Exploring the multiple exposure option on a digital SLR for photomicrography;

Using the Nikon D300 as an example.

 by David Walker, UK

The options available even on the budget to mid-priced digital SLRs can be impressive, sometimes to the point of overwhelming. A feature becoming quite common is 'multiple exposure', where a series of images are taken and the camera automatically overlays the complete set. This feature is distinct from the stitching type mode for creating e.g. panoramic landscapes.  The author's Nikon D300 has such a multiple exposure mode but have never really given much thought to date as to its potential use, either for traditional photography or photomicrography. But recently I've been exploring its potential and offer some experiences below which may be of interest to DSLR owners exploring the feature on their own cameras. In Nikon range, it's also a feature on e.g. the D60.

Combining in-camera images of suitable fluorescent subjects imaged at different exciting wavelengths

My interest in the multiple exposure option was prompted when I studied the complementary triply stained fluorescent slide sent to all entrants of the Nikon Small World 2008 competition. I don't have fluorescence on my Zeiss microscope but a colleague kindly allowed access to his Leitz stand fitted with a Ploemopak. I'm a novice at fluorescence but was pleased to find that the slide readily gave well exposed images with modest camera exposures even when using the 100W halogen brightfield lamp as the epi-light source (a source I preferred to start with rather than the mercury lamp).

The three images obtained in UV, blue and green excitation were saved to the camera's card but had no experience of how these images are combined to create the composites often seen in fluorescent images. I couldn't readily find a macro to do it for my rather dated Photoshop Elements 3.0. So tried the multiple exposure mode on the D300 instead, especially after the comment in manual that it can '... produce results with colors noticeably better than photographs combined in an imaging application because they make use of RAW data from the camera image sensor'.

Multiple exposure in the D300. The feature is readily accessed in the 'Shooting' menu and from 2 to 10 images can be combined. After defining the number of images to combine, the option to adjust the exposure gain is offered, ie a third exposure for each of three combined images etc. The manual notes that gain off is recommended for images with a black background.

The slide is admirably well labelled. By using Molecular Expressions fluorescent resources and wikpedia's entries on the stains, I believe the correct interpretation of the combined image is as follows.

Comments to the author David Walker are welcomed.

Microscopy UK Front Page
Micscape Magazine
Article Library

Please report any Web problems or offer general comments to the Micscape Editor .