HOLOMAR
	High resolution in situ HOLOgraphic recording and analysis of MARine organisms and particles
	MARINE SCIENCE AND TECHNOLOGY PROGRAMME (MAST-III)
	1994-1998
	HOLOMAR Project, contract number MAS3 CT97-0079
	RESEARCH AREA C: MARINE TECHNOLOGY
Sub-area 1.3: Generic Technologies - Underwater viewing
1	Objectives
The objective of this project is to develop a fully-functioning, prototype, underwater holographic camera (holo-camera) for holographic recording of marine plankton and seston within the upper water column. The holo-camera will be capable of either ship deployment or attachment to a fixed buoy and will be appropriately automated and controllable from the ship. It will, furthermore, allow holograms to be recorded with either an in-line (object in transmission) or an off-axis (object in reflection) holographic geometry depending on the local plankton concentrations and the specific requirements of the user (the camera will also be fitted with a variety of other sensors to measure key environmental parameters such as temperature, salinity and water depth). A dedicated, hologram replay facility will be developed in parallel with the holo-camera, containing fully-automated image analysis and data extraction facilities which will allow species identification and measurement of local concentration of a variety of marine organisms. The overriding benefit of developing this unique method of high resolution, 3D imaging is that it allows the non-intrusive, in-situ, recording of living organisms and inanimate particles in their natural environment. Furthermore, it affords the ability to gain knowledge of the behaviour of marine biological communities, their relationship with each other and with the particles with which they interact. This has hitherto been difficult or impossible to obtain.
1.1	Background to the problem
It is widely accepted that the earth’s climate is intimately connected with and dependent on processes occurring in the seas and oceans . Some of the processes are strictly chemical and physical such as dissolution of CO2 at the sea surface; but the underlying controlling forces are nearly always biotic involving the growth, death and sedimentation of the diverse array of plants and animals of the sea. Without the marine biota, the concentration of CO2 in the atmosphere would increase dramatically . The Joint Global Ocean Flux Study (JGOFS)  has attempted to distinguish the various processes responsible for flux of material in the oceans particularly with regard to the drawdown of CO2 from the atmosphere and much progress has now been achieved .
A serious drawback with almost all of these studies has been the assumption that the average properties of the particles (seston) or animals (plankton) contained in a body of water can be considered as representative of that body and thus can be used to determine their relationships and the dynamics of their interactions. The organisms and particles are not, however, randomly or evenly distributed in the water and the relationships between them depend to a large degree on their relative locations. This is apparent on examination of many properties in vertical profiles across chemical and physical gradients  but is also an intrinsic property of planktonic communities even in apparently well mixed bodies of water .
An important goal in oceanographic research is to improve our understanding of these relationships and a first step in this process is to appreciate that average distributions are not an adequate descriptor of the environment. The accuracy of the conceptual and mathematical modelling of these bio-geochemical processes suffers at present from inadequate data. This is largely due to an absence of appropriate techniques for making the necessary observations. Appropriate models cannot be developed unless, and until, reliable and accurate, non-invasive techniques are available to study the spatial relationships of the biotic and abiotic particles of the ocean. Up until now a wide variety of optical and electronic techniques have been employed with varying degrees of success, in the laboratory and in the field, to obtain the necessary data. However, all suffer from some disadvantage or another. Because of the unique advantages offered by holography for high-resolution imaging of large water volumes, the technique offers great promise to marine biologists (as has been confirmed by recent work), chemists, geologists and biogeochemical modellers.
1.2	Benefits of holographic recording of marine organisms
The essence of our project, is that holograms will be recorded, in-situ, of aquatic systems using a pulsed laser and subsequently replayed, in air, in the real image mode of reconstruction. The images, so produced, are life size, fully three-dimensional (retaining parallax and perspective information) and located in the real-image space in front of the observer. Such images can be directly interrogated by measuring microscopy or TV cameras to extract information from the holograms on the particles; for example, their dimensions, shape, identity and position. Viewing of the image using measuring microscopy will provide detailed high-resolution information at any point in a given plane of the image. This ability to select planes of the image for interrogation (“optical sectioning”) is crucial in evaluating the real image; and it is this which sets holography apart from standard photographic or photogrammetric techniques. This process of interrogation of an accurate real image holographic reproduction by direct optical measurement is known, by analogy with photogrammetry, as. It is worth pointing out that the use of holography as a means of high-resolution imaging and mensuration, has been exploited in only a few areas of science and technology and that our application represents a significant advance on the state-of-the-art.
Two basic holographic geometries, viz. in-line (Fraunhofer) and off-axis (Fresnel), are applicable for the recording and high-resolution imaging of marine organisms. These basic geometries have been evaluated for recording of plankton, and compared and contrasted in order to ascertain which method was most applicable for a given situation. Each method has different features and advantages (see Table 1) depending on the situation in which recording is carried out.
	in-line holography	off-axis holography	
Hologram replay	Fraunhofer real image	Fresnel real image	
Subject dimensions	1 m to 1 mm	> 40 m	
Subject transparency	95% unobscured field needed	High reflectivity needed	
Illumination	Single beam, back-lit	Twin beam, variable angles	
Particle concentration	Low concentrations	Medium/high concentrations	
Optical density of medium	Contributes to background noise	Contributes to background noise	
Refractive index mismatch	Not critical	Significant for finite field angles	
Table 1: Comparison of in-line vs. off-axis hologrammetry.
With in-line holography, a single laser beam propagates through the sample volume towards the holographic plate and records the interference between light diffracted by the object and the undiffracted portion of the illuminating beam. No spatially separate reference beam is used. The replayed hologram simultaneously forms two images located on the optic axis; which for a collimated beam are at equal distances on either side of the holographic plate. The organisms are therefore illuminated in transmission and the scene needs to have an overall transparency of about 95% in order that speckle noise does not seriously degrade the image quality; this obviously sets an upper limit on the recordable particle density. A further factor of in-line holography is the need to balance the size of the particles to be recorded with the object-to-hologram distance. The upper limit for good size measurement is about 1 mm at a recording distance of 1 metre. However, particles down to around 5 m dimensions can de identified and measured with ease.
By contrast, off-axis holography utilises a two-beam geometry: one beam illuminates the scene and the other directly illuminates the holographic film. It is the interference between diffuse light reflected from the scene and the spatially separate reference beam which meets the plate at a finite interference angle. Consequently, off-axis holography is usually applied to, primarily, opaque subjects of large volume, making it better suited to recording of more dense aggregates of marine particles (whereas in-line holography finds application for essentially low concentration scenes). The scene can be front, side or back illuminated (or even some combination of all three). Although there is no real upper limit to the size of particles which can be recorded (this is determined by available energy and coherence of the laser), there is a practical lower limit to the resolution which can be recorded (50 - 100 m). Clearly both schemes overlap in their characteristics and the circumstances in which they would be used.
1.3	Specific objectives of this project
Our project is distinct from earlier work in the use of holography in marine science, in that it is dedicated to the application of pulsed laser holography to in-situ 3D recording and analysis of marine organisms and particles. This is coupled with later viewing, identification and mensuration of the images using a completely automated system for data acquisition and analysis by real image reconstruction of the holograms. The proposal advances earlier work which demonstrated that high resolution holograms could be recorded of marine organisms in laboratory observation tanks of 100 l volume; holograms were recorded in both in-line and off-axis geometries and convincingly confirmed the viability of the technique for the study of the behaviour of marine organisms and particles.
Novel aspects of the proposed work include its being completely based around the use of pulsed lasers to record large sample volumes of up to about 105 cm3 (100 l) in the field. The use of pulsed lasers (with a duration of about 10 ns) of course, means that recording is not influenced by water movement. Stand-off distances are determined, primarily, by the output energy of the laser and its coherence length and in our prototype, will be up to one metre from the subject scene (bearing in mind, that the entire recorded volume must fall within the coherence length of the laser). Furthermore, by incorporating both in-line and off-axis holography varied particle sizes and concentrations can be recorded and measured. Although the recording of the holograms take place in water, replay of the image is carried out in air, in the laboratory, to provide identification and measurement of the organisms and particles. Using pulsed holography allows a very considerable increase in the recorded volume that can be studied while still obtaining a high optical resolution (7 µm has already been demonstrated). For the first time, the detailed local interactions between meso--zooplankton and micro-zooplankton, phytoplankton and seston can be observed, recorded and quantitatively determined.
For the above reasons, we will design and build an underwater holographic camera which will be dedicated to the recording of living marine organisms directly in their natural environment. The prototype “holo-camera” will undergo sea trials and will be deployed from a research vessel to establish its use in the field. An associated, high precision, reconstruction facility will be established and will employ sophisticated image processing algorithms to assist in data acquisition and identification and measurement of species concentrations.
It is important to stress that a considerable amount of fundamental background work has been carried out in demonstrating the potential of holographic recording, replay and the extraction of data from the images in tank experiments on plankton samples. Thus we believe that the basic principle is proven and are now seeking funding to develop a system that can be used by marine scientists in the field.
To summarise, the overall aims of this proposed project are:
•	to design, develop and build a precision holographic camera for subsea deployment;
•	to design, develop and build an automated hologram replay facility incorporating sophisticated data acquisition and image recognition facilities
•	to demonstrate the use of this camera for the recording of marine organisms and particles in a variety of subsea systems;
•	to demonstrate the use of the replay facility for characterisation of marine organisms and particles from such holograms and to show the measurement of parameters such as local concentration and mobility.
1.4	Relevance to the MAST III programme and the priority themes
The HOLOMAR project will contribute towards research area C.1.3 - MARINE TECHNOLOGY: Generic technologies - underwater viewing of the MAST-III programme. The holo-camera programme should advance and enhance our knowledge of techniques of underwater viewing and visual measurement of marine organisms. Holography is a method of imaging and mensuration with some unique advantages (three-dimensionality, freedom from perspective distortion, no depth-of-focus restrictions and retention of parallax, high-resolution etc.) not found in other imaging systems. Despite this, it has not yet found widespread application outside some important engineering applications. Our project extends the application of holography to the marine environment and enables, for the first time, observation and quantitative measurements of the detailed local interactions between the marine biota and particles.
Of the four priority themes recently put forward by MAST III, the present proposal clearly addresses Theme IV, (Technological opportunities), forging a strong link, as it does so, between industry and academic research. It applies also to Theme I, (Health of the oceans) by providing the ability to examine “critical biogeochemical processes and fluxes” leading to a better understanding of “small scale physical processes on the dynamics of marine ecosystems”. 
In this respect, this project is a unique application of modern optical technology to the marine sciences. It is our belief that the successful conclusion of this project will result in a widespread use of holography in biological applications.