Before you begin, make sure your sample...

1. is seeded on #1.5 coverglass (thickness = 0.17)

2. is an aqueous solution (ie. fixed samples mounted on a slide will not work - not enough difference in refractive index of the glass and mounting solution for total internal reflection of the laser to occur)

3. are adherent cells (ie. cells in suspension are too far away from the coverslip)

4. has a fluorescent marker on the surface of the cell in order to focus on the sample. The adjacent to the coverslip will be used as a reference to focus on the coverslip (cannot focus or align the laser if there is no fluorescent markers at the coverslip interface)

This training manual was adapted from the guide found at:

http://www.bris.ac.uk/biochemistry/mrccif/tirfguide.pdf

Basic Concepts of Total Internal Reflection

Refractive Index:

Refraction of Light:

A measure in the reduction of the speed of light inside the a medium (compared to the speed of light in a vacuum)

· the bending or change in direction of light as it travels from one medium into another with different refractive indexes

· refraction of light only occurs when the incident light meets the interface at an angle

· light will travel straight through with no change of direction when crossing perpendicular to the interface

· the degree of refraction increases as the angle of the incident light increases

Total Internal Reflection Fluorescence Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRF) is...

TIRF microscopy is a specialized fluorescence technique that images a very thin optical section (50-250nm) adjacent to the coverslip. TIRF microscopy is used to study molecular events at or near the cell surface with speeds and resolution that is not possible with other imaging techniques by using conditions that create total internal reflection that generates an evanescent wave.

Vacuum 1.00Air 1.003

Water 1.33Glass 1.52-1.54

Interface

Aqueous

Solution

(RI=1.33)

Glass

(RI=1.52)

Incident

light

Refraction

Angle

Normal

­ refraction

­ incident

angle

90°

Critical

Angle

Total Internal

Reflection

Critical Angle:

Total Internal Reflection:

the angle of the incident light where the refraction angle is 90°

· occurs when the incident angle is greater than the critical angle· Majority of the light is reflected

Basic Concepts of Total Internal Reflection Continued...

Evanescent Wave:

· during total internal reflection, a small portion of the reflected light penetrates through the interface

· this creates a very thin electromagnetic field (<250nm) adjacent to the interface (evanescent wave)

· this wave has identical frequency to the incident light

· the evanescent wave propagates parallel to the interface

· the intensity decreases exponentially with increasing distance away from the interface

Interface

Aqueous

Solution

(RI=1.33)

Glass

(RI=1.52)

Critical

Angle

Total Internal

Reflection

Evanescent

Wave

} <250nmdecreasingintensity

This evanescent wave is used for excitation in TIRF microscopy

3. Total Internal Reflection Fluorescence Microscopy

For TIRF microscopy...

· The laser is angled within the objective.

· Once the critical angle is passed, total internal reflection of the laser occurs and an evanescent wave is generated.

· The evanescent wave travels along the coverslip exciting the entire sample simultaneously (therefore the image can be acquired with a CCD camera)

· The intensity of the evanescent wave decreases exponentially away from the coverslip

· The evanescent wave only has sufficient energy for excitation within close proximity of the coverslip

· The evanescent wave only has sufficient energy for excitation within close proximity of the coverslip

· Therefore only fluorophores within this close proximity of the coverslip produce emission

Coverslip

Fluorophores

Evanescent

WaveExcited

Fluorophores

Laser

TIRF

Sensor

Objective

Coverslip

Fluorophores

Objective

CRITICAL

ANGLE

Laser

TIRF

Sensor

In the Leica system the returning beam of the total internal reflection is measured on an internal sensor.

This feedback allows precise, fully automatic and reproducible setting of the penetration depth of the generated evanescent field

3. Total Internal Reflection Fluorescence Microscopy continued...

Resolution:

TIRF microscopy is a tool to study the molecular events at or near the cell surface at a speed and resolution that is not possible with other imaging techniques.

More specifically, TIRF microscopy is used to image at or within close proximity...

· distribution · colocalization· trafficking (movement in the membrane, endocytosis, exocytosis, etc.)· changes in the above in response to various stimuli

· Fast image acquisition (up to 30 frames/sec) - the image is capture using a CCD camera since the evanescent wave propagates along the coverslip exciting the entire sample simultaneously

· Very thin optical section (50-250nm) adjacent to the coverslip

· Decrease signal-to-noise (increase contrast)

· Resolution MAY be improved with TIRF microscopy compared to confocal microscopy

the minimum distance between two points required to identify them as separate points

The resolution limit of a microscopy is determined by:

· wavelength of light used for excitation· numerical aperture (NA) of the objective

Advantages of TIRF:

200nm

70

0nm

25

0nm

x

z

Confocal TIRF

200nm 200nm

x

y

Resolve as 2

separate structures

Resolve as 2

separate structures

200nm

70

0nm

25

0nm

coverslip

membrane(~10nm)

Confocal TIRF

200nm 200nm

Unable to resolve as 2

separate structures

Resolve as 2

separate structures

x

y

No difference in

resolution

Improved resolution

with TIRF due to

thinner optical section

R=0.61l/NA

Example: R = (0.61x488nm)/1.4

= ~200nm

Coverslip

Laser

Evanescent

Wave

FluorophoresExcited

Fluorophores

TIRF

Sensor

TIRF

· evanescent wave travels along the coverslip exciting the entire sample simultaneously

· fast image acquisition with CCD camera (30 frames/sec)

· No out-of-focus emission generated

· very thin optical section (50-250nm)

· decrease in signal-to-noise improving the contrast

· May improve resolution at the cell surface compared to confocal

Light

EPI

· entire sample exposed to excitation light simultaneously

· fast image acquisition with CCD camera

· Both in-focus and out-of-focus emission collected

· No optical sectioning (”fuzzy” image)

· High signal-to-noise (poor contrast)

Laser

Focal Plane {

Confocal

· laser passes over the sample point by point

· slow image acquisition (25-30sec/image)

· Out-of-focus emission blocked by pinhole

· optical section (600-900nm)

· decrease in signal-to-noise improving the contrast

Comparison of TIRF microscopy with Epi-fluorescence and Confocal Microscopy

} Focal Plane

focused on coverslip focused on coverslip focused at middle of cell(not the same cell as EPI & TIRF image)

A. THE EQUIPMENT

The Leica DMI 6000B Inverted Microscope...

Toggles between Transmitted

Light (TL) and Incident Light

(IL, fluorescence)

Alters size of field

diaphragm. (leave fully

open)

Adjusts the intensity of

the transmitted light and

fluorescent light

Controls the size

of aperture

diaphragm (leave

fully open)

GFP cube

QAD cube

Light goes to camera

Light goes to eyepiece

RFP cube

Left Side

Front

Right Side

Move objective to

lowest position

TIRF module - contains the

scanner and TIRF sensor

El6000 Fluorescence

light supply

Laser box

Incubation and

laser safety box

SmartMove

Controls z movement

of the objective

Controls x,y movement

of the stage

Precise or fast stage

movement(x,y)

Fine or Course focus

(z movement)

The Leica DMI 6000B Inverted Microscope adapted for TIRF microscopy...

A. THE EQUIPMENT Continued...

1. Set the chamber temperature to 37°C

2. Switch on the system in the following order:

? Turn on Temperature Controller (Bottom Shelf)

? Turn on Heating Unit (On table, left side)

IMPORTANT - The temperature controller MUST ALWAYS be on when the heating

unit is on!!!

The function of the heating unit is to heat and will continue to heat

damaging the system if it is not regulated by the temperature controller

It takes ~2hrs for the temperature of the entire system to equilibrate at 37°C.

Make sure all of the doors and the top of the chamber is closed.

A. Turn on El6000 Fluorescence supply (bottom shelf - right)

B. Turn on the main power switch on the Laser box (green button on the black box left of

the table)

C. Turn on the camera (bottom shelf - left)

D. Start the computer (and login)

E. Move the condenser arm back and then turn on the microscope at the MICBOX (bottom

shelf - middle). The stage will move and it is important to make sure the condenser will

not be hit by the stage.

F. Turn on the laser key switch (black box left of the table)

G. Start the Leica LAS software

Click on OK

H. Intialize the stage? Yes if you want to use the Mosaic or Marking features, No if you do

not. The stage will move if you select yes so make sure the objective and condenser will

not be hit

B. START-UP PROCEDURE

There are 3 portions within the LAS Software:

1. Scan Parameters

Window

2. Configuration Window

3. Image Window

C. SETTING THE CONFIGURATIONS OF THE MICROSCOPE

In the Acquire tab and the Setup window:

1. Select the objective:HCX Plan-Apo 63x, NA 1.47HCX Plan-Apo 100x NA 1.47

NOTE: the 40x is not a TIRF

objective

2. In the Experiment Settings

activate:

þ Use sequencer boardþ Use sequence timestampsþ Single image modeþ Enable Smartmove control

during acquisition

Z movement is irrelevant for TIRF

3. Select the type of Shutter

Control:

þ After each sequence for the fastest image acquisition

4. Set TIRF Configuration to:

þ Automatic mode

D. FOCUSING ON THE SAMPLE

The laser must be aligned at the start of every session. Before the laser can be

aligned, there must be a sample in place and focused on the coverslip. This will be done

using epi-fluorescence (FLUO) .

1. Move the objective to its lowest position

2. Place a SMALL drop of oil on the objection

(too much oil ruins objectives and the TIRF objectives are very expensive!!!!)

3. Place the sample in place and move the objective up with the focus knob until the oil makes

contact with the bottom of the dish.

4. Close the doors on the chamber and let the temperature of the dish equilibrate (~10 min)

5. In the Light Path Settings, set the Contrast Method to FLUO

6. Select the appropriate

filter cube (GFP, RFP, or

QAD)

7. Click on Live to open (and close) the shutter

8. Send the light path to the eye pieces

9. Select Course Focus on the SmartMove and focus the sample

10. Once in focus, send the light path back to the camera and

switch back to Fine Focus on the Smartmove

GFPRFPQAD

D. FOCUSING ON THE SAMPLE Continued...

11. In the Acquisition Tab, adjust the settings of the camera

- Binning

- Exposure

- Gain

- EM Gain

- Intensity (Set to 5 for TIRF)

12. Focus on the coverslip.

NOTE: In order to align the laser properly (essential for good TIRF), you

must focus on the coverslip. It is often very difficult to determine

whether or not you are focused on the coverslip. As a suggestion, try

to focus on the edges of the cells.

13. Click on Live to close the shutter

E. AUTOALIGNMENT OF THE LASER

1. Switch the Contrast Method to TIRF and select the GFP filter cube

2. Select Autoalign

3. Follow the instructions in the Autoalignment

NOTE: All laser lines will be

aligned regardless of

the filter cube selected

F. ACQUIRING A TIRF IMAGE

With the Contrast Method set for TIRF...

1. Select appropriate filter cube (QAD for rapid switching with multi-colour fluorescence)

2. Select the appropriate laser and adjust the laser power (start at 20%)

3. Select the appropriate pseudocolour

4 Select the penetration depth

5. To add another channel, click on “+” and select the appropriate filter cube, laser,

pseudocolour and penetration depth (repeat step #1-4 for the new channel)

NOTE: select the QAD filter cube for multi-colour fluorescence

5. Click on live to preview the image (click on Live again to turn off preview).

GFPRFPQAD

G. OPTIMIZING THE TIRF IMAGE

For each channel, adjust the following to optimize the image

- binning

- exposure

- gain

- penetration depth

- direction of laser

NOTE: In order for high speed image acquisition of multi-colours the filter cube, gain and

direction of the laser must be same for all channels.

Start a time series

Start and Stop preview for the active channel (and open shutter in Fluo)

Capture a single image for the ACTIVE channel

Capture a single image for ALL of the channels

H. CAPTURING A TIME SERIES

Once the settings have been optimized,

1. Click on “t” in the Acquisition Mode

2. Set the criteria for the time series (the interval between images and the duration)

For high speed acquisition, select minimize and synchronize hardware

NOTE: Synchronize hardware is only available if the filter cubes, gain and direction of

the laser are identical for all of the channels.

Start a time series

I. SHUT-DOWN PROCEDURE

1. Close LAS software and shut-down the computer

2. Turn the laser key off, but DO NOT turn the main power switch on the Laser box (green button)

off yet. The laser must cool at least 5 min.

3. Switch off all of the other components in the system:

- El6000 Fluorescence supply (bottom shelf - right)

- Camera (bottom shelf - left)

- Temperature Controller (bottom shelf)

- Heating Unit (on table, left side)

4. Once the laser has cooled for at least 5 min switch of the main power switch on the laser box

5.

· wipe off any oil on the objective with lens paper

· use lens paper damp with lens cleaner to clean the objective

· dry the objective with a clean and dry piece of lens paper

6. Clean up any mess on the stage, in the incubator box, on the table and the desk.

CLEAN THE OBJECTIVE!!!! These objectives are very expensive and are damaged very easily if

not cleaned properly.

To clean:

IF YOU DO NOT CLEAN THE OBJECTIVES PROPERLY, YOUR MICROSCOPE PRIVILEGES

WILL BE TAKEN AWAY.