Chapter 12 Microscopy Applications; Introduction; Mounting The Camera On The Microscope - Princeton Instruments PI-MAX System Manual

Microscopy Applications

Introduction

This chapter discusses the setup and optimization of your digital imaging system as
applied to microscopy.
Since scientific grade cooled imaging systems are usually employed for low light level
microscopy, the major goal is to maximize the light throughput to the camera. In order to
do this, the highest Numerical Aperture (NA) objectives of the desired magnification
should be used. In addition, you should carefully consider the transmission efficiency of
the objective for the excitation and emission wavelengths of any fluorescent probes
employed. Another way to help maximize the transmission of light is to choose the
camera port that uses the fewest optical surfaces in the pathway, since each surface
results in a small loss in light throughput. Often the trinocular mount on the upright
microscope or the bottom port on the inverted microscope provide the highest light
throughput. Check with the manufacturer of your microscope to determine the optimal
path for your experiment type.
A rule of thumb employed in live cell fluorescence microscopy is "if you can see the
fluorescence by eye, then the illumination intensity is too high". While this may not be
universally applicable, it is a reasonable goal to aim for. In doing this, the properties of
your camera should be considered in the design of your experiments.
Hardware binning can also be used to increase sensitivity. If sufficient detail will be
preserved, you can use 2 × 2 binning (or higher) to increase the light collected at each
"super-pixel" by a factor of four or more. This will allow you to reduce exposure times,
thereby increasing temporal resolution and reducing photodamage to the living specimen.
Another way to minimize photodamage to biological preparations is to synchronize a
shutter on the excitation pathway to the intensifier gate/shutter on the camera. This will
limit exposure of the sample to the potentially damaging effects of the excitation light.

Mounting the Camera on the Microscope

The camera is connected to the microscope via a standard type mount coupled to a
microscope-specific adapter piece. There are two basic camera mounting designs: the
F-mount and the C-mount. The F-mount uses a tongue and groove type mechanism to align
the camera with an adapter, while the C-mount employs a standard size thread to connect to
the adapter. Either or both types could be available for a specific camera model.
Chapter 12
143