Una guía para la iluminación estructurada de microscopía TIRF a alta velocidad con colores múltiples

The overall goal of this protocol is to construct and align a structured illumination microscope suitable for high speed live cell super resolution imaging capable of operating in TIRF and optical sectioning modes with multiple colors. This method can help answer key questions in the field of cell biology that cannot currently be answered using standard diffraction limited TIRF microscopy. The main advantage of this technique is its fast time resolution compared to other super resolution microscopy techniques, which make it well suited to imaging dynamic processes in living cells.

Though this protocol describes the steps for construction of a TIRF-SIM microscope, the setup is flexible and easily modified to implement imaging modalities such 3D-SIM, multifocal SIM, and non-linear SIM. To arrange and align the excitation path for a structured illumination microscope, begin by marking the positions of the components on the optical table as indicated here. After inserting the first dichroic mirror, DM4, into the filter cube turret of the microscope frame, insert the second dichroic mirror, DM3, into a one inch square kinematic mirror mount and position it one focal length away from the condenser lens, L5.Next, to accurately define the optical access for the system, remove the objective lens, OB, from the turret, and instead screw in an alignment tool.

Then, use the dichroic mirror DM3 and a temporary alignment mirror positioned at the approximate ultimate location of the SLM to steer a collimated reference beam from laser one through the center of the holes in the two alignment disks. Using three mirrors and dichroic mirror DM2 as depicted here, direct the beam from laser one to the temporary mirror. Once the coarse optical axis has been determined, remove the alignment tool.

Then insert an iris into the beam path before it enters the microscope body and center it on the beam. Attach a piece of white card with a small hole centered on the iris, then reinsert the objective lens. Next, make iterative angular adjustments to the mirrors DM3, and at the SLM position to center the back reflection on the card with the incoming beam.

Then temporarily remove the objective lens and mark the laser spot on the ceiling to create a reference position. Insert the tube lens, L5, roughly one focal length away from the objective, mounting it on a linear translation stage set to translate along the direction of the reference beam. Adjust the tube lens position and angle such that the beam leaving the objective is collimated and hits the reference spot on the ceiling.

Check that the lens is perpendicular to the beam by again checking the back reflection with the iris and white card. Then remove the objective lens and insert the second lens of the image relay telescope, L4.Use the linear translation stage to adjust the position and angle of this lens to maintain collimation, and to ensure the reference beam still hits the marked spot on the ceiling. Replace the objective lens and insert the first lens of the telescope, L3.Adjust the position and angle of this lens to ensure collimation and non-deflection as described earlier.

After mounting the SLM chip according to the text protocol, with the lenses aligned, insert the SLM in place of the mirror. Adjust the position of the SLM such that the reference beam is located at the center of the SLM chip, and adjust the angle so that the beam passes through the two relay lenses. Then check that the reference beam is still centered on the marked spot.

Mount the liquid crystal variable retarder, or LCVR, with its fast axis at 45 degrees to the incident polarization. Then after completing the alignment of the polarization rotator according to the text protocol, using transmitted light through the oculars, focus on a reticle and fix the objective lens at this position. Then prepare a monolayer of fluorescent beads by spreading a drop of 100 nanometer multicolor beads on a number 1.5 coverglass.

Leave the beads to dry to adsorb the beads to the glass before re-immersing in water. Using immersion oil, place the bead sample onto the objective. Finely adjust the position of the camera so that the fluorescent bead layer is in focus.

After generating SIM binary grating patterns as bitmap files and uploading them to the SLM according to the text protocol, load the SLM control software and click Connect. In the Repertoire"tab, click Load"to open the repertoire file, and check the number of running orders contained in the file. Click Send to Board"to upload the repertoire file to the SLM.

Next, select the Status"tab and enter the number of the running order. Click Select"to change the running order to the alignment grating. Insert a spatial mask, or SM, mounted on an XY stage into the beam path at the focal position of L3, and translate its position with respect to the optical axis such that only the desired first orders are passed.

Directly after the spatial filter, only two circular beams will be visible. Check the image of the fluorescent bead layer on the camera. If the two circular beams are not overlapping as depicted here, then reposition the sample plane, iteratively adjusting the objective lens and camera position.

After fine tuning the focus and setting the sample plane according to the text protocol, confirm TIRF illumination by imaging, for example, a solution of 10 micromolar rhodamine 6G dye by bringing the dye into focus. If the two beams are incident at the correct TIRF angle, then single molecules will be visible without high background, and the edges of the circular aperture will be in focus. Fine adjustments to the position of the beams can be made by adjusting dichroic mirror DM3.

To synchronize and calibrate the system, place the bead monolayer sample on the objective, and bring into focus. Use the SLM control software to program the SLM to display each of the three phase shift images in turn for the first pattern orientation. Then switch to running order four of the example repertoire.

In the acquisition software, configure the camera for the global exposure period by choosing Advanced Camera Properties"and setting Output Trigger Kind 1"to positive, and Output Trigger Kind 2"to negative. In the sequence pane under Scan Type, select Hard Disk Record, and set the frame count to three. Then click Start"to acquire three frames.

The SLM pattern will change upon each exposure. After callibrating the system according to the text protocol, in the SLM control software, switch the SLM running order to the full series of nine binary grating images required for TIRF-SIM. This is running order zero in the example repertoire.

After acquiring nine images of the bead sample, in the sequence pane of the camera software, select Hard Disk Record"as the scan type, and change the frame count to nine. Then click Start"to acquire images. Finally, under Save Buffered Images, choose TIFF as the image type, and click OK.Reconstruct a super resolution image according to the text protocol.

Shown here, multicolor 100 nanometer diameter fluorescent beads were imaged to compare standard TIRF to TIRF-SIM. TIRF-SIM clearly had significantly higher lateral resolution compared to TIRF. The estimated resolution of the microscope is 90 nanometers and 120 nanometers for 488 and 640 nanometer TIRF-SIM, respectively.

This corresponds to a two fold improvement in lateral resolution for both wavelengths compared to the theoretical diffraction limited case. As seen here, fluorescently labeled amyloid fibrils were formed in vitro and also used as a test sample for demonstrating doubled resolution. Sub-cellular structures with high contrast, such as emGFP labeled microtubules or LifeAct-GFP are ideal for TIRF-SIM imaging, and yield high contrast, super resolution images.

TIRF-SIM imaging using the demonstrated setup enables observation of a subpopulation of microtubules located in the vicinity of the basal cell cortex, and microtubule polymerization and depolymerization can be seen over time. Low contrast samples without discrete structures lack high resolution information except at the edges of the plasma membrane, and are hence suboptimal for TIRF-SIM imaging. Finally, high modulation contrast is essential for successful SIM imaging.

As demonstrating here, the Fourier transform of the reconstructed image allows visualization of the SIM optical transfer function. Once mastered, this technique can be completed in around one week, if all the necessary parts have been acquired. When attempting this procedure, one must be aware of the most critical issues for successfully implementing TIRF-SIM.

First, precise alignment of the beams is essential. This can be difficult, as they must be located at the edges of the objective lens aperture. Secondly, it is critical that the polarization state of the light is co-rotated with the pattern.

Without this, the pattern contrast of the sample will be low and subsequent image construction will be impossible. After watching this video, you should have a good understanding of the most important steps in the construction and alignment of an SLM based, TIRF-SIM microscope. Don't forget that working with Class 3B lasers can be extremely hazardous.

The laser power must be kept at eye safe levels whilst performing the alignment process with the TIRF objective in place.