General Questions:
Q. What is the function of the Two-Photon Microscopy Laboratory?
A. The Two-Photon Microscopy Laboratory was established as a core facility at ION to provide access to high end systems and to provide training for advanced imaging techniques. Our functions are:
1. To employ the latest technologies in the fluorescence microscopy field.
2. To keep our equipments in good working order.
3. To train new users to use our equipments correctly.
4. To assist experienced users in more advanced applications.
------------------------------------------------------------------------------------------------------
Q. What are the instruments currently available in the Two-Photon Microscopy Laboratory?
A.
1. Zeiss LSM510 confocal with an Axiovert 200M inverted microscope:
The LSM 510 is equipped with four lasers: an argon ion UV laser (351/364nm, 80mW), an argon ion blue laser (458/477/488/514nm, 30mW), a helium-neon green laser (543nm, 1mW), and a helium-neon red laser (633nm, 5mW). The microscope has three photomultiplier tubes (PMTs, detectors) for fluorescent signals and one for transmitted light.
Available objectives:
Plan-NeoFluar 5X/0.15, D=0.17 mm, Ph1, WD=13.6 mm
Plan-NeoFluar 10X/0.30, D=0.17 mm, DICI, WD=5.6 mm;
Plan-NeoFluar 20X/0.50, D=0.17 mm, DICII, WD=2.0 mm;
Plan-NeoFluar 40X/1.30 OIL, D=0.17 mm, DICIII, WD=0.2 mm;
Plan-NeoFluar 40X/1.30 OIL, D=0.17 mm, Ph3, WD=0.2 mm
Plan-Apochromat 63X/1.4 OIL, D=0.17 mm, DICIII, WD=0.19 mm
Alpha Plan-Fluar 100X/1.45 OIL, D=0.17 mm, DICIII, WD=0.11 mm
C-Apochromat 63X/1.20 W CORR, D=0.14 to 0.18 mm, DICIII, WD=0.24 mm;
2. Zeiss LSM510 two-photon with an Axioskop 2FS mot upright microscope:
The two-photon microscope has three visible lasers: an argon ion blue laser (458/477/488/514nm, 30mW), a helium-neon green laser (543nm, 1mW), and a helium-neon red laser (633nm, 5mW). In addition, the microscope is connected via a direct optical-couple to a Coherent integrated two-photon laser system comprising of a Verdi-V5 diode pumped solid state laser and a Mira 900-F modelocked Titanium:Sapphire laser system with X-wave (Mira-F-V5-XW-110). The Verdi is a high power, diode pumped solid state laser that can produce up to 5W continuous power at 532nm. The Mira is a regenerative model-locked Titanium:Sapphire laser and is used in the femtosecond configuration. This combination can be tuned to deliver a pulsed tunable infrared beam anywhere between 700-980nm for two-photon excitation. The system is equipped with two standard fluorescence PMTs/detectors, one META detector, one NDD detector, and one transmitted light detector.
Available objectives:
Achroplan 10X/0.30 W Ph1, WD=3.1 mm;
Achroplan 20X/0.50 W Ph2, WD=1.97 mm;
Achroplan 40X/0.80 W IR DICIII, WD=3.61 mm;
Achroplan 63X/0.90 W IR DICIII, WD=2.0 mm;
3. Instruments for electrophysiological experiments:
Some equipment is also available for performing electrophysiological experiments in conjunction with confocal or two-photon microscopy. These include an EPC-10 triple patch-clamp amplifier, a Master-8 eight channel programmable pulse generator (A.M.P. I.), 2 optically isolated ISO-Flex stimulus isolators (A.M.P.I.), 2 MP-285 motorized micromanipulators (Sutter Instrument Company), a Hamamatsu IR CCD camera (model C2400-79H with camera control unit model C2400-60, Hamamatsu Photonics K.K.) and a JVC color video monitor (model TM-H140PN, JVC Manufacturing Co., Ltd.).
-------------------------------------------------------------------------------------------------------
Q. Who is allowed to use the instruments of the Two-Photon Microscopy Laboratory?
A. We primarily service ION and SIBS faculties, postdoctoral fellows, graduate students and staff members. Schedule permitting, a limited amount of time is available for people from surrounding universities and the local biotechnology industry.
-------------------------------------------------------------------------------------------------------
Q. How can I schedule time on the equipment?
A. At present, you should make reservations by contacting our staff members in Room 419 of the ION building or by phone at 021-54921820. Later on, we will have an online reservation system for trained users to reserve time on specific equipments.
-------------------------------------------------------------------------------------------------------
Q. How can I cancel my reservation?
A. Cancellations must be made one day prior to your scheduled start time. Please call us at 021-54921820 to cancel.
------------------------------------------------------------------------------------------------------
Q. How do I arrange a training session?
A. Due to the complexness of the equipment, we do all training sessions on a one-on-one basis. To arrange a session, please contact Dr. Qian Hu in Room 419 of the ION building or at 021-54921820 to schedule a mutually convenient time. You need to provide a sample stained with the fluorophore(s) used in your experiments. It is advisable to schedule the appointment after you have worked out an effective staining protocol.
------------------------------------------------------------------------------------------------------
Q. What should I bring to training session?
A. You will need a WELL LABELED specimen. This means that you must try out staining protocols and look at your samples prior to arriving at the training session. Verify that the signal is strong (easy to see) and that the background is low. Our systems are based on inverted or upright compound microscopes, so the sample must either be:
n Mounted on a glass slide with a SEALED coverslip of thickness #1 or #1.5 only.
n In a slide chamber or dish with a GLASS COVERSLIP bottom. NO PLASTIC DISHES.
------------------------------------------------------------------------------------------------------
Laser Scanning Confocal Microscopy (LSCM)
------------------------------------------------------------------------------------------------------
Q. What is LSCM?
A. In conventional epi-fluorescence microscopy, light of a defined wavelength is reflected by a dichroic mirror through the objective and bathes the entire specimen in relatively uniform illumination. The dichroic mirror reflects light of shorter wavelength light and transmits longer wavelength light. Emitted light from the specimen (which is of longer wavelength than the excitation light) passes through the chromatic reflector to the eyepiece for visualization. The major problem with conventional microscopy is that up to 70% of the light collected is out-of-focus scattered light, resulting in interference. In thick specimens, this commonly results in an inability to decipher the precise location of labeled proteins.
LSCM was developed to overcome the problems associated with out-of-focus light. In a LSCM system, a single point of excitation light is scanned across the specimen. The point is a diffraction limited spot on the specimen and is usually produced by focusing a parallel laser beam. With only a single point illuminated, the illumination intensity rapidly falls off above and below the plane of focus as the beam converges and diverges, thereby reducing excitation of fluorescent molecules from interfering objects situated out of the focal plane. Fluorescent emissions pass through the dichroic reflector and a pinhole aperture situated in a focal plane conjugate to the specimen. The pinhole acts to restrict light passing to the detector strictly to that emitted from the focal plane. Light rays from below the focal plane come to a focus before reaching the detector pinhole and diverge so that most of the rays are physically blocked from reaching the detector by the leaves of the detector pinhole. In the same way, light reflected from above the focal plane focuses behind the detector pinhole, such that most of that light hits the leaves of the pinhole and is not detected. Light passing through the image pinhole is detected by a photomultiplier tube.
In summary, a confocal imaging system rejects out-of-focus light by two strategies: a) by illuminating a single point of the specimen at any one time with a focused beam, so that illumination intensity drops off rapidly above and below the plane of focus, and b) by the use of a pinhole aperture in a focal plane conjugate to the specimen so that light other than that emitted from the single illuminated point in the specimen does not reach the detector.
-------------------------------------------------------------------------------------------------------
Q. Does LSCM bleach samples faster than conventional epi-fluorescent microscopy?
A. Fading/bleaching of a labeled specimen is a major problem in fluorescence microscopy. The higher power and focused beam of a LSCM acelerates fading of a specimen as compared to conventional epi-fluorescent microscopy which essentially bathes the entire specimen in lower power, wide-beam excitation light. However, this is balanced by the shorter duration with which the laser illuminates the specimen in a LSCM as only a tiny portion of the field is illuminated at any one time in the scanning process.
----------------------------------------------------------------------------------------------------
Q. What are the numerical apertures (NA) of the different objectives on the Zeiss Axiovert 200M inverted microscope and Axioskop 2FS mot upright microscope?
A. The Zeiss Axiovert 200M inverted microscope and Axioskop 2FS mot upright microscope have 12 different objectives:
Plan-NeoFluar 5X/0.15, D=0.17 mm, Ph1, WD=13.6 mm
Plan-NeoFluar 10X/0.30, D=0.17 mm, DICI, WD=5.6 mm;
Plan-NeoFluar 20X/0.50, D=0.17 mm, DICII, WD=2.0 mm;
Plan-NeoFluar 40X/1.30 OIL, D=0.17 mm, DICIII, WD=0.2 mm;
Plan-NeoFluar 40X/1.30 OIL, D=0.17 mm, Ph3, WD=0.2 mm
Plan-Apochromat 63X/1.4 OIL, D=0.17 mm, DICIII, WD=0.19 mm
Alpha Plan-Fluar 100X/1.45 OIL, D=0.17 mm, DICIII, WD=0.11 mm
C-Apochromat 63X/1.20 W CORR, D=0.14 to 0.18 mm, DICIII, WD=0.24 mm;
Achroplan 10X/0.30 W Ph1, WD=3.1 mm;
Achroplan 20X/0.50 W Ph2, WD=1.97 mm;
Achroplan 40X/0.80 W IR DICIII, WD=3.61 mm;
Achroplan 63X/0.90 W IR DICIII, WD=2.0 mm;
--------------------------------------------------------------------------------------------------
Q. I have trouble finding my sample when I look through the eyepieces. Are there any special tricks that I can use to find it more easily?
A. Check the following:
Make sure that the eyepiece position is selected on the beamsplitter, and that you actually have light hitting the sample.
If you are using the mercury arc lamp, also make sure that you have selected a filter suitable for the fluorophore that your sample is labeled with.
Next, make sure that you have the slide oriented the correct way. The coverslip should be facing the objective.
If you're using an oil objective, also make sure that there is just enough oil (not too much oil and no air bubbles) such that the oil is in contact with both the objective and your coverslip.
Try to focus while moving the stage around. It's easier for the eye to detect moving objects than stationary ones.
Also try to focus using transmitted light and the mercury arc light simultaneously, then turn off the transmitted light when you think you have found something.
If you still can't see anything, there is a good chance that your staining did not work. This is especially likely if you have not looked at your sample before coming to the Two-Photon Microscopy Laboratory.
There is also an off chance that the mercury arc lamp has been misaligned. DO NOT try to fix this yourself, but ask our staff members for help. We may also be able to determine if this is the only problem, or if there is anything else that can be done to find your sample.
-------------------------------------------------------------------------------------------------------
Q. Why can't I focus on my specimen?
A. The higher magnification objectives on the microscope all have fairly high numerical apertures (~1.4). This means that the depth of focus (how far into the sample you can focus) is very small, so your sample must be close to the objective. The most common problems are:
1. The coverslip is incorrect for high NA lenses and you cannot see "through" the coverslip to the specimen.
Correction - Use only #1 or #1.5 coverslips.
2. The cells were grown on the glass slide, then mounting medium was applied and then the coverslip. If too much mounting medium is used, the objective cannot focus through to the specimen.
Correction - Apply mounting medium sparingly or grow cells on coverslip and mount on glass slides.
3. Cells were grown in chamber slides with gaskets and the gasket was not removed during mounting. Specimen is too far away from the objective.
Correction - remove gaskets or grow in chamber slides with coverslip bottoms (instead of glass slide bottoms).
-------------------------------------------------------------------------------------------------------
Two-Photon Fluorescence Microscopy
-------------------------------------------------------------------------------------------------------
Q. What is two-photon fluorescence microscopy?
A. The two photon microscope depends on the two-photon effect, in which a chromophore is excited not by a single photon of visible light, but by two lower energy (infrared) photons that are absorbed simultaneously (within 1 femtosecond). Fluorescently-labeled specimens are illuminated by a titanium:sapphire laser that produces very short (less than 200 fs) pulses of infrared light--with a very large peak amplitude (50 kW)--at a rate of 76 MHz.
Fluorescence from the two-photon effect depends on the square of the incident light intensity, which in turn decreases approximately as the square of the distance from the focus. Because of this highly nonlinear (~fourth power) behavior, only those fluorescent molecules very near the focus of the beam are excited. The tissue above and below the plane of focus is merely subjected to infrared light that causes neither photobleaching nor phototoxicity. Although the peak amplitude of the IR pulses is large, the mean power of the beam is only a few tens of milliwatts, not enough to cause substantial heating of the specimen.
Or to answer another way:
The traditional drawback of using fluorescent markers in conventional microscopy is that out-of-focus information is collected, making the precise subcellular localization of proteins within the cell or tissue difficult to achieve. Current solutions to this problem include confocal laser scanning microscopy or wide-field deconvolution technologies to generate optical slices that include only in-focus information.
While these systems provide a solution for some studies, there are two problems that neither can overcome. One is that biological tissues are highly diffractive, making signal determination at significant depth difficult to achieve. The other problem is that the excitation wavelengths of the commonly used fluorophores are often toxic to cells, making time-lapse analyses of live cells virtually impossible for periods longer than several minutes.
A recent technological advance for overcoming these problems is the use of two-photon (2P) excitation produced by an infrared ultrashort pulsed laser beam. The pulsed laser allows the same fluorophores to be excited by photons of twice the wavelength than those used in single photon systems. The longer wavelength photons are not absorbed by the cells, resulting in decreased toxicity to living cells and decreased photobleaching. The infrared wavelength excitation significantly reduces scattering within the tissue as the scattering coefficient is inversely proportional to the square of the excitation wavelength, resulting in deeper penetration into the specimen.
-------------------------------------------------------------------------------------------------------
Q. What are the benefits of 2P excitation?
A.
1. Increased choice of fluorescent molecules: The single photon lasers on confocal systems (Ar, Ar/Kr, HeNe) usually have excitation lines in the range of 488nm - 647nm. This means that investigators wishing to use UV excitable dyes such as DAPI, Hoescht, BFP or CFP are unable to analyze their samples on a single photon confocal system. With 2P, the excitation wavelength is doubled, so UV dyes can be excited with near infrared (NIR) light.
2. Reduced photobleaching: Investigators who want to do fluorescence resonance energy transfer (FRET) with CFP/YFP can do so more effectively, as photobleaching is reduced.
3. No specialized objectives: From a hardware point of view, UV excitation with NIR wavelengths means that special UV optical components are not necessary.
4. Increased signal-to-noise ratio: The larger disparity between the excitation and emission wavelengths increases the signal-to-noise ratio.
5. Bleaching restricted to focal volume: The axial resolution is achieved solely by focal excitation, obviating the need for the pinhole required in confocal systems. This effectively increases light detection and results in bleaching being restricted to the focal volume.
6. Increased penetration into the specimen: Infrared excitation wavelengths are not diffracted by cellular components and thus travel deeper into the specimen.
-------------------------------------------------------------------------------------------------------
Q. How does two-photon microscopy differ from confocal laser scanning microscopy?
A. Problems with normal confocal microscopy:
1. Lasers cause photobleaching of the fluorescent label (chromophore). Because the pinhole aperture blocks most of the light emitted by the tissue, including some light coming from the plane of focus, the exciting laser must be very bright in order to obtain an adequate signal-to-noise ratio. This bright light causes fluorescent dyes to fade with continuous scanning. Thus, the fluorescence signal weakens as subsequent scans are made, during the collection of a z-series or a time series.
2. Phototoxicity is also a problem. Excited fluorescent molecules generate toxic free-radicals. Thus, one must limit the scanning time or light intensity in order to keep the specimen alive.
3. Shorter wavelength photons are diffracted by cellular components and cannot travel deep into tissues.
-----------------------------------------------------------------------------------------------------
General Microscopy
-----------------------------------------------------------------------------------------------------
Q. How can I mount a thick specimen for microscopy without squashing it?
A. You need to support the coverslip to a level at least as high as the level of your cells/sample. People use a variety of tricks, including: a) using tape (scotch tape, electrician's tape) as "spacers" between the slide and the coverslip; b) making a tape "well" by cutting out a small area in the center of the tape, putting the sample in the well, and sealing the coverslip on top.
------------------------------------------------------------------------------------------------------
Q. Can I fix GFP labelled specimens?
A. GFP fluorescence does not survive dehydration or fixation agents such ethanol and acetone. Some reports suggest that the GFP signal is greatly reduced by formaldehyde fixation and that long term storage of samples in aldehydes will decrease the intensity of the GFP signal and increase autofluorescence. Other groups, however, have successfully fixed yeast cells with paraformaldhyde (4% in buffer, 1 hour fixation) without loss of signal. It is recommended, therefore, that you employ low percentage paraformaldehyde for short periods of fixation if possible (5-10 minutes for cells). Samples should be mounted in glycerol based mounting medium (approx 90% buffered glycerol).
Note: if your GFP is not a fusion protein that gets fixed in place, you will lose all signals upon permeabilization by detergents without prior fixation.
Some reports suggest that most nail polishes (actually the solvent in nail polishes, ethyl acetate/acetone) cause loss of the GFP signal. Groups report success with the following: a) Almay Creme (hypo-allergenic, "Canyon" shade); Wet 'n Wild's "Clear Nail Protector" (toluene/ formaldehyde free); b) melted paraffin: Simply melt paraffin and use it as a sealant.
The best quality imaging of GFP is obtained in living cells and tissues. The addition of super radical scavengers in cell cultures systems can help slow down the bleaching rate.
-------------------------------------------------------------------------------------------------------
Q. What do antifade reagents do?
A. When exposed to excitation light, all fluorescent dyes fade (photobleach). The rate of photobleaching is dependent on: 1) the intensity of illumination; and 2) the duration of illumination. Other factors which influence the fluorescence intensity and bleaching of fluorophores include the pH, the embedding medium and the presence of other substances that quench fluorescence.
The photon output of a dye represents the average number of cycles of excitation followed by fluorescence emission that the dye can go through before it is irreversibly photobleached. The average photon output is defined by the ratio of the probability that the dye will fluoresce (fluorescence quantum efficiency or Qf) and the probability that it will photoreact irreversibly to become a nonfluorescent species (photobleaching quantum efficiency or Qb). For example, fluorescein, which is very photolabile, has a Qf /Qb of about 30K in alkaline solution. Both Qf and Qb are properties of the dye that may be affected significantly by the dyes environment. The primary environmental influence on Qb is the presence of singlet oxygen and free radical species.
The main purpose of any antifade reagent is to sustain dye fluorescence, allowing longer observation times. This is usually accomplished by inhibiting the generation and diffusion of reactive oxygen species, thereby reducing Qb (preferably without any accompanying decrease in Qf so that fluorescence can persist).
-------------------------------------------------------------------------------------------------------
Q. Which antifade reagents are recommended?
A. A list of a few commonly used (and most effective) antifade reagents and their advantages/disadvantages is listed below. If available, the commercial source is also provided. References and comments from Confocal Mailing List subscribers are also given.
1. p-phenylenediamine (PPD)
Although it is one of the most effective antifade reagents, it suffers from photo/thermosensitivity, and toxicity - the latter attribute making it unsuitable for in vivo studies. Krenik et al. (1989) suggests the optimal PPD antifade mixture is a solution of 90% glycerol:10% PBS with a PPD concentrations between 2mM and 7mM with a final pH of 8.5 to 9.0.
2. n-propyl gallate (NPG)
NPG is nontoxic, and photo/thermo-stable. While not as effective as PPD (Krenik et al., 1989) it can be used for in vivo studies. Recommended concentrations seem to fall in the range of 3mM to 9mM and a glycerol base also appears to work best. The protocol for making it appears later in this FAQ.
3. 1,4-diazobicyclo[2,2,2]-octane (DABCO)
DABCO is stable, nonionizing, cheap and readily available. Protocol for making it appears later in this FAQ.
4. Ascorbic acid (Vitamin C)
5. Vectashield
Company description: Prevents rapid loss of fluorescence during microscopic examination, retains its anti-fading ability during long-term storage, Inhibits photobleaching of Fluorescein, Texas Red, Rhodamine, AMCA, and other fluorochromes. Unique, stable formula superior to buffered glycerol, polyvinyl alcohol-based mounting solutions, or those containing commonly used anti-fading agents, optically clear.
Vector Laboratories, Inc.
30 Ingold Road,
Burlingame, CA 94010 USA
Tel: (415) 697-3600
Fax: (415) 697-0339
6. Slow Fade
Company description: The original SlowFade formulation (S-2828) was designed to reduce the fading rate of fluorescein to almost zero. Because it provides a nearly constant emission intensity from fluorescein, the SlowFade reagent is especially useful for quantitative measurements and applications that employ a confocal laser scanning microscope, in which the excitation intensities can be extreme and prolonged. The SlowFade reagent can extend the useful fluorescence emission of fluorescein more than 50-fold and can preserve the signal in cell and tissue mounts for up to two years. However, the original SlowFade formulation does substantially quench fluorescein's fluorescence and almost completely quenches that of the Cascade Blue and Alexa Fluor 350 fluorophores.
Molecular Probes, Inc.
P.O. Box 22010 Eugene, OR 97402-0414 USA
Tel: (503) 465-8300
Fax: (503) 344-6504
7. SlowFade Light
To overcome this limitation, Molecular Probes' researchers have developed the SlowFade Light Antifade Kit (S-7461). The antifade formulation in our SlowFade Light Antifade Kit slows fluorescein's fading rate by about fivefold without significantly reducing fluorescein's initial fluorescence intensity, thereby dramatically increasing the signal-to-noise ratio in photomicroscopy. Moreover, the quenching of Cascade Blue, Alexa Fluor 350, tetramethylrhodamine and Texas Red dyes is minimal. In fact, the SlowFade Light antifade reagent reduces the fading rate of the Cascade Blue fluorophore to almost zero, while decreasing its emission intensity by only about 30%.
----------------------------------------------------------------------------------------------------
Q. What is the protocol for making DABCO anti-fade mounting medium?
A. DABCO embedding medium
1. Dissolve 2 g DABCO (antifading reagent) in 90 ml glycerol for 15-30 min at 60OC
2. Add 10 ml 1M Tris-HCl pH 7.5
3. Adjust pH to 8.0 with 5M HCl.
4. Cool to room temperature
5. Add 100 ml 20% thimerosal (in H20)
6. Optional: Add 50 ml PI (stock: 1 mg/ml PI in H20)
7. Store 4OC in brown glass or foil wrapped bottle
Abbreviations:
PI = propidium iodide
PI - excitation/emission similar to Rhodamine - use 488 or 514 nm lines (excitation) - long pass filter 570 or 600 nm (emission)
DABCO = 1,4-diazabicyclo [2,2,2]-octane
Ordering (Sigma) :
D2522 DABCO, 25g
T5125 thimerosal, 10g
P4170 PI, 10mg
--------------------------------------------------------------------------------------------------
Q. What is the protocol for making N-propyl gallate anti-fade mounting medium?
A.5% N-propyl gallate in glycerol (w/v)
1. Add 5% N-propyl gallate to 25 ml glycerol in 50 ml tube,
2. Tumble overnight to mix (room temperature or 4oC)
3. Allow one day for air bubbles to settle out before using.
4. Store at 4oC in brown glass or foil wrapped bottle
---------------------------------------------------------------------------------------------------
Q. The signal collected is very weak. What can I do?
A. The single most critical parameter in determining the quality of the image is the quality of the specimen preparation. A good staining procedure should yield strong signal with very little or no background. The following is a partial list of things to check if your staining is not optimal:
While antibody manufacturers will often cite an "optimum dilution" in their product inserts, you cannot trust that it is correct for all samples prepared with all protocols. The best solution is to perform a dilution series of both primary and secondary antibodies to determine which dilutions provide the best signal without giving a high background.
Try adjusting the blocking step (change composition of block solution, length and number of washes). If samples are fixed with formaldehyde, make sure that you block free aldehydes. While some adjustments can be made in hardware (strength of light source, sensitivity of detector), using these on samples with very low signal will usually result in an unacceptably high level of background.
