Leica DFC495 digital camera recommended settings

The download is a 1.8 megabyte JPEG image file listing my recommendations for settings to use with the 8 megapixel (produces 24 Mb TIFF image files in "interlaced large HQ" 3262 x 2442 pixel mode) Leica DFC495 color camera. This camera uses a Bayer mask on the CCD array.

We have one of these cameras on a Leica DMIRB inverted microscope in the University of Miami image core. The most commonly used objectie lens is a Leica plan-apochromat 20x / 0.6 NA objective lens. Assuming the condenser NA is adjusted correctly (approximately matches condenser NA), the resolution equation is

Airy XY resolution = 0.61 * Lambda / NA

Airy XY resolution = 500 nm (that is, 0.5 um).

I divide Airy XY by 3.5 to get the theoretical optimal pixel size:

Airy XY / 3.5 = (500 nm / 3.5) = 143 nm.

The objective lens to camera field of view / # pixels leads to:

214 nm / pixel for 1.0x optovar 134 nm / pixel for 1.6x optovar

this means to get full theoretical resolution with the 20x/0.6 NA objective lens and camera, we should use the maximum pixel count setting, "interlaced large HQ" 3262 x 2442 pixel mode, AND the 1.6x optovar.

I normally have a long working distance (LWD) condenser on the microscope, so it can be used with T75 and T25 flasks, SBS multiwell plates, as well as microscope slides. The LWD condenser has a maximum NA of about 0.55. We have available a "high dry" NA condenser, that can go to a higher NA (nominally ~0.9). Typically the LWD is "good enough" for most user's needs, as is the 1.0x optovar setting.

Correct microscope settings for optimal imaging: the enemies of excellent imaging with transmitted light are: 1. dirty microscope optics (or specimen). 2. failure to adjust the microscope for Koehler illumination (centered light path, field aperture diaphragm focused using the condenser focus knob with the specimen in focus), and failure to optimize the condenser NA, leading to either: 3. too small a condenser NA setting (dim, too much depth of field). 4. too large a condenser NA setting (too bright, washed out colors).

Optical density warning: the DFC495, like many color cameras, has its internal electronics adjusted so that no light results in zero intensity value. This is most apparent with the "over/under" warning turned on without light going to the camera: the entire image has the blue saturation warning marker. The bottom line is that this camera is not valid for quantitative immunohistochemistry or Feulgen reaction or any other application requiring optical density calculations. If you want to do optical density measurements, use a monochrome CCD camera, correct monochromatic (or near monochromatic) wavelength, optimally adjusted (and clean!) microscope.

Dr. McNamara, AICF Core Manager, thanks the DRIF and JDRF for funding the purchase of the Leica DFC495 color camera.