background image

 

p.

 

11

 

 

 

Open

 

the

 

Channels

 

tab

 

 

 

Check

 

and

 

select

 

only

 

the

 

shortest

 

wavelength

 

track

 

and

 

click

 

the

 

1AU

 

button

  

 

Start

 

scan

 

in

 

continuous

 

mode

 

 

 

Check

 

the

 

Range

 

indicator

 

 

(below

 

the

 

image

 

container

 

in

 

the

 

Dimensions

 

tab).

  

 

Set

 

your

 

image

 

parameters

 

to

 

achieve

 

image

 

without

 

saturation

 

(saturation=red

 

pixels

 

in

 

the

 

image)

 

and

 

the

 

correct

 

black

 

level

 

(black=

 

blue

 

pixels

 

in

 

the

 

image)

 

o

 

Set

 

the

 

PMT

 

gain

 

to

 

550

600.

 

o

 

Set

 

the

 

Laser

 

%

 ‐ 

it

 

is

 

recommended

 

to

 

use

 

the

 

lowest

 

%

 

as

 

possible

 

and

 

to

 

increase

 

gain

 

(master).

 

o

 

In

 

case

 

of

 

low

 

signal

 

with

 

high

 

laser

 

%

 

and

 

high

 

gain

 

you

 

can

 

add

 

digital

 

gain

 

(up

 

to

 

2).

 

o

 

Set

 

the

 

offset

 

level

 

until

 

you

 

reach

 

the

 

PMT

 

threshold

 

(blue

 

pixel

 

will

 

appear

 

in

 

the

 

image

 

while

 

scanning

 

in

 

range

 

indicator

 

mode).

 

 

Stop

 

the

 

scan

 

 

 

Select

 

another

 

track;

 

set

 

its

 

pinhole

 

to

 

the

 

same

 

μ

m

 

section

 

as

 

the

 

first

 

track

 

and

 

set

 

its

 

parameters

 

and

 

so

 

on

  

 

Stop

 

the

 

scan

 

 

 

 

Summary of Contents for LSM 700

Page 1: ...2 Objectives 2 Filter turret 2 Laser lines 3 Light Path 3 Start up 4 Sample mounting and viewing 5 Software 5 Sample viewing 6 Acquisition 8 LSM Channel mode confocal setting 8 Configuration 8 Acquis...

Page 2: ...pectral data point Hardware The microscope The LSM 700 is attached to an upright microscope Axio Imager Z2 Objectives Position Objective lens Magnification NA coverslip Working distance Immersion Parf...

Page 3: ...mission passes through the pinhole aperture and the out of focus light is eliminated 5 6 The variable secondary dichroic beamsplitter VSD is coated with a variable coating that between 420nm 630nm acc...

Page 4: ...ng order In the power strips turn on switches 1 2 Turn on the stage controller 3 Rotate clockwise the silver key in the lasers electronic unit 4 Turn on the computer and log in User name multilabs Pas...

Page 5: ...Light switch between the ocular and the confocal Via the touch pad 1 Choose the preferred objective 2 Choose the proper filter set and open the TL RL shutter Software Log into BookitLab and activate...

Page 6: ...tage to I II or III according to the objective lens see objective lens info on touchpad If you are acquiring transmitted channel set up Kohler illumination 1 Bring the sample into focus 2 Close the fi...

Page 7: ...p 7 2 5 3 4...

Page 8: ...ocal Configuration It is highly recommended to seek BCF staff s advice for new fluorophores exact name or new combinations Choose one Load a saved configuration from the Experiment Manager Reuse setti...

Page 9: ...ThermoFisher Chroma 1 Laser lines 2 Main dichroic mirror 3 PMTs PMT1 and PMT2 4 Variable secondary dichroic beamsplitter VSD 5 Emission Filter selection 6 Emission spectrum with Graphic representatio...

Page 10: ...logy experiments Frame size 512X512 Speed 8 Averaging Method mean in most cases in low signal with low noise Sum method may be useful Number 1 Mode frame for fixative sample for live cells and in case...

Page 11: ...lack level black blue pixels in the image o Set the PMT gain to 550 600 o Set the Laser it is recommended to use the lowest as possible and to increase gain master o In case of low signal with high la...

Page 12: ...ng to the SNR Press Snap in the start buttons Save the image press in the Images and Documents tab right hand side of the window and save in the directory D Users data PI name MM YYYY User name YYYY M...

Page 13: ...uch as brightness correction over z Time series In the Multidimensional Acquisition options check Time series a new tab Time Series will appear under the Multidimensional Acquisition Section Define nu...

Page 14: ...mended option the process can be configured in the processing main tool tab In the Acquisition mode tab Frame size Press optimal Zoom It is recommended to set the zoom above 1 2 Scanning speed 7 Avera...

Page 15: ...After a single scan in the Regions tab choose one of the ROI tools and draw on the acquired image the dimensions can also be set manually in the Width Height textboxes Define which process will be per...

Page 16: ...before moving to high magnification with small field of view Add a position by pressing Positions below the image container and clicking on the image container that was just acquired at the current st...

Page 17: ...lose the ZEN software Log off from your account in the BookItlab window Check if another user has a reservation if yes verify his her arrival If you are the last user for the day or the next reservati...

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