TESM

Description

TESM stands for TIRF Epi-fluorescence Structured Light Microscope. Some specifications can be found here

TESM Startup

Power

Two power switches for TESM

TESM is turned on by the two power switches at the bottom of the equipment rack to the left of the optical table. The order of which you turn them on doesn't matter.

Lasers

The key is in the horizontal position here

Only turn on the lasers required. The lasers should be power cycled once they have electrical power. The keys should moved to the horizontal position, and then moved to the vertical position. It can take as much as 5 minutes for them to go through their start-up cycle. During this time, they will ignore any commands from the computer. They will default to full power, about 100mW.

The 405nm, 491nm, and the 561nm laser control boxes are on the left front side of the optical table. Power levels are set from micromanager.

Unlike the other lasers, the 660nm laser is controlled by a power supply in the equipment rack.

The keys should be moved back to the horizontal position when you are finished using TESM for the day.

Micromanager

Micromanager User Guide has detailed information on how to use micromanager.

Log into TESM's computer, named Germanium, with your BIG credentials. Either start micromanager via an icon on your desktop, or open a terminal and type:

micromanager 

If that fails, you can:

cd /usr/local/Imagej 
./run 

Startup Configuration

Micromanager will then go through a startup sequence. If this is the first time you have started micromanager on TESM, then you will need to select the correct configuration file. It is located here:

/storage/big1/kdb/MicroManager/MMConfigITC18.cfg

Custom Dialog

Micromanager will present you with a custom dialog with several options.

• 16X Slider: There is a rod on the left side of the microscope. When it is pulled out, you will get an increase of 1.6X in magnification, otherwise it is just 1X.
• TIRF or EPR: This sets the default light pathway. This can be changed later in the software.
• Camera Binning: With our default 60X 1.49 Objective, Binning = 2 is appropriate. If you are using a lower magnification objective, you might want to set binning to 1. This can be changed later in the software.
• Vertical Shift Speed: Leave this alone unless your name is Karl

Sutter LAMBDA 10-2

IMPORTANT

The Sutter needs to be reset before using TESM

The Sutter LAMBDA 10-2 must be reset after you have closed the dialog by pushing the reset button on the left. This only needs to be done when you first start TESM for the day. If successfully done right after you close the dialog, you should see the Sutter LAMBDA 10-2 cycling through 0-1-2-3 filters as commanded by micromanager.

TIRF or Structured Light

TIRF or Structured Light (EPI)

TESM has two imaging modes: TIRF and Structured Light. The Structured Light pathway is labelled EPI on the uniblitz. Only one of the two modes should be active at a given time.

Normally, the imaging mode is selected by the computer, but you may override the computer by using two most left toggles on the top UNIBLITZ shutter driver.

IMPORTANT: All toggle switches should be down when under computer control

Laser Shutters

UNIBLITZ Shutter Driver

TESM has 4 lasers: 405nm, 491nm, 561nm, and 660nm.

Each laser shutter can be manually opened by moving their respective toggle switch up. Remember, either the TIRF or the EPI (Structured Light) shutter must be open as well.

Normally, which laser is being used is selected by the computer, but you may override the computer by the appropriate toggle on the UNIBLITZ shutter driver.

IMPORTANT: All toggle switches should be down when under computer control

Laser Power

405, 491, 561

In Micromanager, open the Property Browser window by opening the menu:Open Tools->Device/Property Browser

This is where you can adjust the laser power for the 405, 491, and 561 lasers. The lasers need to have completed their start up first, which takes a few minutes. The units are in watts. For example, if you want 50 mW, then you should enter 0.05. Generally, the lasers power levels run between 0.001W and 0.1W. However, the 491 laser only goes down to 0.002W.

660

This is used to set the power levels of the 660 laser

This laser's power level is set by the big Power Supply in the equipment rack. Select "V-set", then enter a number between 0.5 to 5, finally select "Enter." You might have turn the Voltage on be selecting the "On/Off" button.

Structured Light

500lpi SL grating

The Structured Light (SL) can be created by passing collimated light through a grating on the EPI path of TESM. We use a 500 lines/inch grating. TESM takes three SL images at 1/3 of a period of the grating. It only takes 2-3ms to move the grating between images. A custom TESM imaging protocol needs to be used in order to acquire SL images. The grating needs to be removed if you want to do pure Epi-fluorescence imaging on TESM. However, the TIRF light path produces more even illumination for Epi-fluorescence, when the TIRF beam is set near 0° but not at 0°.

TIRF

TIRF adjustment

TIRF stands for Total Internal Reflection Fluorescence. The three micrometers in the picture adjusts the angle and focus of a converging beam.

The top micrometer moves the beam towards or away from the front of the microscope.

The front micrometer in front moves the beam side to side.

The right micrometer focuses the converging beam on the back focal plane of the objective.

Initial Adjustment of TIRF beam

Once a sample is in focus, one should focus the TIRF beam on the back focal plane of the objective. This is accomplished by adjusting the horizontal micrometer on the right until a point is seen on the ceiling above the microscope. Astigmatism should be minimized by using the other two micrometers. This is called zeroing the TIRF beam, and the beam is considered to be at 0°. The top micrometer should be zeroed.

Using the TIRF beam for Epi-fluorescence

The TIRF beam could be used for Epi-fluorescence when it is near or at 0°. I prefer it to be a few degrees away from 0° to minimize retro-reflections.

Using the TIRF beam for TIRF

The TIRF angle is set by the top micrometer.

Filter Turret

Position 1

520/40, 610/60, 685LP

Position 2

525/50

Position 3

525/50, 580LP

Position 4

50/50 Mirror

Position 5

TIRF 450/60, 525/50, 605/50, 730/120

Position 6

TIRF 525/50, 645/140

Sutter 10-2 Filters

The Sutter 10-2 Filter Wheel is controlled through a custom TESM Imaging Protocol via TTL signals that mimic a parallel port. The driver for the Sutter 10-2 isn't able to be controlled by a user using µManager at this time. You can manually select a filter by selecting the "local" bitton, and then a number. Don't forget to switch back to computer control by selecting "online" button.

Position 0

Open

Position 1

525/50

Position 2

610/75

Position 3

710/51

Position 4

Open

Position 5

Open

Position 6

453LP

Position 7

475LP

Position 8

495LP

Position 9

1m FL Cylindrical Lens

Objectives

TESM's main objective is a 60X 1.49NA designed for TIRF.

If you want to switch objectives, and Karl isn't available, be extremely careful switching objectives.

DO NOT TOUCH THE BOTTOM OF THE OBJECTIVE

Before removing the objective, switch the filter turret to position 4. It isn't labelled, but it is between 3 and 5. Once the new objective is in place, you may move the filter turret back to position 5.

Optivar

1X or 1.6X

The Olympus ix71 has the option of an additional 1.6X magnification when the rod is pulled away from the microscope body. Normally, we use the 1.6X in conjunction with the 60X 1.49NA objective.

Camera

TESM has an iXon^EM+ Andor 885K Camera.

Pixels	1004 x 1002
Pixel Size (µm x µm)	8 x 8
Imaging Area (mm x mm)	8 x 8
Well Depth (e-)	30000
Gain Register Well Depth (e-)	80000
Frame Rate	31.4
Frame Rate 2x2	60.5
Read Noise (e-)	25
Read Noise with EM Gain (e-)	<1

Light Path

Lightpath Dial

The dial lets you select the camera or the oculars. Turn this dial slowly because the IX71 microscope has a weak internal coupler that will cause Karl tremendous grief if he has to fix it again.

Pixel Size Calculation

µm/pixel = 8µm * B/(M * m)

Objective

M

Optivar magnification

m

Camera Binning

B

CCD Pixel Size

8µm

For Example:

8 * 2/(60/1.6) = 0.1666µm/pixel or 167nm/pixel

60X Obj	Optivar
Binning	1X	1.6X
1	0.1333	0.083
2	0.2667	0.167

TESM Imaging Protocols

This is only for very advanced imaging, but this is also the only way to control the piezo positioner for the objective and the structured light grating, as well as the Sutter 10-2 LAMBDA filter wheel

TESM is built around an ITC18 The ITC18 allows TESM to acquire images at high speed with precision, keeping all hardware devices synchronized. TESM Imaging Protocols are written and tested beforehand, and loaded into the ITC18 by µManager by using µManager's BSH scripting engine. Executing TESM Imaging Protocols are done asynchronously of the host computer.

Current TESM Imaging Protocols:

/storage/big1/kdb/Linux/tesm_scripts/protocols

Scripts used to produce TESM Imaging Protocols:

/storage/big1/kdb/Linux/tesm_scripts/