皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

The overall goal of this procedure is to acquire high quality, total internal reflection, fluorescence or turf images of cell wall bearing microorganisms. This is accomplished by first preparing cover slips and cells. The second step is to precisely align the microscope setup for optimal image quality.

Next, the correct incidents, angles, and imaging conditions are chosen for acquisition of images or movies. The final step is post imaging processing of the acquired images. Ultimately, turf microscopy is used to study the dynamics of protein localization at the cell cortex.

The main advantage of this technique over exceeding metals like conventional or confocal for microscopy, is that the image contrast int microscopy it's very high, allowing fast times and low light. As such, This method can help answer key questions in the field of membrane research, such as the mechanisms of lateral protein segregation, or the mobility of membrane proteins. Cover slips for turf microscopy should be cleaned from dust particles prior to use.

Since turf is sensitive to background signals arising from dust on a dirty cover slip. A cleaned cover slip with less background is required to begin the procedure for cleaning cover slips. Use forceps to place cover slips in a ceramic holder with a lid.

Next, fill a glass container with one molar sodium hydroxide. This sodium hydroxide can be reused multiple times. Place the ceramic holder containing the cover slips into the sodium hydroxide and incubate the cover slips for two hours under slow continuous rotation.

Do not incubate for more than eight hours, as this will create opaque cover slips after two hours, wash the cover slips with distilled water twice for five minutes each time under slow continuous rotation, store cleaned cover slips in the ceramic holder covered in 100%ethanol. To prepare bacillus subtlest cells for turf microscopy dilute to an OD 600 of 0.01 to 0.05 in five milliliters of appropriate growth media and grow to exponential phase. Prepare 1.25%aros in synthetic medium dissolve aros powder in a 1.5 milliliter plastic tube at 95 degrees Celsius, and then store in a heating block at 72 degrees Celsius.

Add a small drop of aros to the middle of a slide and with a second slide, press the aros into a flat pad after at least two minutes, carefully separate the slides. Spin down 500 microliters of bacillus subtlest cells in a micro centrifuge at 12, 000 RPM for one minute. Discard supernatant and resuspend pellet in 50 microliters medium.

Add two to four microliters of cells to the center of the aros pad. Next, use forceps to remove a cleaned cover slip from the ethanol filled container and dry it with pressurized air. Carefully place the cover slip on the sample.

Let cells settle for at least two minutes prior to microscopy. For long-term imaging, seal the edges of the cover slip with the heated mixture of Vaseline, lanolin and paraffin or Val app. To prepare Saccharomyces visi cells for turf microscopy, dilute a pre culture and grow in appropriate media for at least five hours.

To exponential phase, take a cover slip from the ethanol filled container and dry it with pressurized air with a pipette spread. Five microliters, conval and a or con a solution on the cover. Slip and dry with pressurized air con A binds to carbohydrates of the yeast cell wall and immobilizes yeast cells.

Next, transfer 4.5 microliters of a yeast cell suspension to one side of the con, a coated cover slip. Carefully place the cover slip sample side down on a microscope slide starting with one edge and letting drops slowly. This will avoid the formation of air bubbles.

Let cells settle for at least two minutes prior to microscopy seal edges of cover slip with val app for long-term imaging. All experiments shown in this video are performed on a customized turf setup based on a fully automated IMEX stand with an Olympus 100 x 1.45 NA objective. The light sources used are 75 milliwatt diode, pumped solid state or DPSS lasers at 488 nanometers and 561 nanometers.

Turf angles and fluorescence. Recovery after photobleaching or frat position can be adjusted instantaneously via two galvanometers. A third galvanometer is used to switch between epi, fluorescence, frap, and turf.

The galvanometers, which are directly controlled from the live acquisition software, allow simple measurement of localization kinetics for objects traced in turf images are collected with an EM CCD camera and a two x magnification lens in front of the camera. Acquisition is also controlled by the live acquisition software. Prior to working with the laser, put on laser safety goggles, project the laser on the ceiling in turf mode and calibrate the zero degree position such that the laser is in a straight line.

With the objective, focus the laser spot to a minimal size after optimal adjustment, the laser profile should form a well-defined spot with a roughly round shape. Perform calibration before every session. To obtain optimal image quality, Alignment of the turf microscope is critical for the success of this procedure.

The correct incidence, angles and parameters for image acquisition must be carefully adjusted for optimal image quality. To begin image acquisition, identify the position of cells using brightfield illumination. Red LEDs are particularly good as they do not induce significant photo damage.

Switch to laser illumination and select appropriate lasers and filter combinations to excite the fluorophores of choice, make sure that the turf angle is subcritical. Otherwise signals arising from GFP fusion proteins will not be detected. Find the top section of the cell, which is side of the cell facing the cover slip.

Gradually increase the incident angle of the laser beam until signals disappear, and then slowly decrease the angle until the point where fluorescence at the cell surface reappears. Adjust the Z focus again for optimal position at the surface. Acceptable turf angles create no blurred halo at the edge of the cell as illustrated by this example of yeast protein.

PMA one GFP imaged with decreasing incidence angles from top left to bottom right. The images show a sudden loss of signal at the critical angle and progressive decrease in structural information and signal to noise ratio, adjust laser intensities and camera gain to maximize dynamic range. Typically, E-M-C-C-D cameras are optimal to detect very low signals at high signal to noise ratios.

Turf microscopy is very sensitive to small height. Differences of the cover slip resulting in only a few cells that can be imaged at the same time. This image of a dense suspension of fluorescent beads is an example of an uneven field of view where only part of the field of view is in focus.

Therefore, for small cells, the acquisition region can often be reduced to a sub region containing the object of choice. For double color turf experiments bleed through of fluorophores must be minimized for R-F-P-G-F-P pairs. Use separate emission filters as RFP is weekly excited by 488 nanometer light.

A typical workflow to avoid bleed through and double color imaging is shown here. After imaging the cells with separate filter sets, the images are devolved and aligned using fluorescent beads before being merged For analysis, a critical parameter influencing image quality is laser alignment and a clean objective. After aligning the laser and focusing on the Z position used for turf imaging, remove the sample and clean the objective of all oil.

Residual oil will lead to diffraction of the laser light and speckles in the beam profile. Turf microscopy can be used to visualize the distribution of actin homologs and cell wall enzymes in bacterial cells. In this representative result, bacillus subtles cells expressing GFP fusions of the actin hoog MBL or the transpeptidase PBPH were imaged by turf and regular epi fluorescence.

The images here represent average projections of a time series to indicate the area covered by motile. MBL patches monitored with different imaging modes. The blue outline in the overlay image indicates the cell boundary visualized from a brightfield image image.

The weekly expressed transpeptidase PBPH localizes to cortical patches that are hardly visible by epi fluorescence, but can be clearly distinguished by turf. The reduced penetration can be best observed for P-B-P-H-G-F-P signal at the SEPTA indicated by the arrows, which appears as lines in epi fluorescence, but as dots in turf. This next set of images show a comparison between epi fluorescence and turf illumination of the TOR complex component, bit 61 in Saccharomyces visi.

This protein is weekly expressed and forms small cortical patches with only a few protein copies per patch. In epi fluorescence only a few patches are visible above the strong background while patches can be imaged with a very good signal to noise ratio in turf. Additional improvement of image contrast can be obtained through various image processing steps, such as subtraction of Gaussian or median blurred images while local background subtraction removes noise and sharpens high intensity structures.

This often comes at the price of a loss of fine structures and exaggerated amplification of high intensity regions. As indicated by the arrow, a superior procedure is 2D deconvolution, which can be performed with free or commercially available software packages. The increase of contrast after deconvolution is illustrated by this time-lapse movie of GFP RAs two that shows alternating raw and devolved turf images.

Since GFP RAs two is a fast moving protein, fast acquisition times are important to resolve. Its dynamic behavior. Image processing is especially important for colocalization analyses as illustrated in this time lapse double color movie of yeast proteins, FET 3G FP, and PMA one RFP.

The first 10 frames represent raw images and the remaining frames are devolved images To make analysis possible, it is critical to maximize contrast and sharpen the blurry raw images. Turf and frap offer a powerful combination for studying the dynamics of cortical proteins. Using this technique in B subtlest cells expressing G-F-P-M-B-L ruled out treadmilling as the mechanism for the observed motility of MBL containing patches.

The chm graph of the moving patch and the intensity profile along the chm graph indicated by the dotted line is shown in a similar fashion turf frap of the yeast plasma membrane. A TPAs PMA one revealed the slow rearrangements of yeast plasma membrane proteins, which distribute into network like domains covering the whole cell surface After its development. This technique paved the way for researches in the field of bacteria cell biology to explore the mechanism of cell wall synthesis in BA subtilis.

After watching this video, you should have a good understanding of how to acquire turf images of microorganisms like yeast and bacteria, and how this technique can help in studying dynamic properties of cortical proteins.