An Imaging Microspectrophotometer (iMSP)
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1
University of Bristol, School of Biological Sciences, United Kingdom
We present a new instrument, an 'imaging microspectrophotometer' (iMSP) which allows the mapping of spatial absorption properties of microscopic heterogeneous biological samples. The instrument has been developed to address questions in visual ecology, and specifically how the spatial arrangment of dichroic pigments and ‘form’ dichroism in retinal mosaics underpins animal senstivity to different polarizations of light.
Conventional spectrophotometry allows the determination of an absorption spectrum from bulk liquid samples. For biological samples this is often impractical as the molecules of intrerest must be extracted from cellular membranes using detergents, centrifuged then resuspended. This destroys any information about how the local environment affects the absorbance and details of the pigment’s spatial arrangement within the cell is lost. The advent of the microspectrophotometer allowed intact cellular specimens to be investigated by focussing the incident light onto a small region of interest. However this technique only generates point measurements. For the first time an imaging microspectrophotometer (iMSP) combines imaging with spectropscopic analysis to explore spatial heterogeneities in absorption whilst also greatly increasing the speed and facility of data collection.
The design of the iMSP is relatively simple. A monochromated Xenon light source (Oriel) is focussed down onto the sample using an achromatic reflecting condenser (Edmund ReflXTM). The sample is held in a XYZ computer controllable stage (Newport) and imaged onto a cooled CCD camera (ProEM1024, Princeton Instruments) using infinity corrected optics (Mitutoyo). To prevent photobleaching of specimens such as photorepctors, the sample is brought into focus under 850nm infra red illumination as this is outside the range of photopigment absorption. During measurements, the position of the stage is adjusted to correct for chromatic aberation. Images are taken at a series of wavelengths and used to compute absorption spectra for regions of interest relative to a referance area. The user defines the wavelength range, exposure time, wavelength increment, region of interest and reference area using custom control software developed in LabviewTM (National Instruments).
The potential applications of this instument are wide ranging. Current projects include investigating the dichroic properties of cryosectioned photoreceptor mosaics and using the iMSP in conjunction with recently developed high pressure microscopy stage [1] to investigate photoreception at pressure in deep sea fishes.
Acknowledgements
The authors acknowledge funding from the Biotechnology and Biological Sciences Research Council.
References
[1] Automated high pressure cell for pressure jump X-ray diffraction; N J Brooks et al, Review of Scientific Instruments, 2010
Keywords:
absorbance spectra,
imaging,
iMSP,
photoreceptors,
Polarization sensitivity
Conference:
Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012.
Presentation Type:
Poster (but consider for Participant Symposium)
Topic:
Sensory: Vision
Citation:
McGregor
JE,
Temple
SE,
Partridge
JC and
Roberts
NW
(2012). An Imaging Microspectrophotometer (iMSP).
Conference Abstract:
Tenth International Congress of Neuroethology.
doi: 10.3389/conf.fnbeh.2012.27.00151
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Received:
27 Apr 2012;
Published Online:
07 Jul 2012.
*
Correspondence:
Dr. Juliette E McGregor, University of Bristol, School of Biological Sciences, Bristol, BS81UG, United Kingdom, juliette.mcgregor@bristol.ac.uk