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

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1 Before you begin, make sure your sample 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:

2 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. Basic Concepts of Total Internal Reflection Refractive Index: A measure in the reduction of the speed of light inside the a medium (compared to the speed of light in a vacuum) Refraction of Light: Vacuum 1.00 Air Water 1.33 Glass 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 Refraction Angle refraction Interface Aqueous Solution (RI=1.33) Glass (RI=1.52) Incident light Normal incident angle Critical Angle 90 Total Internal Reflection Critical Angle: the angle of the incident light where the refraction angle is 90 Total Internal Reflection: occurs when the incident angle is greater than the critical angle Majority of the light is reflected

3 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 Evanescent Wave Interface Aqueous Solution (RI=1.33) Glass (RI=1.52) Critical Angle Total Internal Reflection decreasing intensity } <250nm This evanescent wave is used for excitation in TIRF microscopy

4 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 Fluorophores Evanescent Wave Fluorophores Excited Fluorophores Coverslip Coverslip CRITICAL ANGLE Objective Objective Laser Laser TIRF Sensor 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

5 3. Total Internal Reflection Fluorescence Microscopy continued... 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... Advantages of TIRF: 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 Resolution: 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 R=0.61 /NA Example: R = (0.61x488nm)/1.4 = ~200nm 700nm 700nm z 200nm 250nm membrane(~10nm) 200nm 250nm coverslip x Confocal TIRF Confocal TIRF y 200nm 200nm y 200nm 200nm x Resolve as 2 separate structures No difference in resolution Resolve as 2 separate structures x Unable to resolve as 2 separate structures Improved resolution with TIRF due to thinner optical section Resolve as 2 separate structures

6 Comparison of TIRF microscopy with Epi-fluorescence and Confocal Microscopy TIRF EPI Confocal Evanescent Wave Excited Fluorophores Fluorophores Coverslip Focal Plane { } Focal Plane Laser TIRF Sensor Light Laser 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 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 passes over the sample point by point slow image acquisition (25-30sec/image) Out-of-focus emission blocked by pinhole optical section ( nm) decrease in signal-to-noise improving the contrast focused on coverslip focused on coverslip focused at middle of cell (not the same cell as EPI & TIRF image)

7 A. THE EQUIPMENT The Leica DMI 6000B Inverted Microscope... Left Side Adjusts the intensity of the transmitted light and fluorescent light Controls the size of aperture diaphragm (leave fully open) Alters size of field diaphragm. (leave fully open) Toggles between Transmitted Light (TL) and Incident Light (IL, fluorescence) Front Light goes to camera Light goes to eyepiece Right Side GFP cube RFP cube QAD cube Move objective to lowest position

8 A. THE EQUIPMENT Continued... The Leica DMI 6000B Inverted Microscope adapted for TIRF microscopy... TIRF module - contains the scanner and TIRF sensor Incubation and laser safety box Laser box El6000 Fluorescence light supply Controls z movement of the objective Controls x,y movement of the stage SmartMove Precise or fast stage movement(x,y) Fine or Course focus (z movement)

9 B. START-UP PROCEDURE 1. Set the chamber temperature to 37 C 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. 2. Switch on the system in the following order: 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

10 There are 3 portions within the LAS Software: 1. Scan Parameters Window 2. Configuration Window 3. Image Window

11 C. SETTING THE CONFIGURATIONS OF THE MICROSCOPE In the Acquire tab and the Setup window: 1. Select the objective: HCX Plan-Apo 63x, NA 1.47 HCX Plan-Apo 100x NA 1.47 NOTE: the 40x is not a TIRF objective 2. In the Experiment Settings activate: Use sequencer board Use sequence timestamps Single image mode Enable Smartmove control during acquisition Z movement is irrelevant for TIRF 3. Select the type of Shutter Control: After each sequence for the fastest image acquisition 4. Set TIRF Configuration to: Automatic mode

12 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 GFP RFP QAD 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

13 D. FOCUSING ON THE SAMPLE Continued 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

14 E. AUTOALIGNMENT OF THE LASER 1. Switch the Contrast Method to TIRF and select the GFP filter cube NOTE: All laser lines will be aligned regardless of the filter cube selected 2. Select Autoalign 3. Follow the instructions in the Autoalignment

15 F. ACQUIRING A TIRF IMAGE With the Contrast Method set for TIRF 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). GFP RFP QAD

16 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 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 Start a time series

17 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

18 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. CLEAN THE OBJECTIVE!!!! These objectives are very expensive and are damaged very easily if not cleaned properly. To clean: 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 IF YOU DO NOT CLEAN THE OBJECTIVES PROPERLY, YOUR MICROSCOPE PRIVILEGES WILL BE TAKEN AWAY. 6. Clean up any mess on the stage, in the incubator box, on the table and the desk.

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