Laser-stimulated fluorescence (LSF) imaging is a versatile observational technique that has a multitude of paleontological applications. Both automated and manual systems can be used to scan or otherwise observe fossils under laser illumination. A series of common steps apply to any LSF work, these are detailed below.
Laser light is concentrated on a specimen either as a point source for microscopic work, as a divergent light cone for smaller-sized specimens (with the aid of a laser diffuser), or a collimated beam (in which all light rays are parallel) is raster scanned over very large specimens. Since the laser is very bright, it must be blocked with an appropriate filter that still allows the longer wave fluorescence signal to pass through. Proper precautions using laser-blocking protective glasses and manufacturer’s safety protocol should be followed.
The equipment used with this methodology depends on the exact wavelength of light produced by the laser. Specialized light-blocking longpass filters, often used in astronomy, are best-suited for these methods. These particular filters will allow all wavelengths of light longer than a certain wavelength to pass through the filter, however, it will stop all shorter wavelengths. For instance, a red-orange longpass filter (LP580, MidOpt) will allow 91–95% of light between the wavelengths of 600–1100 nm, however, the transmission sharply decreases between 600–520 nm, and by 510 nm, no light passes through the filter (www.midopt.com ). A 477 nm blue laser would be efficiently blocked by this filter, but will still allow imaging of longer fluorescent wavelengths. The laser wavelengths and filters for each particular specimen were chosen experimentally via the trial and error method, a procedure that we believe is reasonable given the simplicity of this technique. The setup parameters for each of the five case histories presented in this study are given in Table 1 .
Standard UV bulbs can be used in addition to lasers in order to cover a broad range of the light spectrum. Imaging is done in both UVA from 315–400 nm and UVB from 280–315 nm. When working with UV light, photographs can be taken both with and without filters due to the low UV sensitivity of digital camera CCD (charge-coupled device) chips.
No special digital cameras are needed to photograph specimens using laser fluorescence. Typically, digital single lens reflex cameras (DSLRs) capable of manual time exposures (e.g. Nikon D610) with either wide angle or macro lenses are sufficient. Ideally, the photography should be done in a darkroom, basement, or office without windows or with blackout curtains, as any influence of natural light will reduce the clarity of the fluorescence. The use of a tripod is necessary, as the exposure time during photography is typically long—up to several minutes, although this may not be the case for close macro photography. The aperture setting (f-value) should be as low as possible for long-exposure shots.
Multiple types of laser light sources can be used. The more powerful the laser, the better and brighter the fluorescence. For the experiments outlined here, class III lasers in the 300–500 mW category were used. These were well below the threshold that results in radiation damage to the specimens studied. A lab laser, which plugs into the wall and is fairly static, and a high-powered laser pointer that runs off of CR123A lithium batteries, have both been used successfully depending on the locality of the specimen. The benefit of using a lab laser is that it can be used for hours at a time without overheating. It is typically used for precision work and photographing larger specimens. A high-power laser pointer is more portable and adjustable than a lab laser, however it can only be used for ~5 minutes, or else it will overheat and become damaged. If the photographer knows what f-value and shutter speeds are necessary for photography, a laser pointer can be used to great effect. It is excellent for macro photography in the field due to its portability.
A laboratory setup for table-top-sized specimens would typically hold the laser on a fixed mount (Fig 1 ). The laser itself emits a collimated beam, which results in only a small dot of illumination. This beam can be used as is for maximum flux or be expanded using a diffuser (ARF used a 20-degree diffraction diffuser from Thorlabs). The smaller the angle of the diffuser, the better—i.e. 20 degrees would be better than 50 degrees, as it restricts the beam to a narrower angle and results in a brighter and smaller area of illumination, even if the laser is placed further away from the specimen. The laser should illuminate as much of the specimen as possible, and the diffuser’s cone angle changes the area covered by the laser depending on the laser-to-specimen distance.
Larger specimens can be scanned using a custom device (Fig 2A and 2B ). A Powel laser line lens projects a laser line in the Y direction that evenly distributes the laser energy over the length of the line (Fig 2C ). A motor scans the entire assembly in the X direction (Fig 2A and 2B ). This allows specimens of almost any size to be imaged. The exposure time of the DSLR camera should cover one or more of the X direction scans of the specimen.
For a microscope setup, the collimated laser beam is directed through one of the illumination ports or projected directly onto the specimen. The emitted light, laser and fluorescence, comes back through the microscope’s optical train where a longpass filter is placed either before the objective lens or internally in a filter slot to block the intense laser light. The fluorescence can then be observed and photographed in detail.
Specimen sources for each case history:
Case history 1: Burke Museum of Natural History and Culture, UWBM 103073—feather from Green River Fm.; UWBM 103074—feather from Parachute Member of Green River Fm.
Case history 2: Department of Land and Resources of Liaoning Province, LVH 0026—fish specimen from Jiufotang Fm. [20 (link)]
Case history 3: UWBM 103075—microfossils from Brule Fm.; UWBM 103076—microfossils from Hell Creek Fm.
Case history 4: Gobero specimen housed in the University of Chicago Research Collection, G1B2—juvenile female skeleton from mid-Holocene lake deposits
Case history 5: Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China, IVPP V13320—Microraptor skull from Jiufotang Fm. [21 (link)]
Laser light is concentrated on a specimen either as a point source for microscopic work, as a divergent light cone for smaller-sized specimens (with the aid of a laser diffuser), or a collimated beam (in which all light rays are parallel) is raster scanned over very large specimens. Since the laser is very bright, it must be blocked with an appropriate filter that still allows the longer wave fluorescence signal to pass through. Proper precautions using laser-blocking protective glasses and manufacturer’s safety protocol should be followed.
The equipment used with this methodology depends on the exact wavelength of light produced by the laser. Specialized light-blocking longpass filters, often used in astronomy, are best-suited for these methods. These particular filters will allow all wavelengths of light longer than a certain wavelength to pass through the filter, however, it will stop all shorter wavelengths. For instance, a red-orange longpass filter (LP580, MidOpt) will allow 91–95% of light between the wavelengths of 600–1100 nm, however, the transmission sharply decreases between 600–520 nm, and by 510 nm, no light passes through the filter (
Standard UV bulbs can be used in addition to lasers in order to cover a broad range of the light spectrum. Imaging is done in both UVA from 315–400 nm and UVB from 280–315 nm. When working with UV light, photographs can be taken both with and without filters due to the low UV sensitivity of digital camera CCD (charge-coupled device) chips.
No special digital cameras are needed to photograph specimens using laser fluorescence. Typically, digital single lens reflex cameras (DSLRs) capable of manual time exposures (e.g. Nikon D610) with either wide angle or macro lenses are sufficient. Ideally, the photography should be done in a darkroom, basement, or office without windows or with blackout curtains, as any influence of natural light will reduce the clarity of the fluorescence. The use of a tripod is necessary, as the exposure time during photography is typically long—up to several minutes, although this may not be the case for close macro photography. The aperture setting (f-value) should be as low as possible for long-exposure shots.
Multiple types of laser light sources can be used. The more powerful the laser, the better and brighter the fluorescence. For the experiments outlined here, class III lasers in the 300–500 mW category were used. These were well below the threshold that results in radiation damage to the specimens studied. A lab laser, which plugs into the wall and is fairly static, and a high-powered laser pointer that runs off of CR123A lithium batteries, have both been used successfully depending on the locality of the specimen. The benefit of using a lab laser is that it can be used for hours at a time without overheating. It is typically used for precision work and photographing larger specimens. A high-power laser pointer is more portable and adjustable than a lab laser, however it can only be used for ~5 minutes, or else it will overheat and become damaged. If the photographer knows what f-value and shutter speeds are necessary for photography, a laser pointer can be used to great effect. It is excellent for macro photography in the field due to its portability.
A laboratory setup for table-top-sized specimens would typically hold the laser on a fixed mount (
Larger specimens can be scanned using a custom device (
For a microscope setup, the collimated laser beam is directed through one of the illumination ports or projected directly onto the specimen. The emitted light, laser and fluorescence, comes back through the microscope’s optical train where a longpass filter is placed either before the objective lens or internally in a filter slot to block the intense laser light. The fluorescence can then be observed and photographed in detail.
Specimen sources for each case history:
Case history 1: Burke Museum of Natural History and Culture, UWBM 103073—feather from Green River Fm.; UWBM 103074—feather from Parachute Member of Green River Fm.
Case history 2: Department of Land and Resources of Liaoning Province, LVH 0026—fish specimen from Jiufotang Fm. [20 (link)]
Case history 3: UWBM 103075—microfossils from Brule Fm.; UWBM 103076—microfossils from Hell Creek Fm.
Case history 4: Gobero specimen housed in the University of Chicago Research Collection, G1B2—juvenile female skeleton from mid-Holocene lake deposits
Case history 5: Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China, IVPP V13320—Microraptor skull from Jiufotang Fm. [21 (link)]
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