wavelength dyes such as

Cy5. All of the lasers are semiconductor types and can be switched on and off without special care.

All of the lasers are more powerful than those in the previous system (20 mW or

better versus 1 mW for the 543 and 633 lasers of the previous system). Users will find that only a few

percent excitation is required in most circumstances.

Optics

The available objectives (illustrated above, from left to right) are as follows:

Position

Objective

Magnification

n.a.

Working

distance

(mm)

Medium

HC PL Fluotar

10x

0.3

11.0

air

HC PL Apo

20x

0.7

0.59

air,

coverglass

IRAPO

25x

0.95

2.5

water

HC PL Apo

40x

1.30

0.24

oil

IRAPO

40x

1.10 Corr

0.65

water

HC PL Apo

63x

1.40

0.14

oil

*available on request

The air and oil objectives (1, 2, 4, and 6) are similar to those you have used in the past. The two water

objectives are especially good for deep-tissue imaging and are corrected for multi-photon excitation.

Note: the 40x and 63x objectives have spring-loaded front ends that can be inadvertently twisted and

locked in a withdrawn position. Handle them with care.

Disk Drives

The computer has two drives, a 120 Gb solid-state drive (C:) and a 1.8 Tb disk drive (D:). All data should

be saved to the D: drive.

Condenser

A transmitted light condenser is available if high-quality

visualization of unstained tissue or cells is required.

However, there is enough unfocussed light available in

transmitted light mode to enable simple localization of

specimens without a condenser. The Imaging Specialist

will demonstrate the user of the condenser if requested.

The condenser has a short working distance (1.0

mm) and very few options. Condenser centering is via

two screws, as in the Zeiss system. The condenser focus

knob is to the left side. The condenser cannot normally

be brought into focus, but as long as it is properly

centered it will function satisfactorily for the acquisition

of transmitted light images. The Dodt contrast system does not use the condenser.

If you have brought the condenser near to focus (for example, if trying to visualize unstained

cells), please remember to lower it before using the large X-Y stage, otherwise damage to the condenser

may result. For the same reason, install the condenser after the large X-Y stage has been initialized.

The condenser must be removed after use.

The Microscope Controller

Control of Stage Movement

Manual x, y and z stage control is via the knobs attached to the

microscope controller, or by the manual X-Y stage control on the

stage itself. The microscope controller moves the large X-Y stage

only. The knobs allow much finer control than the manual X-Y

stage control.

Touch Screen Control of Microscope Functions

The touch screens on the microscope controller are as follows:

Screen 1: Status and Illumination. Use to adjust

illumination intensity, and the aperture and field stops in

transmitted light (TL). These are in the microscope body,

not the optional condenser.

Screen 2: Illumination Method. Select TL or RL

(Reflected Light, i.e., epifluorescence). Open and close

the shutters to either path. In RL, select the filter cubes

(labeled GFP, RFP and DAPI) for green, red, or blue

emissions.

Screen 3: Objective selection. Some selections will pause

to allow time for oil or water application or removal. The

operation is completed by pressing the selection again.

Screen 4: Nosepiece (i.e., objective) Z-control or stage X-Y

control.

Z-mode: The Home position is the highest point to which

the nosepiece can currently be raised. It is usually set at

the upper limit of travel of the nosepiece. The Focus Stop

position is the approximate focus position (actually, a

little above) for the 10x objective. Both positions are

indicated by –marks. The current position of the

nosepiece is indicated by the = mark and its distance

from the Focus Stop is indicated in mm on the display.

Press and hold the Home button to raise the nosepiece to the Home position. Press and

hold the up/down button to move the nosepiece to any position. Note that this button will allow

the nose piece to travel below the focus position. Careless use of it may drive the objectives into

the specimen.

The Home and Focus Stop positions should not normally be modified. See the section

below for a procedure to set or re-set them.

X/Y Mode: When X/Y mode is selected, the sensitivity of

the X-Y control knobs can be selected (coarse or fine).

Positions in the X-Y plane can be stored for future use.

Maintained touch in the screen will cause the stage to

translate to that position. If the condenser is installed,

be sure to lower it first.

Screen 5: Allows programming of any of the black

buttons for specific functions or combinations of

functions. See us before you program any of them.

Screen 6: Not normally needed by users.

Setting and Restoring Home and Focus Positions (Screen 5).

In some situations, for example when using the large X-Y stage in a lowered position, it may be

desirable to adjust the Home and Focus positions. Here’s how to do it.

First, select Screen 3 on the microscope controller display and select the 10x objective. Then

select Screen 4 and select Z-mode (top right).

Setting the Home position

Using the controls on the microscope controller, raise the

nosepiece all the way to the top, or to a level that allows

you to change your sample without interference from the

objectives. Note the Set/Clear Limits button (bottom left)

which has two parts, the Up/Down symbol and the

word Home .

On the Set/Clear Limits button, push the Home button

on the Set/Clear Limits button (it will turn red). The

button will now be labelled Set/Clear Home Position.

To set the Home position, hit the Set button (it will turn

red). The Home position indicator will appear.

To clear the Home position, hit the Clear button. It will

also turn red. The Home button will go gray.

Setting the Focus Stop position

Raise the nosepiece and set a glass slide specimen on the stage. If you do not have a suitable slide, there

are some H & E stained slides in the top drawer of the desk. The slides are autofluorescent to GFP or

YFP excitation, so you may use fluorescence as an alternative to transmitted light.

Manually lower the nosepiece until the specimen

is in focus through the eyepieces with the 10X lens. Raise

it a small amount.

On the Set/Clear Limits button, push the

Up/Down button (it will turn red). The button will now

be labelled Set/Clear Focus Position.

To set the current nosepiece position as the Focus

Stop position, hit the Set button (it will also turn red). To

clear the Focus Stop position, hit the Clear button. It will

also turn red. The Focus Stop position indicator will

appear.

If you have changed either setting, remember to

restore the Home and Focus positions for the next user.

Positioning a Slide

Raise the objectives using the microscope controller.

Displace the stage to the left manually or using the

stage X-Y manual control. For extra room, rotate the

objectives manually so that the 10x is over the stage. Be

sure that the clips are positioned at the edges of the

slide recess. If the clips are hard to move, use the hi-

tech tool provided.

Place the slide in the slide recess. Usually, placing the

right side first is the most effective method.

Place the clips over the slide, using the tool if necessary.

Manually reposition the stage under the objectives.

Note: dishes can also be accommodated, subject to the

limitations of objective size and working distance. Ask us

for details.

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