Search

Med Sci Imaging Facility

This suite of imaging devices is installed at Indiana University School of Medicine in the Van Nuys Medical Science Building. We currently have a Zeiss LSM 510 NLO (two photon) microscope in Room 305. We also have a fluorescence microscope for digital still photography (Spot camera) in Room 356. All components in this multi-user facility are available to trained users.

Facility Director: Steve Kempson, Ph.D.
Department of Cellular & Integrative Physiology
Indiana University School of Medicine
Indianapolis, IN 46202-5120
Phone: 274-1444



RULES IN THE FACILITY

  • This facility is self-service and requires responsibility and consideration among users.
  • No one gets access to the two-photon microscope without training.
  • The director will help you, but will not run experiments for you.
  • Clean up after you are done. This includes cleaning objectives, counter tops, and removing all your materials.
  • Save all your images/data on your own storage devices, and erase each day's files from the computer. Files older than one month with be purged without notice.
  • Adhere to schedule as closely as possible. Remove your name from the schedule ASAP if you can't make it. If you must cancel within 3 hours of your start time, NOTIFY Lixuan Tackett at 8-0419. (Please see User Fees tab).

Zeiss LSM 510 NLO Two-photon Microscope



OVERVIEW. This Zeiss two-photon microscope and accessories are installed at Indiana University School of Medicine in the Van Nuys Medical Science Building. It is a multi-user facility available to TRAINED users (see Overview tab). The instrument is designed for analysis of multiple dyes (e.g. multicolor immunofluorescence) as well as live cell multi-photon and confocal microscopy (e.g. intracellular regulation of pH and calcium). Current filter sets are designed for DAPI, AMCA, INDO-1, SPQ, Dansyl, FITC, BCECF, Propidium Iodide, SNARF-1, Lucifer Yellow, Rhodamines, Texas Red, Cy3 and Cy5. You can look at individual dyes or any scientifically reasonable combination of these dyes.



SCHEDULING MICROSCOPE USE
If you are a trained user, you can go to the Two Photon web Scheduler which is now maintained on the Internet as part of this site. To obtain the URL, please contact the MSIF director or the webmaster, Stanley Stump.

The Scheduler will allow you to schedule microscope usage. Every user has a separate userID, allowing each user to request time. The USERID and PASSWORD are the same as the ones you use to log-in to the microscope computer. Be aware that it is your responsibility to make sure that your requested time on the calendar does not overlap with anyone else.



OPTICAL CONFIGURATION
The microscope has four lasers providing a number of excitation wavelengths. A Titanium-Sapphire laser (700-1050 nm), Ar laser (458nm, 488nm 514nm), Green HeNe (543nm), and Red HeNe (633nm) are present. Any combination of these lines may be used for your imaging purposes. Of course you must select appropriate filters such that the emission light from your sample is not polluted by the excitation light: this can get tricky if you are using multiple laser lines. The Zeiss 510 has the possibility to rapidly switch between laser lines, which can reduce (but not eliminate) this problem.

Go to this link to see a graphical representation of the filter choices of the microscope. As shown, the system has three fluorescence detectors which are photomultiplier tubes (PMTs). These can be used for confocal fluorescence, confocal reflectance, or two-photon fluorescence collection. There is a separate transmitted light detector for high-resolution laser-excited Nomarski (differential interference contrast) imaging. Any combination of detectors can be used simultaneously to collect images.

The objective lenses available on the INVERTED microscope are a Zeiss C-Apo 40X water immersion lens (numerical aperture 1.2, 220µm working distance), Zeiss plan-Apo 63X oil immersion DIC lens (numerical aperture 1.4) and Zeiss plan-neofluor 100X oil immersion Pol lens (numerical aperature 1.3). Please note that if you must penetrate deeper than about 30µm into an aqueous environment, only water immersion lenses are appropriate for quantitative work with these aqueous samples.



MICROSCOPE SYSTEM STARTUP
1. Turn on Xenon arc lamp and/or Ti-Sa laser. IF YOU DON'T NEED IT, DON'T TURN IT ON. Xenon arc lamp first, making sure it ignites before going further. Then turn on Ti-Sa if needed. Note: HeNe and Ar laser are ignited after computer startup, and are the ONLY lasers that may be turned on or off while the computer is running.

    Turning on Ti-Sa:
  • Find Coherent power supply under air table. Turn key from standby to on. Press MENU SELECT button, select CHILLER SETTINGS, press MENU SELECT button again. Then turn on chiller by pressing on large (POWER ADJUST) knob on power supply. Should hear the chiller turn on. Press MENU EXIT twice to leave the menus.
  • Turn on Verdi pump laser. Press POWER LEVEL #2 button on power supply. Display should indicate 9W. Rotate POWER ADJUST knob on power supply to reach this setting if the preset is not the desired value. You will see LASER SEEKING in the display until the unit reaches approximately 25 amps. If the number of amps does not steadily climb, OR if the final value is greater than 26 amps, then tell Lixuan Tackett.
  • Allow 30-minute warm-up time. BEWARE: you have turned on our most powerful laser. Keep out of the light path!!!!
  • Turn on Coherent accessory electronics on top of air table (on Lucite shelf). Make sure toggle on front of MIRA 900 box is set to CW (not ML). Switch on power to unit (switch on back).
  • Turn on other accessory electronics. Turn on IST-REES analyzer (switch on back). On front, dial in last known Ti-Sa wavelength, and press HI GAIN button (light above button should turn on). Turn on Tektronix oscilloscope with ON/STBY switch.
  • Press SHUTTER OPEN button on Coherent power supply.

2. Press the REMOTE CONTROL rocker switch near the computer monitors to turn on LSM510 electronics. Press the button on the computer (top right round button on tower) to start computer boot.

  • Note and report any error messages that appear during computer boot.

3. You will have to sign in with your userID to gain access to the computer. Please note that charges for the system usage start at time of login.

4. Start the Zeiss 510 program. Make sure to select SCAN NEW IMAGES and EXPERT MODE.

5. Go to ACQUIRE and select LASER button. Turn on the lasers you will need for your experiments. If necessary, input the laser wavelength you will be using for the Ti-Sa (700-900nm is allowed). The computer needs this information to correctly control Ti-Sa laser scanning.

  • For maximum stability of the system, it is advisable to allow 15 minutes for Ar and HeNe lasers to warm up.


PREPARATION FOR IMAGING
1. Checklist: Correct objective lens in place? Ti-Sa laser wavelength correct in Zeiss program if the laser is to be used?

2. Go to ACQUIRE in the Zeiss 510 operating system. Select CONFIG to set up optical path. Some preset parameters for single and multiple dye combinations are available. Start with these to optimize your sample or manually set up each filter.

3. Place sample on stage. Focus in conventional transmitted light or conventional fluorescence. NOTE: we have no computer control of the transmitted light bulb and power supply. You must manually turn it on to use it, and it is best to switch off the lamp when done.

  • Setting up conventional viewing: Use MICRO button to activate microscope motor functions. Select dichroic block desired using computer controls.
  • Optimizing Nomarski imaging: Stop down upper diaphragm. Adjust condenser height for sharp diaphragm edges, and open diaphragm. Rotate polarizer lever on condenser arm towards you, and adjust DIC slider under objective for best background extinction.

4. Move epi-fluorescence filter set to laser scanning position (NONE). Switch to SCANNING mode. Press "Start". If you see an image but it is poor, optimize with contrast and brightness. If nothing appears, then press F9 during scanning to find the image.



SYSTEM SHUTDOWN at end of day
NOTE THAT YOU WILL BE CHARGED FOR CONFOCAL TIME UNTIL YOU LOG OUT OF YOUR USER ACCOUNT.

IF SOMEONE ELSE IS USING THE SYSTEM WITHIN AN HOUR AFTER YOU FINISH, FOLLOW INSTRUCTIONS IN NEXT SECTION

1. Quit LSM program, and if needed turn off the lasers as you exit. While waiting for lasers to cool, ask Windows to shut down. After computer says it is ready to shut down, make sure to wait the 5 min for lasers to cool. Then turn off LSM electronics with REMOTE CONTROL switch. Then turn off Ti-Sa laser first and then arc lamp power supply last. Make sure transmitted light is also off.

  • Ti-Sa laser power down: Turn off IST-REES power and TEKTRONIX power. Flip switch on MIRA900 power supply to CW and then turn power off on unit. On Coherent power supply under table, press POWER LEVEL #1 button on power supply (reading should be 0.01W); Turn key to Standby, WAIT 10 MIN !!! Then press MENU SELECT twice to get to the chiller control then press power adjust knob to turn off chiller. You are now finished.

2. CLEAN UP MICROSCOPE. Clean objectives, counter tops and remove all materials.

3. CLEAN UP COMPUTER. Save all images/data to your own storage device. Flash drives are the recommended storage device type. Then erase any of your files from the confocal computer. Any files older than 30 days will be erased without notice.



SYSTEM LOGOUT when microscope will be used again soon

IF SOMEONE ELSE IS USING THE SYSTEM WITHIN AN HOUR AFTER YOU FINISH, DO NOT TURN OFF ANY LASERS OR THE COMPUTER. LEAVE THE ENTIRE SYSTEM UP AND RUNNING FOR THE NEXT PERSON AND FOLLOW INSTRUCTIONS BELOW.

1. Quit the LSM program and logoff of your user account (from Start menu). If you do not do this you will be charged for extra time until the next person logs on!

2. Follow "clean up" instructions in the previous section (#2 and #3).



CONFOCAL IMAGING TIPS

  • NO IMAGE? Always look at the microscope stage to confirm that the laser beam is at the sample during scanning (to see the Ti-Sa laser you may have to use the IR viewer). If not, check dark slider, dichroic positions and verify that the laser is on. Most times, this is something simple and mechanical.
  • OPTIMIZING MULTIPLE CHANNEL COLLECTION? If you want to eventually collect several channels simultaneously, optimize the setup of each channel while all channels are actively collecting. If you don't want to blast samples with all lasers needed in the final imaging, then optimize using only one laser on at a time (you can only optimize one channel at a time anyway). This strategy is also useful for verifying the amount of cross-contamination between channels.
  • TROUBLE OPTIMIZING CONTRAST AND BRIGHTNESS? Contrast (PMT high voltage) control will not change background in your images, and higher contrast gives brighter (and noisier) images. Video gain also can increase brightness, but background will increase in parallel. Offset is used to control background levels. Be aware that the requirements for quantitative and qualitative imaging affect which of these controls will give you optimal images for your purposes.
  • WHAT IS ON AND OFF-SCALE IN THE IMAGE? There are four default LUTs loaded in the system: using your assigned colors for each channel, normal black and white (B/W), B/W with 0=blue and 255=red ("range indicator"), red-to-yellow color spectrum. Pressing the PALETTE button in the image window allows you to select among these choices. Use the Range Indicator to optimize settings.
  • LOSING TRACK OF YOUR SETTINGS? Look at the graphical interface (CONFIG button in ACQUIRE) to see entire optical setup. When you are happy with the day's settings, SAVE the parameters in a file under your own name (maybe even on your own disk) BEFORE starting data collection. That way if the system crashes in mid-experiment you can reconstruct conditions with minimal hassle.
  • KEEP LOSING YOUR SAMPLE? When you have the sample in focus, set the motor position to zero (using 'Z' button in STAGE control under ACQUIRE). Zero is easy to remember and find again.
  • WHAT IS THE BEST LASER POWER SETTING? Input the lowest power possible to your sample. There are two stages of control for laser power. The first stage is to regulate the laser power supply and thereby control laser output directly. For the Ar and HeNe lasers, this is done by computer using the LASERS button under ACQUIRE. This is the same place where you controls turning these lasers off and on, so it is clear you are manipulating the power supply itself. Here the percent of maximum power indicates how much power is coming out of the laser. The power supply of the Ti-Sa is controlled directly using the buttons and knob on the front of the Coherent power supply. The second stage of control regulates the amount of light entering the microscope. This is performed using the CONFIG button under ACQUIRE. Using these controls, a desired percent of laser output is allowed into the scan head. When this setting is set to 100%, then all of the available laser output is beamed into the microscope. It can only be increased further by using the first stage controls to increase laser output.

DATA STORAGE

RULE: SAVE ALL YOUR IMAGES/DATA ON YOUR OWN FLASH DRIVE or CD DISKS, AND ERASE ANY OF YOUR IMAGE FILES FROM THE CONFOCAL COMPUTER. Any data on these hard disks is eligible to be erased at anytime by anyone (They may need the space for new data collection, etc.).

  • Each user may use a flash drive or CD-RW disk (which can be erased and used multiple times to store up to 550mB of data like a monster floppy disk), and use it at the site to create a database (MDB) file. All images should be saved into your database file on your flash drive or CD-RW disk.
  • PLEASE NOTE: During data collection on the instrument, all files are kept only in the RAM memory of the computer. If you do not SAVE the images, they will be lost when you exit the LSM program. Also, if you collect a new image into the window without saving, it will erase that old image. Use NEW button in SCAN CONTROL to create a new site for collecting images that does not erase whatever is in the old image window.
  • Each lab must decide how to manage mass data storage and data transfer. Two solutions are available.
    • You may use one of the USB 2.0 ports on the front of the confocal computer for high speed data transfer to a flash drive.
    • The confocal computer has a CD drive which can work with CD-RW disks (which can be erased and used multiple times to store up to 550mB of data like a monster floppy disk) or CD-R disks (which can only be written once). You cannot save data directly to these drives during data collection, but can save files immediately after data acquisition.

SUGGESTED METHOD: Save all your images to your flash drive immediately after data collection.


Revised: 8/22/2010 SJS


Cortical F-actin disruption in skeletal muscle (Elmendorf and Strawbridge)


elmendoslide0003_image005.gif

Cells: rat epitrochlearis skeletal muscle

Condition: fixed and labeled with antibodies against caveolin-3 (a plasma and transverse tubule membrane marker) and cortical F-actin

Fluorochromes: (1) FITC (2) Red X

Exc/Em wavelength: (1) Green - 488/520 nm (2) Red - 572/596 nm

Information: Overlap of cortical F-actin and plasma membrane produces a yellow layer. Breakage of the layer indicates disruption of the cortical F-actin meshwork beneath the plasma membrane.


Nephron epithelial cells (Kempson)


kempsonslide0003_image002cropped.gif.jpg

Cells: MDCK cells (epithelial cell line from distal segments of nephron)

Condition: Live; ISO = controls in isotonic medium, HYP = incubated 6 h in 500 mOsm medium

Fluorochromes: (1) betaine transporter tagged with EGFP (EGFP-BGT), (2) FM 4-64 which labels plasma membranes

Exc/Em wavelength: (1) green - 488/500-550 nm, (2) red - 488/620-680 nm

Information: EGFP-BGT is mainly cytoplasmic in isotonic cells, but colocalizes with FM 4-64 in the plasma membrane during hypertonic stress (overlap produces yellow color).


Transformed endothelial cells (Elharrar and Franklin)


elharrarfranklinweb.jpg

Cells: SVEC4-10 (Murine small vessel endothelial cells transformed with simian virus 40) grown on cover slip

Condition: Cells were fixed, permeabilized, and labeled with primary polyclonal antibodies against prostaglandin E2 receptor subtypes, EP1, EP2, Ep3 and EP4 (upper 4 panels) or against selected enzymes of prostanoid biosynthesis, Cox-1, Cox-2, PGES and PGIS (lower 4 panels).

Fluorochromes: (1) anti-rabbit IgG labeled with Alexafluor 488, (2) SYTOX Orange (which stained the nucleus)

Exc/Em wavelength: (1) Green - 488/515 nm, (2) Red - 543/580 nm

Information: While the prostanoid synthesizing enzymes are localized within the cytoplasm (green), prostaglandin E2 receptors are localized within the cytoplasm (green) and the nucleus (yellow = green + red).


To access the Online Schedule please contact the Facility Director or the webmaster.

Facility Director: Steve Kempson, Ph.D.
Department of Cellular & Integrative Physiology
Indiana University School of Medicine
Indianapolis, IN 46202-5120
Phone: 274-1444


 

STANDARD FEES
Zeiss NLO Microscope time $20/hr

M-F, 8am-6pm

$15/hr

off-hours, all weekend hours

CD-RW disks (multi-read/write. formatted. 550mB) $5/disk
CD-R disks (write once. 650 MB) $2/disk
Microscope training $300/person

These are fees incurred by normal usage of the facility, when users adhere to the guidelines running the facility. In most cases, these are the only charges that are made to users. Please note that there is no charge for use of the SPOT camera system.

Microscope training includes two half day sessions of instruction (beginning and advanced) which are separated by several weeks, in which users have up to 10 hours of free microscope time to practice with their own samples. There may be a final session for training on the Ti-Sa laser, but this is included in the original cost as well. After completion of instruction, users are given access to the microscope at any time as fully trained users.


EXTRA FEES
No show fee $20/hr for time signed up
Failure to log out of microscope computer 4 hr laser time or until next user logs on, which ever is less

We have developed a series of additional charges to encourage people to help us run the facility efficiently. These extra charges will only be levied when our records indicate that a problem has arisen. There will be a separate indication in each monthly billing statement of these extra charges. They are not steep charges, but no one likes to be charged more than necessary. A brief explanation of each charge, and how to avoid it, is listed below.


No show fee $20/hour for time signed up

This charge is made when a user signs up for confocal usage but does not show up. This extra charge is because we need to keep the facility operating as much as possible during the week, and must penalize those who "block" the instrument by reserving it, but not using it. It should be noted that NO MATTER HOW LATE YOU CHANGE YOUR MIND, YOU CAN REDUCE THIS CHARGE BY HALF. If you must cancel or shorten your usage on the day you are scheduled, you must call Lixuan 278-0419.


Failure to log out of microscope computer 4 hr laser time or until next user logs on, which ever is less

In order for our records of confocal usage to be accurate, USERS MUST LOG OUT OF THEIR USER ACCOUNT WHEN DONE. If users do not log off, our records are flawed. We are imposing a standard charge if this occurs. To avoid this charge, all you need to do is log off your user account when you are done.


Updated: 8/22/2010 SJS 

 

635 Barnhill Drive, Medical Science Bldg. Room 385 | Indianapolis, IN 46202-5120 | 317-274-7772