Training Guide for Carl Zeiss LSM 510 META Confocal Microscope

Training Guide for Carl Zeiss LSM 510 META Confocal Microscope AIM 4.2 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017)

Power ON Routine 1 2 Turn ON Components and System/PC switches on remote paddle. 2 LSM 510 META Training Guide 3 Turn ON X-Cite epifluorescence light source. Turn ON System PC. Wait for PC to boot into Windows OS and login to LSM User account.

Power ON Routine 4 5 6 Double click LSM 510 icon from desktop to start AIM software. From the Switchboard, select toggle for Scan New Images and then click Start Expert Mode. Wait for system to complete its initialization. 3 LSM 510 META Training Guide

Getting Started with AIM 4.2 The AIM software is contained within the stand-alone toolbar illustrated below. The first two buttons along the top row of options are the main functions used for image collection. They are: File The File Menu contains tools necessary for creating image databases as well as importing/exporting native.lsm files to other formats such as TIFF or JPEG. By default, the AIM software saves natively scanned image data to the.lsm file format. The LSM file format is essentially a TIFF file, but the header of the file contains all the proprietary detail about the settings used to collect the image. When you save a file, not only is an LSM file saved but also a reference to a MS Access database, which defines the location where the image is stored as well as provides a catalog of images and a display of their information. 4 LSM 510 META Training Guide

Getting Started with AIM 4.2 Acquire The Acquire menu contains all the functions for defining your microscope settings, configuring your imaging conditions, defining your scan settings and collecting your image(s). It reads left-to-right in the order that you utilize the tools. 5 LSM 510 META Training Guide

Power On Lasers The first thing we need to do before we mount our sample or configure any settings is to power on all the lasers we will require for our experiment. The LSM 510 META has 4 lasers; an Argon laser (458, 488, 514nm), 2 HeNe lasers (543, 633nm) and a diode (405nm). The Argon laser has a very specific power routine listed below. All other lasers can be quickly switched on/off. 1. From the Acquire mode on the main toolbar, click Laser. 2. Select the Argon laser from the Laser Control dialog. 3. Click Standby. 4. Wait for laser to warm up and reach a standby tube current of between 3.1-3.5A. 5. Click On. 6. Increase Output (%) until Tube Current reads 6.1A. Note: 6.1A is the nominal working current recommended by the laser manufacturer. Running the tube current higher or lower will directly affect the output power and may reduce the lifetime of the laser. 7. Select HeNe543 laser. 8. Click On. 9. Repeat steps 7 and 8 for remaining lasers if required. 6 LSM 510 META Training Guide

Mounting Sample on the Microscope Once the system has been powered on, we can mount our specimen on the stage above the microscope objective. 1. From the Acquire menu on the main toolbar, switch microscope mode to VIS from the default LSM. Note: Changing the microscope over to VIS mode will close the shutters for all laser lines and put the microscope in a state where you can visualize fluorescence in the eyepieces. 2. Click Micro to open the Microscope Control dialog. 3. Mount your sample on the stage carrier. 7 LSM 510 META Training Guide

Mounting Sample on the Microscope 4. From the Microscope Control dialog, use the shortcut buttons to turn on the fluorescence light path to the desired color. GFP generic green filter set DAPI generic UV filter set RFP generic red filter set OFF all light sources off Note: Alternatively, you can select the filter set (A) manually as well as open/close the epifluorescence shutter (B). There is also control over the transmitted light should you require this (C). Note: If you open the Reflected Light shutter and no light comes through the objective, make sure to toggle Start/Stop button on front of X-Cite power supply (see Power ON Routine Step 3). 5. Via the microscope eyepieces, visualize your sample and adjust the focus (Z) and stage position (X,Y) accordingly. 6. Once your imaging location is found, place it in the center of the field of view. 7. To begin imaging, switch back to LSM from VIS in the main toolbar. 8 LSM 510 META Training Guide

Beam Path Configuration The LSM 510 META is equipped with 3 detectors (including a 32 channel spectral array) for up to 8 color simultaneous or sequential image collection. 1. Click Config from the main toolbar to open the light path configuration tool. 9 LSM 510 META Training Guide

Beam Path Configuration The Configuration Control dialog is where we configure our beam path for collecting fluorescence signal. It consists of two modes, Channel Mode and Lambda Mode. Channel mode is used for collecting traditional confocal images. Lambda mode is used for spectral imaging of fluorophores with heavily overlapping emissions. In this example, we will design a 2 channel simultaneous image acquisition for Alexa 568 and Alexa 488 dyes. 2. The system defaults to Single Track which allows image collection of up to 8 dyes simultaneously. 3. Click Excitation to expand the laser line selection box. 4. Choose laser line(s) by placing a check in the box next to the laser wavelength. In this example, we will use 488 for Alexa 488 and 543 for Alexa 568. 5. Set Transmission % to reasonable starting value: Argon 5-10% HeNe 543 100% HeNe 633 100% Diode 405 5-10% 10 LSM 510 META Training Guide

Beam Path Configuration 6. Select primary beamsplitter (HFT) to match laser line combinations used. Note: HFT primary beamsplitter will reflect wavelengths of light noted on the filter. In this example, the HFT 488/543 will reflect wavelengths at 488 and 543nm and transmit all other wavelengths. 7. Select secondary beamsplitter(s) to direct fluorescence emission to desired detector. In this example, we use a Mirror to direct all emission to Ch2 & Ch3 then use an NFT 545 to split the Alexa 488 (Ch2) from the Alexa 568 (Ch3). Note: NFT secondary beamsplitters will reflect all wavelengths < cutoff number and transmit all wavelengths > cutoff number. For example, the NFT 545 will reflect wavelengths < 545nm and transmit wavelengths > 545nm. 8. Select emission filter for each channel. Note: Emission filters come in two varieties. BP (band pass) filters transmit only the wavelengths within their range while LP (long pass) filters transmit all wavelengths > the cutoff number. 9. Place a check in the box next to each detector to activate it for imaging. 10. Choose pseudo-color for each resulting image. 11 LSM 510 META Training Guide

Acquisition Setup 1. Once your configuration has been established, click on the Scan button from the main toolbar. 12 LSM 510 META Training Guide

Acquisition Setup The Scan dialog contains 2 areas, Mode and Channels, which are selected from the top of the dialog. Mode This dialog controls static image settings such as: Objective displays currently selected objective as programmed into microscope. Frame Size image size in pixels. Default is 512x512, however this can be optimized based on objective and zoom settings. Ideal sampling frequency can be quickly selected with the Optimal button. Scan Speed overall speed of the scan during acquisition. Reports pixel dwell time and total scan time. Increasing dwell time (slowing down the scan) will improve signal-to-noise ratio. Bit Depth the number of bits used to indicate the range of signal level in the sample. The default A/D conversion is 12 bit. Averaging selecting Number >1 will scan each X line the number of times and average the result. Used to reduce image noise. Scan Area allows scan area adjustments such as X,Y displacement and rotation of scan in 360 degrees. Zoom can be used to increase magnification without signal loss or objective change. 13 LSM 510 META Training Guide

Acquisition Setup The Channels menu contains the three components used to dynamically control image quality. They are (1) Laser Power, (2) Detector Sensitivity, and (3) Pinhole or Confocal Aperture Diameter. For further discussion on these parameters and how they affect image quality please see Understanding Image Quality starting on page 19. Channels Functions available in the Channels dialog: Lasers controls laser power (%) and wavelength selection. Pinhole confocal aperture diameter that controls optical section thickness. Reports section thickness and pinhole diameter. Detector Gain controls the analog amplification voltage for the PMT (in Volts). The higher this value the more sensitive the detector becomes to the signal. Noise is also amplified by gain, albeit at a slower rate. Amplifier Offset controls the dark current offset for the imaging system. When scanning an image with all lasers off this value set to 0 should report background grey values close to/at 0 grey levels. Amplifier Gain is a digital amplification of signal that is applied post A/D conversion. This amplifies signal at the same rate as it amplifies noise. Leave this value set to 1 there are other locations to control image brightness that are more efficient and less destructive. 14 LSM 510 META Training Guide

Scanning an Image Once the laser(s) have been turned on and the detector(s) have been selected, we can scan an image and begin adjusting our signal level. 1. From the Channels dialog, set the Pinhole to 1 AU for the longest wavelength channel. Note: Setting the pinhole to 1 AU is a compromise between axial resolution and signal level. When you start with an open pinhole, you are at a point where your signal is the highest but your axial resolution is at its lowest. As you reduce the aperture diameter you are increasing your Z resolution but reducing your signal level. This is a reasonable trade off until you reach 1 AU. Reducing the pinhole lower than 1 AU will still linearly increase your Z resolution but now signal will start to drop exponentially. 2. Click Find to have the system scan the image and automatically adjust the gain and offset. Note: The automatic exposure setting works reasonably well with very bright signals. It is not intended as a final optimization, just as a baseline before fine tuning. 15 LSM 510 META Training Guide

Fine Tuning Image Quality In order to find the optimal settings for a particular condition, you must first be able to identify the thresholds of the signal level in the image. From the new image window that is generated with the scan, there are a series of tools along the righthand side of the Image Display. Here you will find a button labeled Palette. In the Color Palette list, you will find Range Indicator which is a lookup table (LUT) that assigns red pixels to areas in the image that are overexposed and blue pixels to background areas that are underexposed. 1. Click on Palette. 2. Select Range Indicator from the list of lookup tables (LUT). Close Color Palette dialog when selection is complete. 3. From the options along the right-hand side of the Image Display, choose Split-xy to view each channel independently. Note: Split-xy is the only way to view the range indicator table on images that have more than one channel. 16 LSM 510 META Training Guide

Fine Tuning Image Quality To fine tune the image intensity to ideal levels for final image acquisition use the following steps: 1. Select one channel at a time begin by selecting the first channel in your list. 2. Click Fast XY to start scanning. 3. Turn on Range Indicator LUT as shown on the previous page. 4. Deselect laser checkbox for that channel. 5. Adjust Amplifier Offset until background areas are just above the display of any blue pixels. 6. While still scanning, reselect the laser line (4). 7. Increase Detector Gain until the brightest areas in the image are just below the display of any red pixels. 8. Stop Scan. 9. Repeat steps 4-7 for each channel independently. 10. Click Single to collect final image. 17 LSM 510 META Training Guide

Fine Tuning Image Quality Ideally, you do not want any area in the final image to contain red or blue pixels with this LUT. For maximum contrast, have the brightest areas fall just below overexposure (red) and the background areas fall just above underexposure limit (blue). In a properly balanced image you should see no red or blue pixels with this LUT. Example of Overexposed (red) and Underexposed (blue) Areas in Image Example of Properly Exposed Image Solution? Reduce Gain (Master) and increase Digital Offset 18 LSM 510 META Training Guide

Understanding Image Quality The result of the final scan above may or may not produce acceptable image quality. Therefore, it is important to have an understanding of the three key factors that play a role in image quality. Pinhole (Confocal Aperture) The confocal aperture controls both axial resolution and signal level. Opening the aperture reduces axial (Z) resolution but increases signal level reported to the detector. Closing this aperture will reduce the signal level but increase axial resolution. Laser Intensity The intensity of the laser illumination source has obvious effects on the signal level. Increasing laser power will increase signal levels but may also introduce nonlinear effects such as phototoxicity and photobleaching. Detector Sensitivity The detector sensitivity is regulated by the high voltage gain applied to the detector. Increasing the gain directly increases detected signal but also amplifies the inherent noise in the system. 19 LSM 510 META Training Guide

Understanding Image Quality Pinhole (Confocal Aperture) Ideally, the confocal aperture should be set to the size of the structure(s) you are trying to resolve. However, for example, some sub-cellular structures of interest may be beyond Abbe s diffraction limit and therefore beyond the microscope s capability to resolve. The confocal aperture has some practical limits that can be used to guide the usage of this setting. Start by closing down the confocal aperture to 1 AU. You can certainly close the confocal aperture below this value and continue to improve resolution, just understand that below 1 AU you will lose signal level at an exponential rate. Conversely, you can increase the confocal aperture diameter to improve the detected signal level if you can sacrifice axial resolution. In some cases, the signal level may be so low that increasing the aperture diameter is the only way to lower the gain enough to get a usable image. In other cases, if the gain is too high (causing excess noise) and the laser power cannot be increased due to photo effects then increasing the confocal aperture is the only option to improve image quality. 20 LSM 510 META Training Guide

Understanding Image Quality Laser Intensity The overall power of the laser will directly affect image quality but most importantly it will have the greatest impact on the health of your sample and fluorophore(s). Increasing laser power will yield more signal but it will also induce negative photo effects such as phototoxicity and photobleaching that can harm the sample. Lasers also generate considerable heat when used at higher powers and that may have unforeseen effects on the sample. For most lasers on this microscope, we recommend laser power settings between 1% and 5% to start. It is recommended to start as low as you can possibly go and still get an image. Then it can be increased as necessary to help balance image quality. The exception are the HeNe laser(s) these are very low power lasers that can be used safely at 100% output. To discover your fluorophore(s) saturation level (i.e. how much laser power you can use before your fluorophore stops absorbing additional photons) you can start imaging at a low laser power and gradually increase the power slider until you reach a point where your image stops getting brighter. Continual increases in laser power will stop yielding more signal. The point where additional laser power stops increasing signal intensity is the practical laser power limit for that particular fluorophore. 21 LSM 510 META Training Guide

Understanding Image Quality Detector Sensitivity The sensitivity of the PMT is a function of the high voltage gain applied to it. The gain setting (as discussed elsewhere in this manual) controls the sensitivity of the detector to photons of light emitted from the sample fluorescence. Increasing gain provides the most flexible and impactful way of improving image quality, but it has some drawbacks. The most obvious is when you increase the gain you also increase the noise in the resulting image. There are various forms of noise that we won t discuss here, but they are readily apparent when using a gain value > 500V. Below 500V it is very difficult to detect much noise at faster scan speeds, however when using the detector above this value you will find that you will need to make some adjustments to reduce this. If laser power is high and the confocal aperture cannot be compromised there are a few methods to dealing with noisy signal due to high detector gain. There are 2 main methods for dealing with high image noise other than laser power and confocal aperture. The first is reducing the scan speed to a slower rate. This will increase the pixel dwell time which will improve the signal-to-noise ratio. The second is signal averaging which will scan the same image multiple times and average the result to produce an image that has an improved signal-to-noise ratio. 22 LSM 510 META Training Guide

Understanding Image Quality Depending on the quality of the signal level in the final optimized image, you may find that the image is somewhat noisy. To compensate for this, the system has averaging functions in the Acquisition Mode tab (page 13) that can be used to clean up the image. Simply select the rate of averaging (1, 2, 4, 8, or 16x) and rescan. No Averaging 2x Averaging 4x Averaging 8x Averaging 16x Averaging Note: Averaging signal will increase the acquisition time by the factor of averaging. For example, a 4x average will increase the time to scan the image by a factor of 4. 23 LSM 510 META Training Guide

Sequential Image Acquisition The example beam path configuration illustrates how to collect 2 fluorophores simultaneously. Often, simultaneous acquisition will cause unwanted cross excitation of fluorophores leading to the effect known as Emission Crosstalk. Emission Crosstalk manifests as the signal from the lower wavelength fluorophore appearing in the image corresponding to the longer wavelength. In our example, we would see the signal corresponding to the Alexa 488 (green) showing up in the channel designated to the Alexa 568 (red) illustrated below: Example of Simultaneous Image Acquisition 24 LSM 510 META Training Guide

Sequential Image Acquisition Emission Crosstalk can be corrected by sequentially acquiring images or via spectral imaging. On the LSM 5 series, sequential imaging is known as Multi-Tracking. Multi-Tracking collects each channel independently, taking twice the amount of time for a 2 channel image as the simultaneous acquisition. Below you can see an example of sequential imaging. Compare this to the same area taken in the simultaneous acquisition above. Example of Sequential Image Acquisition 25 LSM 510 META Training Guide

Sequential Image Acquisition Sequential imaging (or Multi-Tracking) can be accomplished on the LSM 5 series by changing the configuration mode. 1. Select MultiTrack from the Channel Mode in the configuration dialog. 2. Select Switch tracks after each to Line. Note: Choosing Line will allow the system to switch imaging tracks between each X line, minimizing the potential for any sample drift during the scan. Note: By default, the configuration we programmed into the Single Track mode will transfer to the first track of the MultiTrack mode. 3. Give your first track a name. In this example, Alexa 568 will be our first track. 4. Turn on ONLY the detector that corresponds to the fluorophore for the current track. 5. Turn on ONLY the laser line that corresponds to the fluorophore for the current track. 6. Click Add Track. 7. Repeat steps 3-5 for the second track with the settings required for the additional fluorophore. 26 LSM 510 META Training Guide

Sequential Image Acquisition Example of beam path configurations for 2 channel sequential acquisition. Example of Alexa 568 beam path Example of Alexa 488 beam path 27 LSM 510 META Training Guide

Collecting a Z-Stack In order to collect serial sections (Z) we need to first activate the Z-Stack tools in AIM from the Scan button on the main toolbar. 1. Click Z-Stack from top of the Scan Control dialog. This will activate the Z Stack settings. 2. Select Mark First/Last from the Z options. 3. Click XY cont. to begin scanning an actively updated image. 4. Focus the drive in one direction (does not matter which direction you start with) until the image of your sample just goes out of focus. 5. Click Mark First to mark the start position. 6. While continuing to scan, focus the drive in the opposite direction until your sample comes back into focus, then continue focusing until it goes out of focus again. This time on the other side of the sample. 7. Click Mark Last. 8. Click Z Slice to open the slice thickness dialog. 9. From the Z Slice dialog, click Optimal Interval to set the interval size to be equal to ½ the optical section thickness. 10. Click Start to initiate your Z-Stack. 28 LSM 510 META Training Guide

Collecting a Time Series In order to collect a time series (T), we need to first activate the Time Series tools in AIM from the main toolbar. 1. Open the Time Series dialog from the main toolbar. The default mode for Start Series and Stop Series is Manual, which means that the Time Series will start immediately when the user clicks Start T and end when the Number of images has been scanned. To calculate the required number of time points, divide the duration you require for the series by the interval. For example, if we wish to collect for 6 hours in total with an interval of 5 minutes between images we would use the following equation (6x60) / 5 = 72 images. 2. Adjust Number of images to match your required calculations. 3. Adjust Time Interval to match your required calculations. 4. Click Start T to begin collecting the time series. 29 LSM 510 META Training Guide

Saving Images Once your image collection is complete you can save your data to the local hard drive. 1. From the Image Display window, click Save As in the lower right corner of the image. This will open the Save Image and Parameter As dialog. 2. From the Save dialog, select a database from the list of recently opened databases. Note: If you have not yet created a database click Create New Database and navigate to D:\ and create a folder with your name. Give your database a name and click Create to save the newly created database into your folder. 3. Give your file a Name and enter any description or notes you wish to have saved with the file and click OK. Note: The image file is saved into the same location as the database by default. Note: The default format for the AIM system is LSM 5. This file format is essentially a TIFF file that contains all the requisite system information in the file header. This will allow functions such as Reuse to work correctly. 30 LSM 510 META Training Guide

Shutting Down the System 1. Open the Laser Control dialog (See page 6, Power On Lasers) and select each laser you have powered on and turn them off. 2. Close AIM application. 3. Exit from Switchboard. 4. Shut down Windows operating system. Note: Please wait for operating system to shut down completely before proceeding to next steps. 5. Turn off X-Cite epifluorescence lamp (see Power On Routine Step 2). 6. Turn off Systems PC and Components power switches (see Power On Routine Step 1). 31 LSM 510 META Training Guide