Nikon Eclipse Ti TIFT 1454 Bedienungsanleitung

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 1

Source: NIKON

Total Internal Reflection Fluorescence microscope

Nikon Eclipse Ti

Principal:

•Laser is pointed at an angle at specimen

•Due to this angle the laser will totally reflect

on glass/medium interface and creates an evanescent wave above the glass

•Thickness of wave is around 200nm, but decays exponentially

•Possible to only excite fluorophores in membrane, without background signal of the cytoplasm

Benefits:

•Extreme low background signal

•Perfect for membrane studies

•CCD Camera attached will provide in fast imaging

Contact

•Martijn de Gruiter Be-343 tel. 31105

•Gert-Jan Kremers Be-346 tel. 43578

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 2

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 3

Start system

•Turn on the power by the power socket (on the floor under the table)

•Switch the mercury lamp on and then ignite it.

oIf not needed don’t turn the mercury lamp on

oThe unit on the shelf closest to the door

•Turn on all devices with a button on the shelf and the table.

oCamera and laser box are automatically enabled

•Next turn on the device with a key: Laser control panel

•Start the computer and start the program Metamorph.

oSelect suited user account.

Live Cell imaging:

•When imaging at 37oC:

•The heating device to maintain your cells uses water.

ofill reservoir with demineralized water up to level of CO2 tube

•Allow the system to warm up for 15-30 minutes

•Settings for the heating unit are paper on the wall, choose your settings

oGeneral settings for cells on a coverslip:

otop heater 40.5 ° C

obath heater 38.5 ° C

ostage heater 38.5 ° C

olens heater 37 ° C

•Ensure that the cells are grown on a glass coverslip

•Place the slide in the metal ring, tighten firmly but not too hard because it will break the

glass

•Add 1-2 ml of the medium from the appropriate well. There is a convex surface

•Move with a paper towel around the edge of the coverslip from below with a little pressure,

if no medium leaks from the ring your seal is good.

•Wipe clean the underside of the slide with ethanol to prevent contamination of the

objective and microscope

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 4

Finding focus

•Put the objective in the lowest position using button A

oLowest position is ~500 um

•Choose the objective with the right magnification

•Put a little drop emersion oil on the lens

oOnly if it’s an oil emersion objective

•Place the metal ring in the middle of the heating chamber

oSecure with the metal clips

•Centre you cells above the objective with the joystick

oButton on top of joystick will change speed, S (slow) or F

(fast)

•Turn button C until the oil is in contact with the coverslip

•In MetaMorphs Taskbar choose “transmission to eyes” to

illuminate the cells with the halogen lamp

•With button D you can use the fine adjustment control knob

ocourse, fine or extra fine

•Look through the eyepiece and bring cells in focus.

oThe display of the microscope shows the height of the objective

•Finding appropriate fluorescence cells can be done with the mercury lamp

oChoose the illumination settings for your fluorophore in the Taskbar in Metamorph

oIncrease or decrease brightness by changing the ND filters

oND 4, 8, 10 will block intensity, the can be combined, use minimal intensity to reduce

bleaching of your fluorophore

•When your sample is in focus you can focus the beam of the laser if necessary, for detailed

explanation ask a designated OIC member

•To change coverslips, bring objective down with lowest button A. Bring objective back to

previous height with upper button A

Perfect Focus System (PFS)

•If focus is found a LED (E) will turn orange and/or you will hear a beep

oFocus can be kept using the Perfect Focus system

oPress the green blinking button E

•The focus of the PFS is adjustable with a module as shown opposite

•The turntable navigate the lens up and down as shown and with the blue button toggles

between fine (pressed) and coarse

•Focusing with perfect focus should be viewed through the camera

The buttons FGH give the direction of the output signal:

F: eyepiece G: camera left QuantEM:512SC H: camera port right

G H

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 5

Metamorph - Taskbar

•On startup your personal taskbar is loaded

•Metamorph functions can be accessed easily from a

Taskbar and an

Acquire window

•You can restore illumination settings with the

button: "Restore OIC settings"

•Open the Acquire window with Acquire

•Access Acquisition modes with: MDA & Stream

•Select preferred illumination settings

oLeft side: lasers for camera,

oright side: mercury lamp or transmission to

eyepiece

•Open FRAP or laser console

Metamorph - Acquire window

Sidebar left:

•Exposure time

oMinimum interval time at full chip is 90ms, quad chip is 50ms

oExposure time can be lower than interval time

•Full Chip, Quad Chip, Use Active Region

oUse full chip, middle quadrant, or region selected by ROI

•Live bin & bin

oCombine pixels to increase readout speed

oUse only for live viewing or real recording

oNormal state = 1

•Start Live/Stop Live

oStart/stop live mode

Acquire images

•To acquire live mode press "Acquire" button

•Separate images can be recorded as separate images or in stack

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 6

Tab: Display:

•Auto scale option

oEnable Auto scale or change custom auto scale

oFull bit-range is saved to file, this is only for visualization

in the software

•Visualize saturated pixels

Tab Acquire:

•Be sure to set the Illumination choice to

“Current shutter”

Tab Special:

•Gain options:

•10 MHz (EM gain), gain3 3x is standard setting

•Control gain between 0 and 1000

•Use "Frames To Avg" option to average images / or use

longer exposure time

Live image window

•Magnifying glass

•Display image in other LUT

oGrayscale, Color by wavelength

•Histogram of intensities

oManual scaling can be done with the orange arrows

•Auto scaling options

oBest fit range, 8/12/14/16 bit

oAuto scale on/off

oScale within region

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 7

Streaming imaging

Open the Stream Acquisition window with the button in

the taskbar

•Start recording with Acquire button

•Check if chosen configuration is ok

Tab Acquire:

•Acquisition modes:

oRAM save later to hard disk

oHD choose name and location to save

Save as on file or separate TIF files

•Enter the desired number of frames

•Select “Initial illumination”: Current Shutter

Tab Camera Parameters:

•Recording options:

oAverage frames

oNumber of frames to skip, during acquisition

oDon’t show recording (faster when exposure time <

minimal interval time)

•Camera readout default options should be:

oCamera state: HALT

oShutter mode: OPEN PRE SEQUENCE

oClear mode: CLEAR PRE SEQUENCE

Multi-Dimensional Acquisition (MDA)

Combine multiple tasks: z-stack, time-lapse,

streaming, multiple positions, multiple

wavelengths, use of journals.

•Open the MDA window using Taskbar

•Select acquisition mode(s) in Main

•Continue with the tabs shown left:

•Saving: select filename and folder

•Select illumination settings

•… options for acquisition modes

•When setup is done press acquire

•Save or load settings of experiment setup,

with Save/Load State buttons

•Live mode can be started from MDA window

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 8

MDA: Time lapse imaging

•Activate the time-lapse option in MDA’s main window

•Set the desired interval and time

•Choose the desired illumination settings in the wavelength tab

oOr multiple when the option multiple wavelengths are selected

•Start recording by clicking on Acquire

Shut down system

•For live cell imaging:

oRemove Tokai Hit incubator from the stage and place it on the table

oClose the Tokai Hit incubator with a plastic well

•Clean objective carefully

oRemove oil with lens paper:

oHold lens paper tight between your fingers and wipe over objective

oClean lens with 2-propanol on your lens paper

•Cover stage with lens paper box

•Save data to OIC network storage (O:\ drive)

oTo collect the data from your own pc, connect to network storage drive via the address:

\\oic-station\oic

user: guest, password: guest

oWhen the hard disk of the microscopes pc gets full, data will be deleted

oYou are responsible for your own data, the networkdrive is only for transport

•If you are the last user of the day

•Shut down all devices

oShut down mercury lamp as last

oCamera power is switched at the adapter box at the floor

•Switch off the power socket located at the floor

Contact

•Martijn de Gruiter Be-343 tel. 31105

•Gert-Jan Kremers Be-346 tel. 43578

Manual: TIRF Microscope

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Martijn de Gruiter Erasmus OIC 9

FRAP on the Fly Calibration

Before you can use FRAP the laser must be calibrated

•Find an empty piece of glass without cells/fluorescence

•Set the exposure time around 100 - 200ms

•Open ILAS2 with icon

•Open calibration tab and put the power of selected laser to 100% (in ILAS2 window)

•Activate the laser [G] and press “ Show Live”

•Place laser in upper left corner by moving the red cross, press [H]

•Repeat for lower right corner

oIf the spot isn’t visible or too big and bright change power and/or exposure time

oview is rotated, so move laser in other direction

okeep in mind to move red cross into left and right corners of the calibration screen

•Calibrate with button [i]

•To check if your calibration was successful open “ on the fly” tab

•Change time to 3000

•Increase the power of your laser and press “ Show Live”

•Click in the image and check if laser pulse in at the position of your mouse pointer

Troubleshooting - FRAP

FRAP calibration error

•Notification: Calibration canceled (exceeded 15 bits)

Possible errors

•Emitting fluorophore or auto fluorescence

oEnsure that the area of the slide really empty

•Too much laser power or exposure time

oReduce laser power in the ILAS2 window and/or exposure time in Acquire window

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