Optical Layout: Confocal and 2 Photon Microscopy:
Optical
layout of
the microscope:
A major advantage of our design for a laser scanning microscope is
that it applies equally to the excitation pathway of both the confocal
and 2-photon microscope. Besides the excitation laser, the major
difference between systems is the detection of the emission signal.
For confocal
microscopy, the
scanning mirrors are used to de-scan the emission fluorescence and
direct the signal though a dichroic mirror to a confocal aperture. This
eliminates the out-of-focus light to restrict the image plane.
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For 2-photon microscopy, the emission fluorescence does not need to passed through a confocal aperture because there is no out-of focus light and it also does not have to be focused to form an image. Consequently, the emission signal need only be separated from the excitation light with a dichroic mirror behind the back-aperture of the objective before detection with a photomultiplier. Thus, the 2-phton optics are actually simpler than the confocal optics.
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Basic Components:
The basic components
of the system are addressed in the order they appear in the excitation
optical pathway.
Confocal Laser: The illumination is generated by either 20 mW solid-state laser (488 nm). Lasers of this type are compact, cool-running and have long-life times. A laser with a lower power rating can be seriously considered because we rarely use this laser at greater than than 20 % power even with beam attenuation with a neutral density filter of 2.0. The calculated laser illumination is in the order of 0.2 mW. Lasers of other wavelengths can be used.
We original used a 100 mW Argon gas laser but this had the disadvantages of being large, requiring strong air-cooling (with a noisy fan and air extraction problems) and a relatively short life-time. One advantage of a gas laser is that multiple laser lines be available. In this case, specific line filters are required to select the appropriate excitation wavelength.
2-Photon Laser: We use a Tsunami (Spectra-Physics) Ti-Sapphire laser pulsed with a 5 W Verdi laser. The laser output is about 800 mW. Be aware that this is a 2-piece and very large laser that requires a separate power supply and water-cooling system. These can be placed under a large optical table. Newer versions of this type of laser exist as self-contained instruments.
Shutter: A Uni-Bltz shutter from Vincent associates is used to control the specimen exposure. Design considerations were reflective shutter blades to avoid laser damage during prolong closure, opening speed or response time and shutter status monitoring for electronic control. The shutter can be manually controlled with a switch on the control box or by software via a computer IO board delivering TTL.
Top Adjustable Mirror: The laser is aligned with numerous steering mirrors. The exact placement of components will determine the numbers required. The important ergonomic feature is the placement of the adjustment controls above the mirror for easy manual access. The mirrors should be front-surface with a highly reflective spectrum relative to the position and wavelengths of light. The mirror used to reflect the 2-photon beam differs from the one used to reflect the emission wavelengths. We commonly use protected aluminum coating.
Beam Splitter: The power of Tsunami 2-photon laser is in considerable excess of the the requirements for biological imaging (<50 mW). This provides the opportunity to use a single laser to simultaneously drive multiple 2-photon microscope set ups.
Neutral Density Filter: The excess laser power is attenuated by a variable neutral density filter. However, accurate manual placement of the filter is difficult. Discrete step neutal density filters offer a better way to reliable reproduce intensity settings but variable intensity control can also be implemented by varying the laser power with digital accuracy. Because laser light is polarized, a rotatable polarizer can be used but this was found to unevenly affect the beam profile.
Plano-concave Lens: Principle function is to expand the laser beam to fill the back aperture of the objective lens and in doing so serves to extrapolate the origin of the laser as a point source at the focal point behind the lens. For the confocal microscopy the focal length of this lens (500 mm) plus the distance from the lens to the dichroic mirror should be about equal to the the distance of the confocal aperture from the same dichroic mirror. A lens is not required for the 2-photon because an aperture is not used and beam expansion is not essential (but could be used) because the cross-section size of the 2-photon laser is generally larger than that of the confocal laser.
Line Filter: (optional - depends on laser spectrum) The laser beam is selectively filtered for the required wavelength and band-pass.
Confocal Dichroic mirror: (Required for confocal, replaced with a steering mirror for 2-Photon) This dichroic mirror performs the critical function of both reflecting the excitation laser light to the scanning mirrors (M3H and CRS) and the transmission of the returning emitted fluorescence light to the the confocal aperture. A sharp cut-off near the excitation wavelength is desirable.
Vertical Scan Mirror (M3H or M3S): A mirror is attached to a scanner module generates a vertical scan of the laser and reflects this to the horizontal mirror. This mirror moves at rates of 15 to 120 Hz and is driven by a saw-tooth command signal . The mirror is offset from the scanner axis on the end of an arm extension or paddle. The M3H module has been recently replaced by the M3S module.
Horizontal Scan Mirror (CRS): The horizontal scan of the laser is generated by a counter rotation scanning (CRS) or resonant scanner mirror that oscillates at approximately 8 KHz. This mirror oscillates with a sinusoidal waveform. The M3H and CRS also de-scan the emitted fluorescence.
Mirror Bracket: The M3H/S mirror and CRS mirror are held orthogonally by a custom-built mounting bracket. The bracket allows the mirrors to be rotated as well as to be moved axial.
Scan Lens (Microscope eye piece): The scan lens which is simply a microscope eye-piece focuses the line scan generated by the rotating mirrors at a conjugative image plane of the microscope. This raster scan is relayed by the microscope optics to the conjugate plane within the specimen to excite the fluorescence dye.
2-Photon Dichroic: (absent in confocal) This dichroic mirror transmits the 2-photon excitation light and reflects the emitted fluorescence to a side port immediately below the objective back-aperture.
Blocking Filter: This filter (although of different specifications) is required for both the confocal and 2-photon systems to prevent or block any excitation laser light from reaching the light-sensitive photomultipliers but allowing all the emitted fluorescence light to reach the PMTS. The blocking ability of the filter is critical because even though only a small amount of excitation light reaches this filter, this is considerably strong than the emitted fluorescence light. Any light leakage is amplified by the PMTs to the detriment of the required signal. For the confocal system this is a long-pass filter. For the 2-photon system this is basically a low pass filter.
Confocal Aperture: Because the design has not compressed the optical axis, the size of the confocal spot is relatively large and aperture does not need to be microscopic. A variable zero-aperture iris is used. Variable depth of the confocal plane can be obtained by altering the aperture size. This has the advantage of being able to compromise between light sensitivity and axial resolution.
Selective Dichroic Mirrors and Filters: To provide the ability to detect fluorescence from a variety of dyes, either simultaneously or individually, a combination of dichroic filters and band pass filters can be placed in front of the PMTs but behind the confocal aperture. The filters are arranged to reflect short wavelengths and pass longer wavelengths. Ideally, the dichroic mirrors should be removable so that light losses are not encountered when wavelength separation is not required.
Photomultipliers (PMTs): A key element in the system is the PMT that detects the low level of fluorescence light. A separate PMT is used for each wavelength and up to 4 PMTs can be used simultaneously with the current design. Currently, for the confocal we use a side-on PMT. This has high sensitivity and a fast response time. The detection area is oblong but this is not a problem for detecting the light emanating from the stationary confocal aperture.
For the 2-photon, we currently use circular end-on PMTs. The large (1" diameter) face-plate simplifies light collection from the moving beam. However, these PMTs appear less sensitive than side-on PMTS. A potential solution to increase sensitivity are the channel PMTs from Perkin-Elmer that claim an order or two higher sensitivity. We are testing these as well as side-on PMTs in the 2-photon.
For simultaneous transmission microscopy, an additional PMT is placed above the specimen in the position above the microscope condenser to collect the transmitted laser light. With either phase-optics or differential interference optics, a non-confocal transmitted image is obtained.