Appendix 1 Practical Tips for Two-Photon Microscopy Mark B. Cannell, Angus McMorland, and Christian Soeller INTRODUCTION blue and green diode lasers. To provide an alignment beam to which the external laser can be aligned, light from this reference As is clear from a number of the chapters in this volume, 2-photon laser needs to be bounced back through the microscope optical microscopy offers many advantages, especially for living-cell train and out through the external coupling port: studies of thick specimens such as brain slices and embryos. CAUTION: Before you switch on the reference laser in this However, these advantages must be balanced against the fact that configuration make sure that all PMTs are protected and/or commercial multiphoton instrumentation is much more costly than turned off. the equipment used for confocal or widefield/deconvolution. Given Place a front-surface mirror on the stage of the microscope and these two facts, it is not surprising that, to an extent much greater focus onto the reflective surface using an air objective for conve- than is true of confocal, many researchers have decided to add a nience (at sharp focus, you should be able to see scratches or other femtosecond (fs) pulsed near-IR laser to a scanner and a micro- mirror defects through the eyepieces). The idea of this method is scope to make their own system (Soeller and Cannell, 1996; Tsai to cause the reference laser beam to bounce back through the et al., 2002; Potter, 2005). Even those who purchase a commercial optical train and emerge from the other laser port. To do this, select multiphoton system find that it helps to understand a bit more about filter settings that will allow some of the light from the internal how to optimize the performance of the fs laser system.1 This laser to exit the chosen coupling port. In order to bring two laser Appendix has been added to the Handbook to provide the basic beams to co-linearity, a beam-steering device is essential. A single- alignment and operating information that such people need. mirror beam steerer provides angular control while changing the First, the safety announcement... separation between the mirrors of a 2-mirror steerer provides beam translation (Fig. A1.1). It is also possible to achieve beam translation with a second LASER SAFETY angular control mirror. After adjusting the incoming near-IR beam to an intensity where it can be viewed without totally overwhelm- 2 Light sources for multiphoton microscopy are almost without ing the reference beam, adjust one mirror to make both laser spots exception very powerful pulsed lasers (laser class IV). It is vital merge at the surface of the other (angle-adjustable) mirror. Then that any personnel who perform alignment or other operations that that mirror is adjusted to bring the beams to co-linearity. We find carry a risk of beam exposure are familiar with and follow laser it useful to use a piece of light-blue paper as this shows the dimmed safety regulations. During routine operation one MUST ensure infrared beam well. If the laser has been tuned to the far part of that accidental exposure to the pulsed laser beam is prevented by the spectrum, you may have to use an IR viewer or viewer card to providing proper shielding and interlocks. visualize the beam. During alignment, protective eyewear is not an option — it is essential! See http://www.osha.gov/SLTC/laserhazards/ for US TESTING ALIGNMENT AND guidelines. SYSTEM PERFORMANCE On a regular basis and particularly subsequent to laser alignment, LASER ALIGNMENT the performance of the multiphoton microscope should be tested. The prime indicator of proper alignment of an imaging system is Just as in any other type of microscopy, correct optical alignment its point-spread function, as measured by using a sample contain- is crucial for achieving optimal, diffraction-limited performance in ing sub-resolution fluorescent beads. A test slide can be prepared 2-photon microscopy. The alignment of external lasers such as the by letting a drop of diluted beads dry onto a coverslip. The beads Ti:S or similar 2-photon sources into a laser scanning microscope are then embedded in a drop of Sylgard elastomer (Dow Corning, can be simplified if a well-aligned “internal” or reference laser is USA) with a microscope slide placed on top. We usually use available. In commercial confocal microscopes, typical candidate 0.2mm beads from Molecular Probes (Eugene, OR). These are lasers include Argon-ion or green HeNe lasers or, more recently, available in a range of colors suitable for 2-photon microscopy. It 1 The Multiphoton Users Group e-mail network at ·mplsm-users@ 2 As is explained below, this can be achieved by over-closing the slit and/or yahoogroups.comÒ, operated by Steve Potter at Georgia Tech, enrolled its reducing pump power, because mode-locking is not required. We typically 500th member in 2003. use <20mw @ 800nm and <10mW at 720nm. Mark B. Cannell, Angus McMorland, and Christian Soeller • Department of Physiology, FMHS, University of Auckland, New Zealand 900 Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. Practical Tips for Two-Photon Microscopy • Appendix 1 901 AB C FIGURE A1.1. (A) 2D simplification of the beam alignment process using a conventional beam-steerer. A vertical translation of a tilted mirror is used to bring the two beams to a common point on a second, tiltable mirror. (B) Rotation of the second mirror at the point of the common spot makes the two beams co-linear. (C) The co-linear beams after alignment. takes only about 30 minutes to prepare 10 slides in this way. Once ticularly if the system is to be used for 2-photon flash photolysis the elastomer has set, these slides will last for months if kept in a or combined confocal and multiphoton co-localization studies. dark drawer. As a result, they provide a good standard to check the In our laboratory we perform a basic system test with a pre- microscope sensitivity and resolution provided you have recorded pared bead sample on a daily basis. This check (usually conducted microscope and laser settings (including center wavelength, laser following system startup) is well worth the ~5 minutes it takes, power and bandwidth/pulse length) with each reference image. especially if it helps avoid debugging signal problems later when With proper alignment, the beads should blur approximately a precious biological sample is on the stage. evenly as you focus above and below them. Asymmetric blurring above-and-below best focus indicates spherical aberration while motion of the centroid of intensity means that the objective LASER SETTINGS AND OPERATION aperture is filled asymmetrically. The spatial resolution (without a pinhole) should be similar to confocal performance, values Historically, the mode-locked lasers used for 2-photon imaging between 0.2–0.4mm in plane, full-width at half maximum could be quite temperamental and ensuring that proper laser oper- (FWHM) and 0.5–0.8mm out of plane (in the z direction) should ation was a large part of the challenge of running a multiphoton be attainable when using a high-numerical-aperture (NA ~1.3) microscope. With the advent of fully computer-controlled turn-key objective. laser systems, this has become less of an issue. In any case, as the A very weak and noisy signal can have a number of causes. If most versatile source for 2-photon imaging is still the tunable there is no problem with the detectors or emission filters (most of Ti:S laser in the femtosecond configuration, we will focus on it which would also be apparent when operating the microscope with here. Regardless of whether you are using a fully automated or a conventional [1-photon] laser excitation), check that the laser manually adjusted Ti:S system, it is important to monitor and beam fills the objective rear aperture fully and evenly by rotating optimize the laser output before imaging. the objective turret to an empty position, placing a lens tissue over The choice of center wavelength is generally determined by the opening and inspecting the pattern of illumination (using an IR the fluorochromes to be excited. As a general rule of thumb you viewer if necessary). The beam should be accurately centered in should try to use the longest wavelength compatible with the dyes the empty socket and should form a uniform circle of light that in your sample as this will help minimize photodamage and also will cover the rear aperture (~8–10mm wide) of a typical objec- reduce scattering of the excitation light. Data on excitation spectra tive lens. If the light intensity at the rear aperture is low (<10mW) is now available from many sources in the literature and, if in make sure that no IR-opaque optical items are obstructing the illu- doubt, there are mailing lists where one can ask other researchers mination path.3 It is also possible that the beam is so badly mis- for advice (see http://groups.yahoo.com/group/mplsm-users/ and aligned that only scattered light is being observed. You can check http://listserv.acsu.buffalo.edu/archives/confocal.html). for this by ensuring that adjustments of the alignment mirrors have the expected effects on the spot in the BFP. If the microscope is a combined confocal/multiphoton system, MONITORING LASER PERFORMANCE the bead slide is also a useful tool to disclose alignment offsets between the 2-photon laser system and any other lasers. In partic- During tuning and imaging, laser operation can be very conve- ular you should check for any axial offsets (i.e., focus shifts), par- niently monitored using a spectrum analyzer.