What camera and lens should I use for my whole animal Chemiluminescence imaging?
Field Of View
The formula to use is fov = ccd x wd /fl
Fl = focal length mm.
Ccd = horizontal / vertical length mm
Wd = Working Distance mm
Fov = Field Of View mm
I. Assuming you use Xenon 0.95/25, 1" standard lens, which has a minimum working distance of 0.30m with 2/3" format ccd (6.6 x 8.8 mm), 1344(H) x 1024(V) effective no of pixels.
Animal Horizontal dimension = 6.6 x 300 / 25 = 80mm
Animal Vertical dimension = 8.8 x 300 / 25 = 106mm
Above Field Of View is maximum magnification achievable by using Hamamatsu C4742-98-24ERG with above Xenon lens. You can't fill in your screen with a smaller animal, yet you can fill in your screen with a larger animal by increasing WD (working distance).
II. Assuming you use 50mm f/1.2 Nikkor Al-S lens, which has minimum focus distance of 1.7 ft (561mm) with array size, 13.3 x 13.3 mm, 1024 x 1024 pixels.
Animal Horizontal / vertical dimension = 13.3 x 561 / 50 = 150mm
Above Field Of View is maximum magnification achievable by using above set up. You can't fill in your screen with a smaller animal, yet you can fill in your screen with a larger animal by increasing WD (working distance).
The larger is the Field Of View, the lower is the magnification.
RESOLUTION
Chemiluminescence imaging
I. Assuming your set up is with Hamamatsu C4742-98-24ERG,
resolution will be
80mm / 1344 = 60u (H)
106mm / 1024 = 104u (V)
Since the digitization interval must be small enough to sample the smallest spacing with 2.5 to 3 pixels ( Nyquist Sampling Theorem), effective resolution will be better than 180u (H) and 312u (V)
II. Assuming you are using the other set up,
resolution will be
150mm / 1024 = 146u (V) and (H)
Since the digitization interval must be small enough to sample the smallest spacing with 2.5 to 3 pixels ( Nyquist Sampling Theorem), effective resolution will be better than 440u (H) and (V).
Fluorescence Imaging
I. Assuming you have a modern microscope set up with a 50x N.A. 1.4 objective, coupled to a CCD camera through a 1.5x relay lens. Assuming a wavelength of 500 nm (green), the objective is expected to deliver a resolution of approximately 0.3u. With an overall magnification of 1.5x50 = 75, the smallest resolved spacing will represent 15u at the camera plan. The digitization interval must be small enough to sample the smallest spacing with 2.5 to 3 pixels ( Nyquist Sampling Theorem). Hamamatsu C4742-98-24ERG cell size is 6.45u x 6.45 u, just the correct range for most demanding microscope. Any bigger pixel will deteriorate resolution.
Hamamatsu C4742-98-24ERG is suitable for both Fluorescence and Chemiluminescence imaging.
II. Assuming you are using the other set up with pixel size of 13 x 13u, resolution is inadequate for fluorescence with a high quality microscope.
Light Collection
I. Assuming you use Xenon 0.95/25,1" standard lens, which has a minimum working
distance of 0.30m and 39mm dia. The lens sees each element of the animal in an angle of lens dia / w.d = (39/300) = 130 m.rad
II. Assuming you use 50mm f/1.2 Nikkor AI-S lens, which has a minimum focus distance of 1.7 ft (561mm). The lens sees each element of the animal in an angle of lens dia / w.d = 50/561 = 89 m.rad
Lens I collects (130/89) ^2 = 2.13 more light than lens II, hence set up I needs about half the time needed for set up II. This has an important impact on time dependent noise.