Using lasers for fluorescence applications requires special consideration for the optical path. This special attention centers around the coherent nature of the light, the small beam diameter, and the power. In this note the emphasis is on the filters and dichroic mirrors used in the beam path.
Using lasers for fluorescence applications requires special consideration for the optical path. This special attention centers around the coherent nature of the light, the small beam diameter, and the power. In this note the emphasis is on the filters and dichroic mirrors used in the beam path.Synopsis
In standard epi-fluorescent microscopy the excitation filter is designed to reject all light, from deep ultraviolet (UV) to 1200 nm, other than the band needed for the excitation of the fluorochrome, which is typically a design with 30–60 nm of full-width, half-maximum transmission (FWHM). This optic in standard wide-field microscopy has very low specifications for optical quality and is designed for a 0 degree angle of incidence (AOI) on float glass.
For laser applications, the specifications are significantly different and much more stringent. While there are still many users that believe that lasers do not require clean-up filters, this is rarely true. All lasers produce some noise outside the primary line. Therefore, all laser systems should be tested for signal-to-noise with, and without, a clean-up filter. This optic is typically 10–20 nm FWHM, is ground and polished, and has an anti-reflection (AR) coating on any surface not used for the coating. The clean-up filters are designed for 3–5 degree AOI to insure that none of the reflected laser light goes back into the laser cavity.
The second optic in the configuration is the dichromatic mirror, which in wide-field epi-fluorescent systems is typically specified with low to moderate optical quality parameters. The dichroic mirror, also called the beamsplitter, should be made of fused silica and is traditionally designed for a 45 degree AOI.
In laser applications the dichroic mirror must be made to much more exacting specifications, especially concerning surface flatness and transmitted wavefront distortion. All laser mirrors require AR coating on any uncoated surface to reduce the laser reflections and scatter.
An important note regarding all dichroic mirrors is that any coating applied to a substrate, such as fused silica, will induce stress. This stress may be relatively minor and unnoticeable, but it may also be severe enough to exhibit extreme astigmatisms in the beam. Most manufacturers have some proprietary method for counteracting this stress.
The final optic in the configuration is the emission filter, also frequently called the barrier filter. The primary function of this optic is to block the excitation wavelengths of light from reaching the detector, with the secondary function to transmit the desired emission. This optic can be either a bandpass or a longpass.
For laser applications, the minimum requirements of the emission filter can be nearly identical to those in wide-field, except that the blocking at the laser line(s) should be a minimum of optical density 6 (OD 6), and they should be AR-coated in all cases.
In summary, laser optics must be designed and made differently from standard wide-field filters and mirrors. While it is true that most laser optics will work fine in wide-field applications, the inverse is clearly not true. Differences in design and construction may not be apparent in every laser application, but will be painfully obvious in most, especially the more demanding such as total internal reflection microscopy (TIRM), raman, and multiphoton microscopy.
Address correspondence to C. Michael Stanley, Ph.D., Chroma Technology Corp., 10 Imtec Lane, Rockingham, VT 05101, Tel.: 802-428-2500, Fax: 802-428-2525, [email protected] Chroma Technology Corp., PO Box 489, Rockingham, VT USA, [email protected], 800-824-7662, www.chroma.com.
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